ZA200506371B - Treatment of excessive osteolysis with indolinone compounds - Google Patents

Description

v

Treatment of Excessive Osteolysis with Indolinome Compounds

Background of the Invention

Bone is a dynamic tissuae, subject to a constant remodeling process that operates to . maintain skeletal strength and health. This remodeling process enta-ils two phases: an osteolysis phase and an osteogenesis phase. In osteolysis, osteoclast cells invade bone andl erode it by releasing a cocktail of acids and enzymes that dissolve collagen and minerals.

This creates a small cavity in thie bone. In osteogenesis, osteoblast cells deposit new collagzen and minerals into the cavity. VWVhen osteolysis and osteogenesis are din balance, no net chanmge in bone mass results. Each year, the bone remodeling process replaces approximately 2045 of a healthy individual’s bone throughout the skeleton.

In certain disease states , osteolysis is more active than osteogenesis, resulting in a ret loss of bone. Such excessive o steolysis may occur in localized areas of the skeleton or mo=re broadly throughout the skeletom. ‘Regardless, bone loss has serious health consequences, including fractures, hypercalceania, nerve compression syndromes, deformity and pain.

One particularly serious cause of localized excessive osteolyssis is cancer metastasis to bone, Cancer cells often secrete factors, such as macrophage colony~ stimulating factor (M—

CSP), Mat promote osteoclast evelopment and activity. When sucha cancers establish themselves in bone, they promote extensive osteolytic damage. Suck tumor-associated osteolysis coincides with many~ types of malignancies, including hennatological malignanciwes (e.g. myeloma and lymphoma) and solid tumors (e.g., breast, prostate, lung, renal and thyroid). In fact, 70% of patierats dying of breast cancer have bone metastases, and bone is the most common site of first d istant recurrence for breast cancer. C ancer patients with boxe metastases may survive for sev eral years, which highlights the need for therapies that reduce the effects of bone metastases. } When excessive osteolysis occurs throughout broad areas of ®he skeleton, it falls : under the generic description osteoporosis. Common types of osteoporosis include age- . related, post-menopausal, glucocorticoid-induced, diabetes-associate d and disuse osteoporosis. Worldwide, osteoporosis presents a staggering problerm. In the United States . alone, millions of individuals seaffer from the disease and its attendarxt pain, deformities and® debilitating fractures.

WO 2004/075775 PCT/US2004/00528=3 v

Osteoclasts, the cells that meadiate excessive osteolysis, operate under the control of numerous cytokines and growth factors (1, 2). They are multinuacleated cells that derive from monocytic precursors (3,4). Differertiation of the monocytic precursors into osteoclasts is a . compl-ex process that requires both NA-CSF and RANKL (receptor activator of the NF-«I3 ligand) (3,4). These factors, and all eother cellular and humoral r—equirements for osteoclast y differe=ntiation exist in the micro-env-ironment of a skeletal meta_stasis (17).

Macrophages, which are relasted to osteoclasts, are a maj-or component of the host cellulamr response to cancers, and can contribute to tumor growth-. In particular, macrophaages, as wel 1as tumor cells, secrete M-CSF, a key cytokine for devel=opment of osteoclasts froem monocyte precursors (18-22). Macrophages, as well as monocytes and some tumor cells, also expresss M-CSF receptors (23-28).

Inhibiting osteoclast developmment and function is a desir-able approach to treating excesssive osteolysis. However, the currently available substanc-es that do so have limitec3 utility. and often cause significant sicie effects.

Calcitonin, a peptide hormon e secreted by the thyroid in response to elevated seriam calcium, is a well-characterized inhibitor of osteoclast formatiorn and function (5). Howe=ver, chronic exposure to calcitonin leads to loss of its inhibitory effects on osteoclasts, througth down regulation of the calcitonin rec=eptor MRNA and calcitonit receptors on the surface= of osteoc lasts (6). Additionally, becausse calcitonin is a protein, it cannot be taken orally, as it would be digested before it could work. While it does not affect other organs or systems in the boedy, injectable calcitonin may c ause an allergic reaction aned unpleasant side effects includ-ing flushing of the face and hamds, urinary frequency, nau=sea and a skin rash.

Like calcitonin, TGF-P and e«chistatin, a snake venom, block osteolysis in vitro (7 ).

However, TGF-f and echistatin lack specificity, and echistatin bwlocks platelet adhesion, thereb y potentially causing life-threa_tening bleeding. . } Monoclonal antibodies that b-ind antigens expressed on owsteoclast cells also can block osteolysis (8-10). However, such an-tibodies can induce an immune response in patients.

Bisphosphonates also inhibit osteoclast activity (11), andl extensive data exists regard. ing their use. Despite their bemefits, bisphosphonates are gooorly absorbed from thes gastroJntestinal tract and often induces gastrointestinal discomfort. Moreover,

v bisphosphonates remain in the bone for years, creatingg a potential of blocking ncomal bone repair mechanismss if too much is taken for too long.

Immune ce=1l products such as interferon-gamrma (IFN-y), interferon-alpma (IFN-a), oncostatin M, and taxol, suramine and nitric oxide inkibit osteoclast activity as vovell (12-14). ) However, all of theese agents have significant side effescts that limit their utility. Interferons can induce flu-like= illness, taxol and suramine frequently have severe toxicities &associated with gastrointestin_al and/or hematopoietic side effects, and nitric oxide can indu_ce vasodilation and low blood pressure.

Estradiol iss yet another well-known inhibitor of osteolysis (15) that induces apoptosis, or programmed cell death, in osteoclasts. Similarly, retinoic acid inhibits osteolysis (16).

Unfortunately, thesse compounds also cause systemic effects that make them less— acceptable.

For example, long—term estrogen therapy poses an incweased risk of breast, uterirae, and ovarian cancer. Additionally, estradiol therapy can cause vaginal bleeding, breas=st tenderness, mood disturbancess and gallbladder disease. Selective estrogen receptor modulat-ors (SERMS) mimic e strogens in some tissues and anti-es trogens in others. They haave the advantage of causi—mg fewer unwanted side effects, but still provoke hot flashes and deep vein thrombosis in somee patients.

Thus, a coratinuing need exists for effective anci practical treatments for excessive osteolytic conditiomns. Based on this continuing need, #he present inventors devel®>oped a method of treating excessive osteolytic conditions by administering an effective samount of a compound of form ula I or II, which are described belo w.

Summary of the Inv ention

One embodiment of the invention relates to a nmethod of treating excessiv—e osteolysis by administering a-n effective amount of a compound of Formula I:

-

X

NC

2 oo . H (Ra) i

XX

0 (Re)p H @ wherein

R is independently H,. OH, alkyl, aryl, cycloalkyl, hetesroaryl, alkoxy, hetero-cyclic and amino; each R; is independerntly selected from the group consisting of alkyl, halo, aryl, alkoxy, haloalkyl, haloalkoxy—, cycloalkyl, heteroaryl, heterocyclic, hydroxy, -C(O)-Rg, -NRgR 10, -I™NR9C(0)-R1z and —C(O)NRgR 0 3 each R; is independerntly selected from the group conssisting of alkyl, aryl, hacteroaryl, —C(0)-Rs, and SO;R> ’°, where R’’ is alkyl, aryl, heteroaryl, NRgN;q or alkoxy; each Rs is independeratly selected from the group consisting of hydrogen, alB«yl, aryl, haloalkyl, cycloalkyl, heteroaryl, heterocyclic, hydroxcy, -C(O)-Rg and (CHR)R;;

XisOorS; B pis 0-3; q is 0-2; ris 0-3;

Rg is selected from thee group consisting of OH, alky, aryl, heteroaryl, alko xy, cycloalkyl and heterocyclic;

Ry and Ryo are independently selected from the group consisting of H, alkyl, aryl, aminoalkyl, heteroaryl, cycloalkyl and heterocyclic, or Rg and Ryo together with N may feorm a ring, where the ring atoms are selected from the group consisting Of C,N, O and S; ) R;; is selected from tte group consisting of ~OH, amimo, monosubstituted armino, disubstituted amino, amlkyl, aryl, heteroaryl, alkoxy, cycloalkyl and heterocyc=lic;

Ry; is selected from tle group consisting of alkyl, aryl , heteroaryl, alkoxy, csycloalkyl and heterocyclic;

Z is OH, O-alkyl, or —NR3Ry, where Rj and R4 are ind ependently selected from the group

AY

A 4 consisting of hydrogen, alk=yl, aryl, heteroaryl, cycloalkyl, zand heterocyclic, or Ry ancd R4 may combine with N to form a mring where the ring atoms are sel ected from the group conssisting of

CH2, N,O and S or 1 (Yn R r3 — Ng | ve

AN ~N ond \ | R*

R'/ nm wherein Y is independentl=y CH, O, Nor §,

QisCorN; nis independently 0-4; ancl mis 0-3; or a salt thereof, to a patiertin need of such treatment.

In a preferred embodiment, the compound administered to the patient is a cormnpound of Formula II:

F ee

H Ry)

N

H

AN

, = ©

N

Rib H am, where the variables are as previously defined.

In another embodirment of the invention, R; is halo «e.g., F and Cl) and Z is —_INR3Ry ’ wherein R; and Ry are indespendently selected from alkyl ard hydrogen in Formula I or II as administered to a patient imn need thereof.

In another embodiment, Z of Formula I or II is ~NR23R4, wherein R3 and R4 fom a morpholine ring.

In another embodirment, Z of Formula I or II is:

RT

Yn R3

ONLY

\ A 104 N ~ - : ); | R

R'"/m ’ wherein each Y is CF, each n is 2, m is 0 and R; amnd Ry form a morphol ine ring.

In any of the gpreviously recited embodiments, preferably R; is me=thyl and q is 2, wherein the methyls zare bonded at the 3 and 5 posit ions of Formula I or T1.

In a particulax- embodiment of the invention, the compound admin istered is selected from the group consissting of 0 I o

M3C NTN HiC NN [ BB

N CHj CHa x / H . / 0 ’ . By N 0 > 0

I J) I

H3C N=" N HiC N 0 / ) CH. [ ) NE _° x [A x [oh

N O ! N 0 and

H H

0

HaC N

B v \

N CHa 0 x _— \_ 0)

N

H bl wherein X is F, Cl, I «or Br. In a preferred embodimesnt, X is F.

In another par-ticular embodiment of the inve=ntion, the compound of formula I is selected from the growip consisting of:

‘wv 0 ro ©

HC NN HC NN

S$ BD

N Hy N CH, . 7 . BE . (¢} , y ; [o} ,

Compound 1 o Compound 2 (CO Wy

H,C. N————N HC. Ne NH / b. CH, [ ) CH, . I . A [¢] [o}

Compound 3 Compound 8 ls) [=o]

HC N wn \ HiC N vO

BB NR BB TT Le

N : N ’

F / H a / H 0} ’ ’ oO B

N N

H H

Compound 6 Compound 7 [e} [0]

H,C. N N \ HiC N ; S A TIT N° J \ ou an 3 3 Oo : BB Co [H [7 (¢} (o] y N

Compound 4 Compoumd 9 [o}

H,C \ / \ cH = (7

N 3

F / H and

[8] - N

H

Compound 5

.

In certa-in embodiments of the inventior, the patient suffering from excessive osteolysis has Osteoporosis, cancer that has metastasized to bone, canceer that secretes M-CSF, and/or is post-rmenopausal. . Yet another embodiment of the inventicon relates to a method o=f treating a cancer that expresses CSF1R, the M-CSF receptor, by adninistering an inhibitory amount of a ’ compound of Formula I or Formula II, as described above, to a patient_. In one embodiment, the cancer is determined to express CSF1R pricor to administration of a. compound of Formula

Tor Formula II.

Anothem embodiment of the invention pwertains to a method of ixnhibiting phosphorylation of CSF1R by administering ar inhibitory amount of a compound of Formula

Tor Formula IT, as described above, to a patient.

Brief Descriptio of the Drawings

Figure 1 shows a Western Blot demonsstrating that compound L_ of the invention inhibits phospimorylation of M-CSF receptors.

Figure 2a shows a graph demonstrating that compound 1 of thes invention inhibits osteoclast deve lopment in a dose-dependent maxnner in vitro.

Figure 2b shows a graph demonstrating that compound 1 of thes invention inhibits an early stage of o-steoclast development, but not 1=ater stages.

Figure _3 depicts, via bioluminescence, ®hat compound 1 of the invention inhibits the growth of breasst cancer metastases in vivo.

Figure -4 is a graph demonstrating that rice with breast cancer metastases to bone exhibit less oste=olysis, as measured by pyridino=line levels, when treatec3 with compound 1.

Detailed Descripticon of the Invention ) The pre sent inventors have made the suprising discovery that ¢ hemical compounds having the structure of Formula I and Formula MJ, as set forth herein, inBhibit phosphorylation of the M-CSF receptor, CSF1R. Moreover, the inventors have discover—ed that the compounds inh_ibit osteoclast development in viZvo and significantly dec-rease osteolysis associated with_ tumor metastases to bone.

Accordingly, the compounds of Formula I arid Formula I are useful im the treatment of patients with exce=ssive osteolysis. In this contex®, “osteolysis” refers to a “breakdown of bone by cells that se~crete acids and/or enzymes. Os teoclasts are a primary example of such ; cells, but the inventi on embraces inhibition of osteo ysis mediated by other cell types as well, including tumor cell s and osteoclast precursors. “Excessive osteolysis” referss to an : imbalance between steolytic and osteogenetic activities that results in a net Moss of bone, either locally or systemically.

One embodirnent of the invention relates to za method of treating exce:ssive osteolysis that comprises administering an effective amount of a compound of Formula I

I

YOR R)p—2Z 28

H [X..

N

H lc == ©

N

(Rip H (Io, wherein

R is independently Ed, OH, alkyl, aryl, cycloalkyl, heteroaryl, alkoxy, heteroc yclic and amino; each R; is independ ently selected from the group cosnsisting of alkyl, halo, aryl, alkoxy, haloalkyl, haloalkoxy, cycloalkyl, heteroaryl, heterocyclic, hydroxy, -C(O)-Rg, -NRoR op, -NRoC(O)-Ri2 and —C(O)NRgR_ yp; each R; is independ_ently selected from the group consisting of alkyl, aryl, heteroaryl, -C(O)-

Rgand SO,R”’, whe=re R”’ is alkyl, aryl, heteroaryl, “NRgN, or alkoxy; - each Rs is independ_ently selected from the group consisting of hydrogen, alk yl, aryl, haloalkyl, cycloalkyl, heteroaryl, heterocyclic, hydr oxy, -C(O)-Rg and (CHR Ry; - XisQorS; pis 0-3; qis 0-2; ris 0-3;

Rg is selected from the gr-oup consisting of OH, alkyl, aryl, heteroaryl, alko=Xy, cycloalkyl and heterocyclic;

Ry and Ry; are independently selected from the group consisting of H, alkyl, aryl, aminoalkyl, : heteroaryl, cycloalkyl ancl heterocyclic, or Rg and Rg t=ogether with N may f-orm a ring, where the ring atoms are selecte=d from the group consisting o fC, N, O and S;

Ry; is selected from the group consisting of ~OH, amin: 0, monosubstituted arzmino, disubstituted amino, alkyl, aryl, heteroaryl, alkoxy, cyc=loalkyl and heterocyclic

Riz is selected from the group consisting of alkyl, aryl, heteroaryl, alkoxy, c=ycloalkyl and heterocyclic;

Z is OH, O-alkyl, or -NR_3R4, where R3 and R4 are indespendently selected fr=om the group consisting of hydrogen, a 1kyl, aryl, heteroaryl, cycloalk=yl, and heterocyclic, «or R3 and Rs may combine with N to form = ring where the ring atoms ares selected from the group consisting of

CH, N,Oand Sor (Vn Rr 5 — Hi

Sn re

R' /m wherein Y is independently CH,, O, Nor S,

QisCorN; n is independently 0-4; ard m is 0-3; or a salt thereof, to a patieent in need of such treatment.

In a preferred embodiment, the compound administered to the patient is a compound of Formula II; ) C——NRg———(CHR),———Z 2%

H Pa GHY

N

H

0 4 = N (Ry)p H (ID where the variables are as previously defined.

In another embodiment ofthe invention, R; is halo (e.=, F and Cl) and Z is -NR_3R, wherein Rj and Ry are independ ently H or alkyl in Formula I oor II as administered to a patient in need thereof.

In another embodiment, Z of Formula I or II is -NR3R_, wherein Raand Ry form a morpholine ring.

In another embodiment, Z of Formula l or II is: 1 (Y)n R R?

Za NI \ au c N ~ v0 Rr!

R'/m wherein each Y is CHp, each n iss 2, m is 0 and Rj and R4 form_ a morpholine ring.

We 2004/075775 PCT/US2004/005283

In a particular embodiment of the invention, the compound amdministered is selected fron the group consisting of . Q rr Ow

Ha NTN HiC NN [ h CH | 3 . f N 3 N CHa x H X / H

O *

N N © ; ° 0 ) oO

HaC N——~——N HaC. N \ oS LO

X / y X / N o e} 4 N 0 and

H . H 0

HaC N [ De, 0) x / \__° lo

N

H Eb] wherein X is F, Cl, I or Br. In a preferred embodiment, X is F.

In another embodiment of the invention, the therapeutic methmod involves adm 1nistering to a patient having excessive osteolysis an effective amount of a compound selected from the group consisting of: 5-(5-Fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-d mmethyl-1H-pyrrole-3- carb oxylic acid (2-diethylamino-ethyl)-amide (compound 1); 5-(5-Fluoro-2-0x0-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dmmethyl-1H-pyrrole-3- ‘ carb oxylic acid (2-pyrrolidin-1-yl-ethyl)-amide (compound 2); : 5-(5-Fluoro-2-0xo0-1,2-dihydro-indol-3-ylidenemethyl)-2,4-d mmethyl-1H-pyrrole-3- carb oxylic acid (2-morpholin-4-yl-ethyl)-amide (compound 3); (8)-5-(5-Fluoro-2-o0xo0-1,2-dihydro-indol-3-ylidenemethyl)-2 ,4-dimethyl-1H-pyrrole- 3-ca:xboxylic acid (2-hydroxy-3-morpholin-4-yl-propyl)-amide (compoound 4);

(R)-5-(5-Fluoro-2-0 x0-1,2-dihydro-indol-3-ylidene=methyl)-2,4-dimethyl- 1 Fl -pyrrole- 3-carboxylic acid (2-hydrox y-3-morpholin-4-yl-propyl)-am_ide (compound 5); 5-(5-Fluoro-2-oxo-1 ,2-dihydro-indol-3-ylidenemethayl)-2,4-dimethyl- 1 H-pyrmrole-3- : carboxylic acid (2-hydroxy- 3-morpholin-4-yl-propyl)-amid e (compound 6); ] 5-(5-Chloro-2-oxo0-1 ,2-dihydro-indol-3-ylidenemetiayl)-2,4-dimethyl-1H-pyr—role-3- carboxylic acid (2-hydroxy- 3-morpholin-4-yl-propyl)-amid e (compound 7); 5-(5-Fluoro-2-oxo-1 _2-dihydro-indol-3-ylidenemetha yl)-2,4-dimethyl- 1 H-pyrr—ole-3- carboxylic acid (2-ethylamixo-ethyl)-amide (compound 8); and 3-[3,5-dimethyl-4-(4--morpholin-4-yl-piperidine-1-caarbonyl)- 1 H-pyrrol-2-me-thylene]- 5-fluoro-1,3-dihydro-indol-2-one (compound 9).

To clearly set forth tte compounds of Formula I andl I, useful in the inventiv ¢ method, the following defin-itions are provided. "Alkyl" refers to a saturated aliphatic hydrocarbon raadical including straight chain and branched chain groups of 1 to 20 carbon atoms (whenever a_ numerical range; e.g. "1—20", is stated herein, it means that the group, in this case the alkyl group, may contain 1 cartoon atom, 2 carbon atoms, 3 carboon atoms, ec. up to and includ—ing 20 carbon atoms). Alkyl groups containing from 1 to 4 carbon atoms are referred to zas lower alkyl groups. When said lower alkyl groups lack subsstituents, they are referred to as unsubstituted lower alkyl groups.

More preferably, an alkyl group is a medium size alkyl havi_ng 1 to 10 carbon atoms e.g. , methyl, ethyl, propyl, 2-progoyl, n-butyl, iso-butyl, tert-butyl , pentyl, and the like. Most preferably, it is a lower alky~1 having 1 to 4 carbon atoms e.=., methyl, ethyl, propyl, 2-propyl, n-butyl, iso-butyl, or tert-butyl, and the like. The alkyl groump may be substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more, mmore preferably one to three, evera more preferably one or two suBsstituent(s) independently selected from the group con sisting of halo, hydroxy, unsubs-tituted lower alkoxy, aryl optionally substituted with one or more groups, preferably one, two or three groups which are independently of each other halo, hydroxy, unsubstituted lowwer alkyl or unsubstitutecd lower alkoxy groups, aryloxy optionally substituted with one or m ore groups, preferably on_e, two or : three groups which are indegpendently of each other halo, hydroxy, unsubstituted lowe=r alkyl or unsubstituted lower alkoxy groups, 6-member heteroaryl having from 1 to 3 nitrogzen atoms in the ring, the carbors in the ring being optionally sumbstituted with one or mor—e - groups, preferably one, two or three groups which are indep-endently of each other hao,

hydroxxy, unsubstituted lower alkyl or unsubstituted lower alkoxy grooups, 5-member heterozaryl having from 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, the carbon and the nitrogen atoms in the group be=ing optionally : substit-uted with one or more groups, preferably one, two or three groups which are independently of each other halo, hydroxy, unsubstituted lower alkyl = or unsubstituted lower ) alkoxy groups, 5- or 6-member heterocyclic group having from 1 to 3 heteroatoms selected from thhe group consisting of nitrogen, oxygen and sulfur, the carbon and nitrogen (if present) atoms in the group being optionally substituted with one or more gro ups, preferably one, two or thre=e groups which are independently of each other halo , hydroxy~, unsubstituted lower alkyl or unsubstituted lower alkoxy groups, mercapto, (unsubstituted lower alkyl)thio, arylthE o optionally substituted with one ox more groups, preferably ome, two or three groups which are independently of each other halo, hydroxy, unsubstituted I-ower alkyl or alkoxy groupss, cyano, acyl, thioacyl, O-carbamyl, N-carbamyl, O-thiocarbar-nyl, N-thiocarbamyl, C- amido-, N-amido, nitro, N-sulfonamido, S-sulfonamido, RS(O)-, RS(=O),-, -C(O)OR,

RC(0O)O0-, and —-NR;3R 4, wherein Ry; and Ry, are independently selected from the group consisting of hydrogen, unsubstituted lower alkyl, trihalomethyl, cyc=loalkyl, heterocyclic and aryl ogptionally substituted with one or more, groups, preferably one, two or three groups which_ are independently of each other halo, hydroxy, unsubstituted 1 ower alkyl or unsub stituted lower alkoxy groups.

Preferably, the alkyl group is substituted with one or two sub. stituents independently selecteed from the group consisting of hydroxy, 5- or 6-member heter-ocyclic group having from 71 to 3 heteroatoms selected from the group consisting of nitrogeen, oxygen and sulfur, the ca_rbon and nitrogen (if present) atoms in the group being optionamlly substituted with one or mo=Te groups, preferably one, two or three groups which are indepeendently of each other halo, Thydroxy, unsubstituted lower alkyl or unsubstituted lower alkoxy groups, 5-member heteroaryl having from 1 to 3 heteroatom s selected from the group ceonsisting of nitrogen, oxygen and sulfur, the carbon and the nitrogen atoms in the group being optionally substi tuted with one or more groups, preferably one, two or three groups which are indep endently of each other halo, hydrox y, unsubstituted lower alkyl or unsubstituted lower alkox_y groups, 6-member heteroaryl having from 1 to 3 nitrogen atozms in the ring, the carbo 1s in the ring being optionally substituted with one or more groups, preferably one, two or thr-ee groups which are independently ©f each other halo, hydroxy—, unsubstituted lower alkyl or unsubstituted lower alkoxy groups, or ~NRi3R14, wherein Rg3 and Ry4 are independently selected from the group consisting of hydrogen ard alkyl. Even meore preferably the alkyl group is sub-stituted with one or two substitixents which are inedependently of each other hydroxy, dimethyl amino, ethylamino, diethylamin_o, dipropylamino.. . pyrrolidino, piperidino, morphoRino, piperazino, 4-lower alkylpi_perazino, phenyl, imidazolyl, pyridinyl, pyridazinyl, pyrimidimnyl, oxazolyl, triazinyl, and the Kike. "Cycloalkyl" refers to a 23 to 8 member all-carbon monocyclic ring, an all- carbon 5- member/6-member or 6-membe=1/6-member fused bicyclic ring «or a multicyclic fiased ring (a “fused” ring system means that each ring in the system shares am adjacent pair of carbon atoms with each other ring in th_e system) group wherein one or more of the rings may contain one or more double bormds but none of the rings has a completely conjugated pi- electron system.

Examples, without limit-ation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cy=clohexane, cyclohexadiene, adamantane, cyclohepotane, cycloheptatriene, and the like. £A cycloalkyl group may be substituted or unsubsti tuted. When substituted, the substituent groiap(s) is preferably one or more, rnore preferably omne or two substituents, independently selected from the group consisting ef unsubstituted lower alkyl, trihaloalkyl, halo, hydroxy, unssubstituted lower alkoxy, aryl op tionally substitute=d with one or more, preferably one or two groups independently of each other halo, hydroxy=, unsubstituted lower alkyl or un_substituted lower alkoxy groups-, aryloxy optional ly substituted with one or more, psreferably one or two groups indespendently of each other halo, hydroxy, unsubstituted lower alkyl or unsubstituted lower alko=xy groups, 6-menber heteroaryl having from 1 to 3 ritrogen atoms in the ring, the ca_rbons in the ring toeing optionally substituted with one= or more, preferably one or two groups independe ntly of each other halo, hydroxy, unsubstitiated lower alkyl or unsubstituted lower alkoxy gro ups, 5- member heteroaryl having frorm 1 to 3 heteroatoms selected from the group cons=isting of nitrogen, oxygen and sulfur, thme carbon and nitrogen atoms of —the group being optionally substituted with one or more, oreferably one or two groups ind_ependently of eac -h other halo, hydroxy, unsubstituted lower alkyl or unsubstituted lower alkoxy groups, 5- or 6&-member heterocyclic group having frormn 1 to 3 heteroatoms selected from the group conssisting of ‘ nitrogen, oxygen and sulfur, tle carbon and nitogen (if presentc)atoms in the group being optionally substituted with ones or more, preferably one or two groups independe=ntly of each other halo, hydroxy, unsubstit-uted lower alkyl or unsubstituted lower alkoxy groups, mercapto, (unsubstituted lowenr alkylthio, arylthio optionally substituted with orme or more,

preferably one or two gro=ups independently of each other halo, hydroxy, urmsubstituted lower alkyl or unsubstituted low=er alkoxy groups, cyano, acy=], thioacyl, O-carbarryl, N-carbamyl,

O-thiocarbamyl, N-thiocamrbamyl, C-amido, N-amido, mitro, N-sulfonamido , S-sulfonamido, : RS(0)-, RS(0),-, -C(O)O®R, RC(0)O-, and -NR13R14 zre as defined above. ] "Alkenyl" refers teo a lower alkyl group, as defi ned herein, consistin_g of at least two carbon atoms and at least one carbon-carbon double bond. Representative e=xamples include, but are not limited to, eth-enyl, 1-propenyl, 2-propenyl, 1-, 2-, or 3-butenyl, and the like. "Alkynyl" refers to a lower alkyl group, as defined herein, consisting of at least two carbon atoms and at least: one carbon-carbon triple bord. Representative ex—amples include, but are not limited to, eth_ynyl, 1-propynyl, 2-propynyl, 1-, 2-, or 3-butynyl_, and the like. "Aryl" refers to am all-carbon monocyclic or fimsed-ring polycyclic Ci.e., rings which share adjacent pairs of camrbon atoms) groups of 1 to 122 carbon atoms havirmg a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl. The aryl group may be= substituted or unsub stituted. When substituted, the substitutesd group(s) is preferably one =or more, more prefer=ably one, two or three, even more preferably one or two, independently. selected from the group consisting of unsubstituted lower alky1, trihaloalkyl, halo, hydroxy, unsubstituted lower alkoxy, mercapto,(unsubstituted lower alkyl)thio, cyano, acyl. thioacyl, O-carbamya/l, N-carbamyl, O- thiocarbamyl, N-thiocartbamyl, C-amido, N-amido, nitro, N-sulfonamido, SS-sulfonamido,

RS(0)-, RS(0),-, -C(O)R, RC(0)O-, and ~NR13R14- with Ry3 and Rig as defined above.

Preferably, the aryl grou p is optionally substituted wish one or two substitt ents independently selected from halo, unsu_bstituted lower alkyl, trihaloaalkyl, hydroxy, mercampto, cyano, N- amido, mono or dialkylammino, carboxy, or N-sulfonarmido. "Heteroaryl" refesrs to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group of =5 to 12 ring atoms containing one, two, or three rirg heteroatoms selected from N, O, or 5, the remaining ring atoms being C, and, in additicon, having a completely conjugated pi-electron system. Examples, without limitation, -of unsubstituted heteroaryl groups are pyw/ole, furan, thiophene, imid=azole, oxazole, thiazowle, pyrazole, pyridine, pyrimidine, qirinoline, isoquinoline, purine and carbazole. The Ineteroaryl group may be substituted or umsubstituted. When substitute=d, the substituted grcoup(s) is preferably one or more, more preferably one, two, or three, evern more preferably ones or two, independently selected from the group consisting of umsubstituted lower a_lkyl, trihaloalkyl,

halo, hydroxy, umsubstituted lower alkoxy, mercapto,(unsubstituted lower al kyl)thio, cyano, acyl, thioacyl, O—carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N- amido, nitro, N-ssulfonamido, S-sulfonamido, RS(O)-, RS(0);-, -C(O)OR, R_C(O)O-, and — : NRi3R 14, with R_13 and Ri4 as defined above. Preferably, the heteroaryl grougp is optionally substituted with one or two substituents indepermdently selected from halo, unsubstituted ) lower alkyl, trihzloalkyl, hydroxy, mercapto, cy~ano, N-amido, mono or dialkylamino, carboxy, or N-sutaifonamido. "Heteroc=yclic" refers to a monocyclic or- fused ring group having in “the ring(s) of 5 to 9 ring atoms in which one or two ring atoms ares heteroatoms selected from "N, O, or S(O)n (where n is an integer from 0 to 2), the remainirag ring atoms being C. The wings may also have one or more double bonds. However, the arings do not have a completely conjugated pi- electron system. Examples, without limitation, «f unsubstituted heterocyclic groups are pyrrolidino, pip eridino, piperazino, morpholino , thiomorpholino, homopipe razino, and the like. The hetero cyclic ring may be substituted o-1 unsubstituted. When substituted, the substituted grovap(s) is preferably one or more, mnore preferably one, two or three, even more preferably one or two, independently selected farom the group consisting of —unsubstituted lower alkyl, trithaloalkyl, halo, hydroxy, unsubs-tituted lower alkoxy, mercapoto, (unsubstituted lower alkylthio, cyano, acyl, thioacyl, O-carba-myl, N-carbamyl, O-thiocar bamyl, N- thiocarbamyl, C-amido, N-amido, nitro, N-sulfeonamido, S-sulfonamido, RS(0)-, RS(0);-, -

C(O)OR, RC(D)0-, and -NR3R 14, with Ry3 an d Ry as defined above. Pref erably, the heterocyclic group is optionally substituted wit™h one or two substituents inclependently selected from hi alo, unsubstituted lower alkyl, torihaloalkyl, hydroxy, mercagoto, cyano, N- amido, mono ox dialkylamino, carboxy, or N-suilfonamido.

Preferably, the heterocyclic group is op tionally substituted with ones or two substituents independently selected from halo, unsubstituted lower alkyl, tr-ihaloalkyl, hydroxy, mercapto, cyano, N-amido, mono or clialkylamino, carboxy, or N -sulfonamido. "Hydroxy" refers to an -OH group. "Alkox y" refers to both an -O-(unsubst-ituted alkyl) and an -O-(unsweibstituted cycloalkyl) group. Representative examples include, but are not limited to, e.g., methoxy, ethoxy, propox<y, butoxy, cyclopropyloxy, cycBobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. ) ¥

"Arylox_y" refers to both an -O-aryl and an -O-heteroaryl group, as «defined herein.

Representative examples include, but are not li-mited to, phenoxy, pyridiny loxy, furanyloxy, thienyloxy, pyr-imidinyloxy, pyrazinyloxy, and the like, and derivatives the=reof. : "Mercagpto" refers to an -SH group. "Alkylthhio" refers to both an -S-(unsubsstituted alkyl) and an -S-(un substituted cycloalkyl) group. Representative examples include, but are not limited to, e.g., methylthio, ethylthio, prop -ylthio, butylthio, cyclopropylthi o, cyclobutylthio, cyclopent ylthio, cyclohexylthio , and the like. " Arylthwio" refers to both an -S-aryl and. an -S-heteroaryl group, as clefined herein.

Representative: examples include, but are not 1amited to, phenylthio, pyridinylthio, furanylthio, thi entylthio, pyrimidinylthio, and the like and derivatives thereof. "Acyl" refers to a -C(O)-R" group, where R" is selected from the group consisting of hydrogen, unsubstituted lower alkyl, trihalomesthyl, unsubstituted cycloalk yl, aryl optionally substituted with one or more, preferably one, two, or three substituents sel=ected from the group consistimg of unsubstituted lower alkyl, trihalomethyl, unsubstituted lower alkoxy, halo and -NR3R 4 groups, heteroaryl (bonded thro ugh a ring carbon) optionall-y substituted with one or more, pereferably one, two, or three subsstituents selected from the gmroup consisting of unsubstituted Tower alkyl, trihaloalkyl, unsubs-tituted lower alkoxy, halo ard —NR3R14 groups and heterocyclic (bonded through a ring carbown) optionally substituted with one or more, preferably ones, two, or three substituents selected from the group consistirig of unsubstituted lower alkyl, trihaloalkyl, unsubstituted lower alkoxy, halo and —NR;3R14 gromips. Representative acyl groups include, but are not limited sto, acetyl, trifluoroacety ll, benzoyl, and the like. "Aldekyde" refers to an acyl group in ~which R" is hydrogen. ‘ "Thioacyl" refers to a -C(S)-R" group, with R" as defined herein. "Ester-" refers to a -C(O)O-R" group with R" as defined herein exc-ept that R" cannot : be hydrogen. "Acetyl" group refers to a -C(O)CH3 group. "Halo * group refers to fluorine, chlorizne, bromine or iodine, prefemrably fluorine or chlorine.

WO» 2004/075775 PCT/US2004/0(B5283 "Trihalomethyl" group refers to a-CXj group whereina X is a halo group as defined herein. : "Methylenedioxy" refers to a- -OCH;0- group where tlhe two oxygen atoms are bonde=d to adjacent carbon atoms. : "Ethylenedioxy" group refers to a -OCH,CH,O- wher-e the two oxygen atoms are bonde=d to adjacent carbon atoms. "S-sulfonamido” refers to a —S(0);NR3R14 group, witzh Ris and Ris as defined herein. "N-sulfonamido” refers to a —NR;3S(0),R group, witka Ry; and R as defined herein. "Q-carbamyl" group refers to a -OC(O)NR3R 14 grougpo with R;3 and Ry4 as defined hereim. "N-carbamyl" refers to an R<OC(O)NR4- group, with R and Ry as defined he rein. "O-thiocarbamyl” refers to a -OC(S)NR3R4 group with Ry3 and R4 as define=d hereim. "N-thiocarbamyl" refers to @ ROC(S)NR 4- group, with R and Ry4 as defined herein. " Amino” refers to an —NR;3R4 group, wherein Riz ard Ry4 are both hydrogera.

"C-amido" refers to a -C(O»NR3R14 group with R;3 &and Ry, as defined hereir.

"N-amido" refers to 2a RC(C®NR4- group, with R anc Ry, as defined herein.

"Nitro" refers to a -NO, gro up.

"Haloalkyl" means an unsulbstituted alkyl, preferably= unsubstituted lower alk—yl as defied above that is substituted with one or more same or different halo atoms, e.g. 5 -CH_2Cl, -CFs, -CH,CF,, -CH,CCl3 , and the like.

"Aralkyl" means unsubstitumted alkyl, preferably unsubstituted lower alkyl as defined abowve which is substituted with an aryl group as defined above, e.g, -CH;phenyl, -(CETL).phenyl, -(CH,)sphenyl, CH ,CH(CH3)CHyphenyl, an_d the like and derivative s thereof.

"Heteroaralkyl" group mea-ns unsubstituted alkyl, pr eferably unsubstituted lower alkyl

: as dl efined above which is substituged with a heteroaryl groump, e.g., -CHpyndinyl, -(CHz)pyrimidiny, -(CH,)simidazolyl, and the like, and derivativess thereof.

"Monoalkylarmino" means a radical -NHR’ where R’ is an unsubstitute=d alkyl or unsubstituted cycloalkyl group as defined above, e.g., mesthylamino, (1-methyMethyl)amino, cyclohexylamino, aned the like. "Dialkylamin_o" means a radical -NR’R’ where e=ach R’ is independent_ly an unsubstituted alkyl ox unsubstituted cycloalkyl group as defined above, e.g., dmmethylamino, diethylamino, (1-mefthylethyl)-ethylamino, cyclohexylm ethylamino, cyclopentylmethylammino, and the like. "Cyanoalkyl'™ means unsubstituted alkyl, preferalbly unsubstituted lower alkyl as defined above, whickn is substituted with 1 or 2 cyano greoups. : "Optional" om "optionally" means that the subseq uently described event or circumstance may bit need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example=, "heterocycle group optionally substituted with an alkyl group” means that the alkyl may buxt need not be present, and the description includes situations where th. € heterocycle group iss substituted with an alkyl group and situations where the heterocyclo group is not substitu ted with the alkyl group.

A "pharmaceutical composition” refers to a mixture of one or more of the compounds described herein, or physiologically/pharmaceutically acceptable salts or prodrugs thereof, with other chemical components, such as physiologicall y/pharmaceutically acceptable carriers and excipiemis. The purpose of a pharmaceuticzal composition is to fa_cilitate administration of a scompound to an organism.

Prodrugs of acompound of Formula I or Formula Ii are within the scope of this invention. Additiornally, a compound of Formula I or F orrula II itself may asct as a prodrug.

A "prodrug" refers —to an agent that is converted into a pearent drug in vivo. Pr—odrugs are often useful because, in s ome situations, they may be easier t<o administer than the gparent drug.

They may, for instamnce, be bioavailable by oral administration whereas the paarent drug is not.

The prodrug may also have improved solubility in pharmaceutical compositicons over the parent drug. An excample, without limitation, of a prodrug would be a compound of the ] present invention tka is administered as an ester (the "porodrug") to facilitate transmittal across a cell memb rane where water solubility is detrinciental to mobility but —then is metabolically hydreolyzed to the carboxylic acid, the active entity, once inside the cell where water solubility is toeneficial.

A further example of a prodrug might be a short polypeptide, for examples, without {imitation, a 2-10 amino acid polypeptide, bonded thro ugh a terminal amino grousp to a carboxy group of a compoeund of this invention, wherein the polypeptide is hydrolyzed or metabolized in vivo to release the active molecule.

Additionally, it is econtemplated that a compourd of Formula I or Formulaa IT would be metabolized by enzymes in the body of the organism s.uch as human being to gererate a metabolite that can moduli ate the activity of the proteir kinases. Such metabolites are within the scope of the present irmvention.

As used herein, a * 'physiologically/pharmaceutically acceptable carrier” r-efers to a carrier or diluent that doess not cause significant irritati on to an organism and doe=s not abrogate the biological ac-tivity and properties of the aciministered compound.

An "pharmaceutically acceptable excipient” re ers to an inert substance a dded to a pharmaceutical composition to further facilitate administration of a compound. HExamples, without limitation, of excipients include calcium carbonate, calcium phosphate, =various sugars and types of starcim, cellulose derivatives, gelati_n, vegetable oils and polyethylene glycols.

As used herein, thee term “pharmaceutically acceptable salt” refers to thosse salts that retain the biological effectiveness and properties of thes parent compound. Such salts include: @) aci_d addition salt which is obtai .ned by reaction of the frees base of the parent compound with inorganic acids such as hydrochloric acid, hydrob-romic acid, nitric acid, phosplnoric acid, sulfuric acid, and poerchloric acid and the like, or with organic acids sucka as acetic acid, oxalic acid, (D) or (L) malic acid, male=ic acid, methanesulfonic eacid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid or maloric acid and the like, prefer-ably hydrochloric acid. or (L)-malic acid such as the L-malate salt of 5-(5-fluoero-2-0xo0-1,2- dihydroindol-3-yRidenemethyl)-2,4-dimethyl- L H-pyrrole-3-carboxylic ac=1d(2- } diethylaminoethy~1)amide; or (in) salts formed when an acidic proton present in the parent czompound either is replaced by a metal ion, e.g, an alkal® metal ion, an alkaline ear—th ion, or an aluminum ion; or- coordinates with an organic base such as ethanolamine=, diethanolamine, triethanolamine, tromethamin._e, N-methylglucamine, an«d the like.

"Method" refers to manners, means, techniques and proc-edures for accompl_ishing a given task including, but not limited to, those manners, means, t echniques and proc=edures either kno -wn to, or readily developed from known manners, me=ans, techniques andi ‘ procedure=s by, practitioners of the chemical, pharmaceutical, biological, biochemic=al and medical amts. "Tan vivo" refers to procedures performed within a living organism such as, without limitation, a mouse, rat or rabbit. "Treat", "treating" and "treatmemt" refer to a method of alleviating, amelior=ating, abrogatin gor relieving a disease condition and/or any of its attesndant symptoms. "Patient" refers to any living entity comprised of at least= one cell. A living organism can be as simple as, for example, a single eukaryotic cell or as ccomplex as a mammal, includingz a human being. "MW herapeutically effective amoutant" refers to that amount of the compound Wbeing administered which will prevent, allevi ate, ameliorate or relieve to some extent, orme or more of the sigzns or symptoms of the disorder being treated. “CCSF1R” denotes the macrophage colony stimulating factor receptor, and i_ncludes what ma=y be designated CSF-1 receptor, M-CSF receptor and/or c-fins gene products. Also included are any constitutive parts or e lements of a macrophages colony stimulating factor receptor.

Osteolytic conditions that can be treated according to thme present invention_ include the systemic condition known as osteoporosis. The osteoporosis nay be attributed to (1) menopatse in women, (2) aging in mem or women, (3) suboptimmal bone growth during childhoosd and adolescence that resulted in failure to reach peal=< bone mass, and/or— (4) bone loss secondary to other disease conditions, eating disorders, medications and/or medical treatmemts. Another systemic condition that may be treated is _Pagets disease, whi ch comprisees an excessive osteolytic component.

Other osteolytic diseases that can be treated according wo the present invenation are more localized. A particular example is metastatic tumor-indu. ced osteolysis. In t=his conditiosn, bone cancers or bone metas tases induce localized osteolysis that causess pain, bone weaknesss and fractures. Such localized osteolysis also permitss tumors to grow lamrger by creatingz more space for them in the bone and releasing growth factors from the bcone matrix,

Cancers presently kno -wn to cause tumor-induced osteolysis include hematological malignancies (e.g., myeloma and lymphoma) and sol®d tumors (e.g., breast, prosstate, lung, renal and thyroid), all of which the present invention contemplates treating.

As noted abowe, the inventors have discovere=d that compounds of Formula I and . Formula TI inhibit phcsphorylation of M-CSF receptors. Thus, the invention inecludes methods of inhibiting M-CSF receptor phosphorylatien by administering a com_pound of

Formula I or Formula II to a patient.

Additionally, #he invention includes methods of treating cancers that exgpress CSF1R.

Examples of such cancers include, but are not limited to, breast cancers and carcers of the female reproductive tmract such as ovarian cancer and endometrial cancer. Othemr cancers include myelodysplas-tic syndromes (MDS), acute myeloid leukemia (AML) an_d acute promyelocytic leukenmia (APML). Therefore, the cornpounds of Formula I or Formula II may be administered to tre=at patients with M-CSF recepto=r positive cancers.

Prior to admiristering a compound of Formul aI or Formula Il to a canc-er patient, the cancer may be tested to determine whether it express es CSF1R. Such testing may directly detect CSF1R proteirms (e.g-, immunological assays such as ELISA, RIPA, THC- staining) or may do so indirectly «(e.g., detecting gene transcripts via hybridization methods such as ISH).

Such procedures are ccommonly known in the art, ancl have successfully been paerformed for

CSF1R by Kacinski ext al. (23, 26-27), Tang et al. (321), and Toy et al. (28). The fact thata cancer expresses CSEZ1R indicates that treatment with a compound of Formula Tor Formula IT will be useful. _Administration and Pharmaceutical Composition

The claimed =nethods involve administration of a compound of Formula I or Formula

II or a pharmaceutically acceptable salt thereof, to a human patient. Alternatively, the compounds of Formula I or Formula IT can be admirnistered in pharmaceutical ecompositions in which the foregoirng materials are mixed with suit able carriers or excipient(ss). Techniques : for formulation and =administration of drugs may be ~found in "Remington's Phamrmacological

Sciences,” Mack Pubolishing Co., Easton, PA., latest edition.

As used herein, "administer" or "administratZon" refers to the delivery of a compound of Formula I or Formula Il or a pharmaceutically acceptable salt thereof or of &2 pharmaceutical composition containing a compound. of Formula I or Formula I'Tor a pharmaceutically acceptable salt thereof of “this invention to an orga. nism for the purpose of treating excessive osteolysis or cancer.

Suitable Toutes of administration may include, without limit. ation, oral, rectal, transmucosal or intestinal administration or intramuscular, subcutareous, intramedullary, intrathecal, direct intraventricular, intraveneous, intravitreal, intraperitoneal, intranasal, or intraocular injec=tions. The preferred routes of administration are or—al and parenteral.

Furthernmore, one may administer the drug in a targeted druge delivery system, for example, in a ligposome coated with turnor- specific antibody. The 1 iposomes will be targeted to and taken up selectively by the tumor or osteoclast progenitor.

Pharmaczeutical compositions of thes present invention may We manufactured by processes well Bknown in the art, e.g., by means of conventional mixing, dissolving, granulating, dreagee-making, levigating, emulsifying, encapsulating=, entrapping or lyophilizing processes.

Pharma_ceutical compositions for use in accordance with th-€ present invention may be formulated in am conventional manner using one or more physiologmcally acceptable carriers comprising excipients and auxiliaries which facilitate processing o={ the active compounds into preparatiomns which can be used pharmaceutically. Proper forrmulation is dependent upo=n the route of admministration chosen.

For injection, the compounds of thue invention may be formulated in aqueous solutions, preferably in physiologically cosmpatible buffers such as Hanks' solution, Ringer's solution, or phu ysiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulat ion. Such penetrants are generally known in the art.

For orzal administration, the compounds can be formulated _ by combining the active compounds w-ith pharmaceutically acceptable carriers well know in the art. Such carriers enable the cornpounds of the invention to» be formulated as tablets, pills, lozenges, dragees, : capsules, liquids, gels, syrups, slurries, staspensions and the like, Sor oral ingestion by a patient. Pharmmaceutical preparations for oral use can be made usmng a solid excipient, ] optionally gri nding the resulting mixture, and processing the mixture of granules, after addang other suitable= auxiliaries if desired, to ob tain tablets or dragee comres. Useful excipients are_, in particular, filllers such as sugars, includimg lactose, sucrose, manrmitol, or sorbitol, cellulose preparations such as, for example, maize starch, wheat starch, ric e starch and potato starch and other materials such as gelatin, gum tragaxcanth, methyl cellulose, Laydroxypropylmethyl- cellulose, sod_ium carboxymethylcellulose, arad/or polyvinyl- pyrrolido ne (PVP). If desired, disintegratinge agents may be added, such as cross-linked polyvinyl pyr=tolidone, agar, or alginic acid. #A salt such as sodium alginate nay also be used.

Drageze cores are provided with suitable coatings. For this purpose, concentrated sugar solutioms may be used which may optionally contain gum arabic=, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxid-e, lacquer solutions, and suitable sorganic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dramgee coatings for identification or to characterize different= combinations of active compound dsoses.

Pharmaceutical compositions that cam be used orally include p ush-fit capsules made of gelatin, ass well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. “The push-fit capsules can contain the active ingredients ir: admixture with a filler such as lactose, a binder such as starch, and/or a lubricant such as tales or magnesium stearate and, optionamlly, stabilizers. In soft capsules, the active compounds m ay be dissolved or suspended imn suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Stabilizers also may be added in th_ese formulations.

Pharmaceutical compositions that m ay also be used include haard gelatin capsules. As a non-limiti_ng example, compound 1 in a capsule oral drug product feormulation may be as 50 and 200 meg= dose strengths. The two dose strengths are made from tae same granules by filling into «different size hard gelatin capsules, size 3 for the 50 mg capsule and size 0 for the 200 mg caposule.

The= capsules may be packaged into brown glass or plastic bottles to protect the active compound from light. The containers containing the active compourd capsule formulation must be stored at controlled room temperat ure (15-30°C).

For— administration by inhalation, th_e compounds for use according to the present invention =are conveniently delivered in the form of an aerosol spray usinga pressurized pack or a nebuli_zer and a suitable propellant, e.g, without limitation, dichmlorodifluoromethane, ’ trichloroflmioromethane, dichlorotetra- fluo roethane or carbon dioxidlle. In the case of a pressurized aerosol, the dosage unit may be controlled by providing a valve to deliver a metered arnount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator ma-y be formulated containing a p-owder mix of the compound and a suitable powder base such as lactose or starch.

The cosmpounds may also be formulated for parenteral administration, e.g., by bolus injection or cosntinuous infusion. Formulatiors for injection may be pressented in unit dosage form, e.g., in ampoules or in multi-dose cont ainers, with an added pres:ervative. The compositions “may take such forms as suspemsions, solutions or emulsieons in oily or aqueouss vehicles, and mmay contain formulating materials such as suspending, s®abilizing and/or dispersing agents.

Pharm_aceutical compositions for parenteral administration incl ude aqueous solutions of a water solwible form, such as, without lim_itation, a salt, of the actives compound.

Additionally, suspensions of the active compounds may be prepared irm a lipophilic vehicle.

Suitable lipop=hilic vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl eoleate and triglycerides, or mat erials such as liposomes. Aqueous injection suspensions nay contain substances which imncrease the viscosity of thee suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers and/or agents that increase the solubility of ~ the compounds to allow for the goreparation of highly concentrated solutions.

Altermatively, the active ingredient nay be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free w- ater, before use.

The compounds may also be formulamted in rectal compositions= such as suppositoriess or retention enemas, using, e.g., conventionaal suppository bases such zas cocoa butter or othe=r glycerides.

In adc3ition to the formulations described previously, the compeounds may also be formulated ass depot preparations. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by imtramuscular injection.

A compound of this invention may be formulated for this route of administration with suitable polymeric or hydrophobic materialss (for instance, in an emulssion with a pharmacolog ically acceptable oil), with ion exchange resins, or as a spoaringly soluble derivative suach as, without limitation, a spar-ingly soluble salt.

A nor-limiting example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a noenpolar surfactant, a water-miscib- le organic polymer and an aquezous phase such as the VPID co-solvent system.

VPD is a solution of 3% w/v benzyl alcohmol, 8% w/v of the nonpolar surfactant Polysorbate 80, and 65% w/v polyethylene glycol 300 , made up to volume in absolute ethanol. The VPED co-solventk system (VPD:D5W) consists o-f VPD diluted 1:1 with a 59-4 dextrose in water ’ solution. “This co-solvent system dissolve=s hydrophobic compounds wwell, and itself produces low toxicity upon systemic administratiora. Naturally, the proportions of such a co-solvent system may be varied considerably withowut destroying its solubility amnd toxicity characteristics. Furthermore, the identity of the co-solvent componerats may be varied: for example, other low-toxicity nonpolar surfactants may be used insteadll of Polysorbate 80, thes fraction si_ze of polyethylene glycol may te varied, other biocompatitole polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone, and other su_gars or polysaccharides=s may substitute for dextrose.

Al ternatively, other delivery systems for hydrophobic pharmamceutical compounds may be ermployed. Liposomes and emuls-ions are well known examples of delivery vehicless or carrierss for hydrophobic drugs. In add3tion, certain organic solvents such as dimethylssulfoxide also may be employed. although often at the cost Of greater toxicity.

Aciditionally, the compounds may be delivered using a sustaired-release system, suczh as semipe=rmeable matrices of solid hydro phobic polymers containing the therapeutic agent.

Various s—ustained-release materials have “been established and are we=1l known by those skilled in the art. Sustained-release capsules may, depending on theim chemical nature, release th e compounds for a few weeks upp to over 100 days. Depend_ing on the chemical nature and the biological stability of the therapeutic reagent, addition al strategies for proteira stabilizat® on may be employed.

Tkne pharmaceutical compositions herein also may comprise suitable solid or gel phase carriers or excipients. Examples off such carriers or excipients include, but are not limited tc, calcium carbonate, calcium ph_osphate, various sugars, stamrches, cellulose derivatives, gelatin, and polymers such ass polyethylene glycols.

Examples of formulations for use in the present invention are in Tables 1-3 (below), which cam be found in U.S. Patent Appliczation Serial No. 10/237,966, filed September 10, : 2002, now a provisional application, which is expressly incorporated in its entirety by references.

. TABLE 1 1H-pyrrole-3+-carboxylic acid (2-dicthylammino-ethyl)-amide hard geelatin capsules

Ingredient N ame | Concentration | Amowmntin 50 | Amount in 75 | Amount in 200

Ee % wiw meg mg mg (API [650 [soo 1750 12000

Fd BS

Sodium* ‘Povidone (K—25) [5.0 [38 |s7 |i52

Fo a a A LJ

Stearate

Capsule |- |simed |Swe3 [Size0

TABLE 2 : yliedenemethyl)-2,4-dimethyl-1 BH -pyrrole-3-carboxylic acd (2—diethylamino-ethyl)-amide I. -malate hard gelatin capsules

Ingredient Concentration | Amountin SO mg % wiw™)

Fa LN CL

Sosdium®

Magnesium Stearate [0.5 |1445 (Capsule [- [sized

TABLE 3

Compositiomn of 5-(5-fluoro-2-ox0-1,2-dih -ydro-indol-3-ylidenemethayl)-2,4-dimethyl- capsules

Ingredient Concentration | Amo-untin 25 | Amountin 580 | Amount in 100 % wiw mg mg mg : AFF [400 [33400 66800" [200°

Bt J A e Sodium®

TPovidone (E<25) [50 [4175 [8350 ~~ [16700

Stearate

Canale |= ISwes [Smel _ [Sime0

* Drug subsstance quantity required for the batch will be adjusted to have 100% of labeled stremmgth for capsules. Appropriate adjustment will be made to mannitol quantity to l<eep the same fill weight for each strength. . ® Quantity ezquivalent to 100 mg free base © Quantity exquivalent to 50 mg free base. ¢ Quantity esquivalent to 25 mg free base. ¢ Half intra-granular half extragranular.

Many of the compounds of Formula 1 and Formula II may be provided as physiologically acceptable salts wherein the compound may form the ne=gatively or the positively charged species. Examples of salts in which the compound foms the positively charged mom ety include, without limitation, «quaternary ammonium, saltss such as the hydrochloride, sulfate, carbonate, lactate, taxtrate, malate, maleate, succinate wherein the nitrogen atoem of the quaternary ammonium group is a nitrogen of the sezlected compound of this inventicon which has reacted with the appropriate acid. Salts in which a compound of this invention forms the negatively charged species include, without limitatEon, the sodium, potassium, calcium and magnesium salts foamed by the reaction of a ca—rboxylic acid group in the compor=ind with an appropriate base (e.g sodium hydroxide (NaOH), potassium hydroxide (KOH), Calcium hydroxide (Ca( OH),), etc.)

Phamrmaceutical compositions suitat»le for use in the present invention include compositiosns wherein the active ingredient s are contained in an amount sufficient to achieve the intende=d purpose, i.e., a therapeutically” effective amount.

De-termination of a therapeutically effective amount is well withhin the capability of those skilleed in the art, especially in light ofthe detailed disclosure pro-vided herein.

Fo-r any compound used in the methods of the invention, the theerapeutically effective amount or— dose can be estimated initially £rom cell culture assays. Theen, the dosage can be formulatead for use in animal models so as to achieve a circulating concentration range that includes tThe ICs as determined in cell culture (i.e., the conceniration of the test compound ) which achieves a half-maximal inhibition of phosphorylation of CSF1 R). Such information can then toe used to more accurately deterxmine useful doses in humans.

Toxicity and therapeutic efficacy of the compounds described herein can be determine=d by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by d_etermining the ICsp and the LDso, wherein the LDsg is the co ncentration of test : compourmd which achieves a half-maximal inhibition of lethality, for = subject compound.

The data obtained ~from these cell culture assays ard animal studies can bes used in formulating a rang e of dosage for use in humans. “The dosage may vary depending upon the dosage form employed and the route of administramtion utilized. The exact formulation, route } of administration and dosage can be chosen by thes individual physician ira view of the patient's condition . (See e.g., Fingl, et al., 1975, im "The Pharmacological Basis of

Therapeutics", Ch- 1 p.1).

Dosage ammount and interval may be adjusted individually to provi de plasma levels of the active species “that are sufficient to maintain the kinase modulating eff=ects. These plasma levels are referred to as minimal effective concent rations (MECs). The MEC will vary for each compound buat can be estimated from in vitroe data, e.g., the concentr—ation necessary to achieve 50-90% irhibition of a kinase may be asceertained using the assays described herein.

Dosages necessarsy to achieve the MEC will deperad on individual charact=eristics and route of administration. HEPLC assays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen that maintains plasrmna levels above the ME&C for 10-90% of the time, preferably between 30-90% and most prefer ably between 50-90%.

At present, the therapeutically effective armounts of compounds o Formula I or

Formula II may range from approximately 25 mg~m2 to 1500 mg/m2 per day; preferably about 3 mg/m2/daay. Even more preferably 50mg/=qm qd till 400 mg/qd.

In cases o—f local administration or selectiv—e uptake, the effective 1 ocal concentration of the drug may mmot be related to plasma concentration and other procedures known in the art may be employed to determine the correct dosage= amount and interval.

The amount of a composition administereed will, of course, be depsendent on the subject being treated, the severity of the affliction, the manner of adminisstration, the judgment of the prescribing physician, etc.

It is contesmplated that the inventive methcad could be used in combination with other : therapies, including chemotherapies, radiation therapies and surgical ther—apies for cancer.

For combination therapies and pharmaceutical co mpositions described hesrein, the effective amounts of the compound of the invention and ofS the other agent can be edetermined by those of ordinary skill #n the art, based on the effective amounts for the compowunds described herein and those known or described for the othemr agent. The formulations and route of administration foer such therapies and compositiom can be based on the in _formation described herein for compositions and therapies comprising the compound of the invention as the seole active agent and on information. provided for the chemotherapeutic and other agent in combination therewith.

Specifically, it is contenplated that the described compounds may be combined vevith bisphosphonate or with hormormmal therapy (e.g., aromatose inhibit-ors) to prevent bone breakdown in breast cancer. It is further contemplated that the co-mpounds may be comb ined with all-trans retinoic acid (ATERA) in the treatment of AML and other cancers.

General Synthetic Procedure

The following general maethodology may be employed to gorepare the compounds of this invention:

The appropriately subst-ituted 2-oxindole (1 equiv.), the appropriately substituted aldehyde (1.2 equiv.) and a bas (0.1 equiv.) are mixed in a solvemt (1-2 ml/mmol 2- oxindole) and the mixture is then heated for from about 2 to abou t 12 hours. After coolirag, the precipitate that forms is filtered, washed with cold ethanol or ether and vacuum dried to give the solid product. If no precipitate forms, the reaction mixture is concentrated and tlhe residue is triturated with dichlo romethane/ether, the resulting soli dis collected by filtratieon and then dried. The product may optionally be further purified bx chromatography.

The base may be an orgsanic or an inorganic base. If an organic base is used, preferably it is a nitrogen base. Examples of organic nitrogen basses include, but are not limited to, diisopropylamine, trimethylamine, triethylamine, anili ne, pyridine, 1,8- diazabicyclo[5.4.1]undec-7-enes, pyrrolidine and piperidine.

Examples of inorganic bases are, without limitation, amm_onia, alkali metal or alk=aline earth hydroxides, phosphates, carbonates, bicarbonates, bisulfatess and amides. The alkal: 1 metals include, lithium, sodiunm and potassium while the alkaline earths include calcium, magnesium and barium.

In a presently preferred. embodiment of this invention, whaen the solvent is a proti c solvent, such as water or alcohol, the base is an alkali metal or arm alkaline earth inorganiec base, preferably, a alkali metal or an alkaline earth hydroxide.

Tt will be clear to those skilled in the art, based both on kraown general principles of organic synthesis and on the dasclosures herein which base would be most appropriate fowr the reaction contemplated.

The solvent in w~hich the reaction is carried out may be a protic or an apxzotic solvent, preferably it is a protic solvent. A "protic solvent” is a ssolvent which has hydrogen atom(s) covalently bonded to oxzygen or nitrogen atoms which mrenders the hydrogen atoms appreciably acidic and thus capable of being "shared" with a solute through hydrogen bonding, Examples of gorotic solvents include, without= limitation, water and alc=ohols.

An "aprotic solvrent” may be polar or non-polar= but, in either case, does not contain acidic hydrogens and therefore is not capable of hydro _gen bonding with solutes=. Examples, without limitation, of n-on-polar aprotic solvents, are psentane, hexane, benzene, toluene, methylene chloride andl carbon tetrachloride. Examples of polar aprotic solven®ts are chloroform, tetrahydro— furan, dimethylsulfoxide and climethylformamide.

In a presently preferred embodiment of this invention, the solvent is a p=rotic solvent, preferably water or an =alcohol such as ethanol.

The reaction is carried out at temperatures greater than room temperatume. The temperature is generally from about 30°C to about 150°C, preferably about 80°C to about 100°C, most preferable about 75°C to about 85°C, which is about the boiling p oint of ethanol. By "about" is- meant that the temperature ran ge is preferably within 10 degrees

Celsius of the indicated temperature, more preferably within 5 degrees Celsius of the indicated temperature and, most preferably, within 2 Clegrees Celsius of the indicated i temperature. Thus, fox example, by "about 75°C" is rmeant 75°C + 10°C, prefe=rably 75°C = 5°C and most preferably, 75°C = 2°C. 2-Oxindoles ard aldehydes, may be readily synthesized using techniqu-es well known in the chemical arts. Itt will be appreciated by those sk=illed in the art that other synthetic pathways for forming the compounds of the inventior are available and that th_e following is offered by way of example and not limitation.

Compounds of the present invention are prepared according to the following methodologies and as. described, e.g., in U.S. Patent Application Serial No. 09 /783,264 and - WO 01/60814, WO 0=0/08202, U.S Provisional Appli_cation No. 60/312,353, faled August 15, 2001, now U.S. Paterat Application Serial No. 10/281_,985, filed August 13, 20802, U.S.

Provisional Application No. 60/411,732, filed Septermber 18, 2002, U.S. Provisional

Application No. 60/3 28,226, filed October 10, 2001, now U.S. Patent Applica_tion Serial No. 10/268,082, filed October 10, 2002 and U.S. Patent Application Serial No. 10./076,140, filed

February 15, 2002, all of which are incorporated by reference in their entirety . Additionally,

the disclosures of U.S. provisional applications No. 60/448,874 and No. 60/448,922, filed

February 24, 2003, are incorporated herein bay reference.

Synthetic Methodologies

Method A: For mylation of pyrroles

P<OCl; (1.1 equiv.) is added dropwise to dimethylformamide (3 equaiv.) at 10°C followed by addition of the appropriate pyrr-ole dissolved in dimethylformaamide. After stirring feor two hours, the reaction mixture i s diluted with H,O and basifie dto pH 11 with 10 - NKOH. The precipitate which forms is coll ected by filtration, washed with H,O and dried in a vacuum oven to give the desired aldehyde -

Method B: Saponification of pyrrolecarboxylic acid esters / mixture of a pyrrolecarboxylic acd ester and KOH (2 — 4 equiv-)in EtOH is refluxed until reaction completion is indicat-ed by thin layer chromatograp hy (TLC). The cooled reaction mixture is acidified to pH 3 with 1 N HCL The precipitate= that forms is collectecl by filtration, washed with H,O aned dried in a vacuum oven to gi ve the desired pyrrolec arboxylic acid.

Method C: Amidation

To a stirred solution of a pyrrolecarlooxylic acid dissolved in _dimethy-1formamide(0.3M) is added 1-ethyl -3-(3-dimethylamino- propyl)ecarbodiimide (1.2 equiv.), l-hydroxybenzotriazole (1.2 equiv..), and triethylamine (2 equiv.) . The appropriate amine iss added (1 equiv.) and the reaction sstirred until completion is indicated by TLC. Ethyl acetate dis then added to the reaction mixturee and the solution washed witla saturated NaHCO; and brire (with extra salt), dried over anhydrous MgSO, and concentrateci to afford the desired amide.

Method D: Condensation of aldehydes and oxindoles containing carboxylic acid su bstituents

A mixture of the oxindole (1 equivalent), 1 equivalent of the aldelhyde and 1-3 equival ents of piperidine (or pyrrolidine) ir ethanol (0.4 M) is stirred at 0-100°C until reactior completion is indicated by TLC. "The mixture is then concentrat ed and the residue acidifie=d with 2N HCl. The precipitate tha® forms is washed with H>O an_d EtOH and then dried ir a vacuum oven to give the product.

Method E: Conclensation of aldehydes and o—xindoles not containing carboxylic acid snbstitue nts

A mixture ofthe oxindole (1 equivalent), 1. equivalent of the aldehyde and 1 — 3 . equivalents of piper-idine (or pyrrolidine) in ethanol (0.4 M) is stirred at 90- 100°C until reaction completiom is indicated by TLC. The mixture is cooled to room termperature and the solid that forms is collected by vacuum filtration, ~washed with ethanol and ried to give the product. If a precipi tate does not form upon coolirmg of the reaction mixture, the mixture is concentrated and prarified by column chromatogra—phy. skkaxkk

The followimng examples are given to illustmrate the present invention. It should be understood, howevesr, that the invention is not to b-e limited to the specific conditions or details described in these examples. Throughout t"he specification, any and all references to publicly available dRocuments are specifically incozxporated into this patent agoplication by reference.

Synthetic Ex.amples

Example 1 - Synth esis of (3Z)-3-{[3,5-dimethyl—4-(moerpholin-4-yl)piperidin-1- ylcarbonyl]-1H-py~rrol-2-ylmethylidene}-5-fluomro-1,3-dihydro-2H-indol—2-one (Compound 9)

Q

STO

F an: y 0

Step 1

To a stirred mixture of 4-amino-1-benzylpaperidine (Aldrich, 1.53 mL, 7.5 mmol),

K,CO; (2.28 g, 16.5 mmol), and DMF (15 mL) he=ated at 50 °C was added dropwise over 60 min bis(2-bromoetkayl) ether (Aldrich, tech. 90%, =0.962 mL, 7.65 mmol). After stirring 6 h at 80 °C, TLC (90:10=1 chloroform/MeOH/aq. conc. NH4OH) indicated formation of a new - spot. Heating was continued as the solvent was ewaporated by blowing witla a stream of nitrogen over 2 h. “The crude material was relativesly pure, but subjected to a relatively short silica gel column (1% to 6% gradient of 9:1 MeORd/aq. NH,OH in chloroform). Evaporation of the pure fractiorms gave ~1.7 g of the diamine 4—(morpholin-4-yl)-1-benzy~Ipiperidine as a waxy solid.

"HNMR (400 MH==, d¢- DMSO) 6 7.31 (m, 4H), 7 .26 (m 1H), 3.72 (t, J = 4.7 Hz, 4H), 3.49 (s, 2H), 2.94 (br d, J = 5.9 Hz, 2H), 2.54 (t, J= 4.7 Elz, 4H), 2.19 (tt, J= 11.5, 3.9Hz, 1H), 1.96 (td, J = 11.7, 2.2 Hz, 2H), 1.78 (br d, J = 12.5 Iz, 2H), 1.55 (m, 2H).

Step 2 - A stirred mixture of PAd(OH)2 (20% on carbon (<=50% wet), 390 mg, 25 wt%), methanol (50 mL), and <&.7 M HC1 (3 eq, ~10.6 mL — iracluding water added la~ter when ppt was seen) under nitrogen —was exchanged to 1 atm. hydros gen atmosphere by flushing (~20 sec) using a balloon of nitrogen into the vessel and out through an oil bubbler. After 20 min. the reaction mixture under hydrogen was heated to 50 °C and 4-(morpholin-4-yR)-1- benzylpiperidine (1.56 g, 6.0 mmol) in methanol (8 mL) was added dropwise ower 30 min.

After 10 h, tlc indicated a_11 starting amine was consumed to a more polar spot (zainhydrin active). The reaction mix=ture was then filtered through CCelite and evaporated to yield the 4- (morpholin-4-yl)piperidire dihydrochloride as an off-white solid. This material was subjected to free-basing using excesss basic resin (>16 g, Bio-Rad WM aboratories, AG 1-X8,. 20-50 mesh, hydroxide form, methano»1washed two times) and a metlhanol mixture of the anmine hydrochloride. After swi rling with the resin for 30 min. _, the methanol solution was decanted and evaporated to yield 9 32 mg of 4-(morpholin-4-yl)pigperidine free base as a “waxy crystalline solid. "HNMR (400 MIz, dg-DMSO) 8 3.53 (br s, 4H)», 3.30 (v br s, IH(+H,(D)), 2.92 (br d,

J=11.7 Hz, 1H), 2.41 (s_, 4H), 2.35 (~obscd t, J = 11.7 Miz, 2H), 2.12 (br t, 1H) , 1.65 (brd, J =11.7 Hz, 2H), 1.18 (br q,J= 10.9 Hz, 2H); LCMS-AFCl m/z 171 [M+17".

Step 3 (32)-3-(3,5-Dim ethyl-4-carboxy-1 H-pyrrol-2-y_Imethylidene)-5-fluoro—1,3-dihydro- 2H-indol-2-one (120 mg_, 0.40 mmol), prepared as described in PCT Publication No 01/60814, and BOP (221. mg, 0.50 mmol) were suspended in DMF (5 mL) wit good stirring at room temperature and triethylamine (134 nL, 0.96 m-tmol) was added. After 10-15 min, to , the homogeneous reaction mixture was added the 4-(morpholin-4-yl)piperidines (85 mg, 0.50 mmol) all at once. The reaction mixture was stirred for 48 h (might be done much earlier), ’ then transferred to a funmel containing chloroform-isopmropanol (5/1) and 5% acy. LiCl. The cloudy-orange organic p«hase was separated, washed with additional 5% aq LiCC1(2X), 1 M aq

NaOH (3X), satd aq NaCCl (1X), and then dried (Na;SO_4) and evaporated to yield the crude product (96.3% pure; treace HMPA by 'HNMR). This c=rude product was then —further purified by passage through a ve-xy short column (3 cm) of silicam gel (S to 15% gradient ©f MeOH in

DCM) where a trace of faster moving 3E-isomer was reemoved. The pure fracticons were evaporated and recrysta lized overnight from a satd EtCOAc soln which was dilused with Et,O * (~3-fold) and chilled at 0 °C. The mother liquor was de=canted to yield after full. vacuum the g desired compound as or-ange crystals (153 mg 85%). 'HNMR (400 MHz, de-DMSO0) & 13.60 (s, 1H) 10.87 (s, 1H), 7.72 (dd, J= 9.4,2.7

Hz, 1H), 7.68 (s, 1H), 6.91 (td, J=9.3, 2.6 Hz, 1H), 6.582 (dd, J= 8.6, 4.7 Hz, 1 H), 3.54 (app brt, J =43 Hz, 4H), 3. 31 (2x s, 3H+3H), 2.43 (br s, 4K), 2.36 (m, 1H), 2.25 (brm, 6H), 1.79 (brs, 2H), 1.22 (br s, 2); LCMS m/z 453 [M+1]".

Proceeding as described in Example 1 above bit substituting (3Z)-3-(3,=5-dimethyl-4- carboxy-1H-pyrrol-2-y-lmethylidene)-5-fluoro-1,3-dihxydro-2H-indol-2-one for «(3Z2)-3-(3,5- dimethyl-4-carboxy-1H-pyrrol-2-ylmethylidene)-1 ,3-Alihydro-2H-indol-2-one g=ave (32)-3- {[3,5-dimethyl-4-(mor-pholin-4-yl)piperidin-1-ylcarbomnyl]- 1H-pyrrol-2-ylmethylidene}-,3- dihydro-2H-indol-2-omme. "HNMR (400 MHz, de-DMS 0) 8 13.55 (s, 1H), 10.84 (s, 1H), 7.74 (d,J=17.6 Hz, 1H), 7.59 (s, 1H), 7.11 (t, J=7.6 Hz, 17H), 6.97 (t, J = 7.6 Hz, 17TH), 6.86 (d, J = 7.4 Hz, 1H), 3.54 (app brt,J = 4.3 Hz, 4H), 3.31 (2x ss, 3H+3H), 2.43 (brs, 4HT), 2.35 (m, 1H), 2.28 (br m, 6H), 1.79 (brs, 2H), 1.22 (brs, 2H); LCMS m/z 435 [M+17".

Proceeding as «described in Example 1 above b-ut substituting (3Z)-3-(3 5 5-dimethyl-4- d carboxy-1H-pyrrol-2-~yimethylidene)-5-fluoro-1,3-dih_ydro-2H-indol-2-one for (3Z)-3-(3,5- dimethyl-4-carboxy-1_H-pyrrol-2-ylmethylidene)-5-chaloro-1 ,3-dihydro-2H-ind . ol-2-one gave (32)-3-{[3,5-dimethyl -4-(morpholin-4-yl)piperidin-1 —ylcarbonyl]-1H-pyrrol-2 - ylmethylidene}-5-chloro-1,3-dihydro-2H-indol-2-one=. "HNMR (400 MHz, de-DMSO) 8 13.56 (s, 1HI), 10.97 (s, 1H), 7.95 (d, J=2.0Hz, 1H), 7.74 (s, 1H), 7.1 1 (dd, J = 8.2, 2.0 Hz, 1H), 6.85 (d,/=8.2 Hz, 1H), 3.549% (app brt,J = ~4 Hz, 4H), 3.31 (2x s, 3H+3H), 2.43 (brs, 4H), 2.37" (m, 1H), 2.25 (br m, 6HC), 1.79 (brs, 2H), 1.23 (brs, 2H); LCMS m/z 470 [M+17%.

Proceeding ass described in Example 1 above Wout substituting 4-(morploolin-4-yl)- piperidine with commercially available 4-(1-pyrrolidSinyl)-piperidine gave (3Z2)-3-{[3,5- dimethyl-4-[4-(pyrro didin-1-yl)piperidin-1-ylcarbony~1]- 1H-pyrrol-2-yl)methyRidene]-5- fluoro-1,3-dihydro-23-indol-2-one.

HNMR (400~ MHz, d-DMSO) 8 E/Z isomer mixture; LCMS m/z 437 [M+17".

Synthesis of the above exampless can proceed according to the procedure of U.S.

Provisi_onal Application No. 60/328,2265, filed October 10, 2001 zand U.S. Patent Applicat ion

Serial No. 10/268,082, filed October 1(®, 2002, incorporated by reference in its entirety.

Exampple 2 - Synthesis of (32)-3-{[3,5—dimethyl-4-(morpholin—4-yl)azetidin-1- ylcarb- onyl]-1H-pyrrol-2-yhnethylide=ne}-5-fluore-1,3-dihydrce-2H-indol-2-one

Step 1

A solution of 1-azabicyclo[1.1.@]butane, prepared from 2,3-dibromopropylamine= hydrotoromide (58.8 mmol) according t-o a known procedure described in Tetrahedron Letters 40 (19299) 3761-64, was slowly added toa solution of morpholinee (15.7 ml; 180 mmol) and sulfuric acid (3.3 g of 96% soln.) in an"hydrous non-denaturated ethanol (250 ml) at 0 °C. The reaction mixture was stirred on ice bath for 30 min., then at roon temperature for 8 h.

Calcivam hydroxide (5.5 g) and 100 ml of water was added and tle obtained slurry was st=irred for 1 Mh and then filtered through a pad of cellite. The filtrate was concentrated and distilled at reduceed pressure (20 mm Hg) to remove water and an excess of morpholine. The distilleation reside was re-distilled at high vacuurx using a Kugelrohr apparatus to obtain a pure 4- (azeti_din-3-yl)morpholine in 33% yiel-d (2.759 g) asa colorless oily liquid. 3C.NMR (CDCls, 100 MHz): 66.71(2C), 59.37 (1C), 51.46 (2C), 49.95(2C) 'H (CDCl, 400 MHz): 3.727 (t, J=4.4 Hz, 4H), 3.619 (t, J=8Hz, 2H), 3.566 (t, J=8Hz, 2H), : 3.227 (m, J=7Hz, 1H), 2.895 (br s, IHX), 2.329 (br s, 4H)

Step 2 1-(8-Azabenztriazolyl)-ester o-f (3Z)-3-({3,5-dimethyl-4—carboxy] 1-H-pyrrol-2- yl} ma ethylene)-5-fluoro-1.3-dihydro-2H-indol-2-one (0.5 mmoM, 210 mg) [prepared by activ-ating (3Z)-3-(3,3-dimethyl-4-camboxy-1 -H-pyrrol-2-ylmet_hylene)-5-fluoro-1.3-dih_ydro- 2H-i-ndol-2-one (480 mg; 1.6 mmol) with the HATU reagent (5770 mg, 1.5 mmol) in the preseence of Hunig base (3.0 mmol, O».525 ml) in DMF (5ml) ard isolated in pure form toy precipitation with chloroform (5ml) aand drying on high vacuurnr in 92% yield (579 mg) HW was suspwended in anhydrous DMA (1.0 mal). A solution of 4-(azeti din-3-yl)-morpholine; (1 42.5 mg, 1mmol) in anhydrous DMA (1.0 ml) was added in one poxrtion and the obtained so lution was stirred at room temperature for 2.0 min. The reaction mixtwire was evaporated at rocom ’ temperature using an oil pump, the thick residue was diluted with 6 ml of a mixture of methanol plus diethyl amine (20:1; v/v), inoculated mechanica®ly and placed into a refrigerator (+3 °C) for 8 hours. Thes= precipitates were filtered (with a brief wash with aan ice-

cold methanol) and dried on high vactaum to give the desired product. 71.5% yield (152 mg of an orange solid) . I_.C/MS: +APCI: M+1=425; -#APCIL: M-1=423

L 9% NMR (d-DMSO, 376.5 MHz): -122.94 (m, 1F)

RH (-DMSO, 400 MHz): 13.6551 (s, 1H), 10.907 (s, 1H), 7. 754 (dd, J=9.4 Hz, J=2.4

Hz, 1H», 7.700 (s, 1H), 6.935 (dt, J=8.2 Hz, J=2.4 Hz, 1H), 6.841 («dd, J=8.6 Hz, J=3.9Hz; 1H), 3.963 (br s, 2H), 3.793 (br s, 2H), 3.581 (br t, J=4.3 Hz, 4H), 3.133 (m, 1H), 2.367 (s, 3H), 2.340 (s, 3H), 2.295 (br s, 4H) “Proceeding as described in Ex<ample 2 above but substitutimmg (32)-3-(3,5-dimethyl-4— carboxxy-1 H-pyrrol-2-ylmethylidene) -5-fluoro-1,3-dihydro-2H-indol-2-one with (3Z)-3-(3,5— dimeth=y1-4-carboxy- 1 H-pyrrol-2-ylnmethylidene)-5-chloro-1 ,3-dihysdro-2H-indol-2-one gave (32)-3— {[3,5-dimethyl -4-(morpholin—4-yl)azetidin-1-ylcarbonyl]-1_H-pyrrol-2- ylmeth-ylidene}-5-chloro-1,3-dihydro-2H-indol-2-one as an oranges solid. : LC/MS: +APCT: M+1=441; —APCI: M-1=440,441 'H (d-DMSO, 400 MHz): 13 .607 (s, 1H), 11.006 (s,1H), 7. 976 (d, J=2.0Hz, 1H), 7.756 Cs, 1H), 7.136 (dd, J=8.2 Hz, ¥=2.0 Hz, 1H), 6.869 (d, ]=8.2 Hz, 1H), 3.964 (br s, 2H)», 3.793 br s, 2H), 3.582 (br t, ]=4.3 Fz, 4H), 3.134 (m,1H), 2.369 (s, 3H), 2.347 (s, 3H), 2.296 . (br s, 44H)

Proceeding as described in FExample 2 above but substituting 4-(azetidin-3- yl)momrpholine with 4-(azetidin-3-y1-cis-3,5-dimethylmorpholine «(prepared in a procedure analogzous to the preparation of 4-(a zetidin-3-yl)-morpholine but \asing cis-3,5- dimetRaylmorpholine (20.7g; 180 mr mol) in place of morpholine) grave (3Z)-3-{[3,5-dimethy~1- 4-(2,5 -dimethylmorpholin-4-yhazetmidin-1 -ylcarbonyl]-1H-pyrrol—2-ylmethylidene} -5-fluor o- 1,3-di hydro-2H-indol-2-one as an o-range solid

LC/MS: +APCL M+1=453;. -APCIL: M-1=451 192 NMR (d-DMSO, 376.5 MHz): -122.94 (m, IF) 4 (d-DMSO, 400 MHz): 1 3.651 (s, 1H), 10.907 (s; 1H), 7.758 (dd, J=9.4 Hz, J=2.23 . Hz; 1 H), 7.700 (s, 1H), 6.935 (dt, J=8.6 Hz, J=2.7 Hz, 1H), 6.842 (dd, J=8.2 Hz, J=4.3 Hz, 1H), 3.961 (br s, 2H), 3.790 (br s, 2H), 3.546 (br m, 2H), 3.092 (xx, 1H), 2.690 (br s; 2H), 2.364 (s, 3H), 2.338 (s, 3H), 1.492 (br m, 2H), 1.038 (brs, 6H)

Proceeding as described in Example 2 above but su_bstituting (32)-3-(3,5-dinnethyl-4- carboxy-1H-pyrrol-2-ylmeth ylidene)-5-fluoro-1,3-dihydro—2H-indol-2-one with 3Z2-)-3-(3,5- dimethyl-4-carboxy-1H-pyrrol-2-ylmethylidene)-5-chloro— 1,3-dihydro-2H-indol-2-One and 4- (azetidin-3-yl)morpholine with 4-(azetidin-3-yl)-cis-3,5 -dimmethylmorpholine gave (3Z)-3- {[3,5-dimethyl-4-(3,5-dimethylmorpholin-4-yl)azetidin-1-ylcarbonyl]-1H-pyrrol-2- ylmethylidene}-5-chloro-1,3-dihydro-2H-indol-2-one as amn orange solid.

LC/MS: +APCL M+ 1=469, 470; -APCI: M-1=468 ,469 'H (d-DMSO, 400 MHz): 13.606 (s, 1H), 11.008 (s5, 1H), 7.979 (4, J=2.0Hz. 1H), 7.758 (s, 1H), 7.138 (dd, J=8.2Hz, J=2.0Hz, 1H), 6.870 (d., ]=8.2Hz, 1H), 3.964 (br s, 2H), 3.790 (br s, 2H), 3.547 (br m, 2H), 3.095 (m, 1H), 2.691 (ors, 2H), 2.366 (s, 3H), 2 .345 (s, 3H), 1.494 (br m, 2H), 1.039 (brs, 6H)

Proceeding as described in Example 1 above, but ssubstituting 4-(morpholin—4-yl)- piperidine with 2-(R)-pyrrolidin-1-ylmethylpyrrolidine prepared as described below provided (3Z)-3-{[3,5-dimethyl-2R-(pyrrolidin-1-ylmethyDpyrrolidin-1 -ylcarbonyl]-1H-pyrr—ol-2- ylmethylidene}-5-fluoro-1,3-dihydro-2H-indol-2-one

Synthesis of 2(R)-pyrrolidin-1-ylmethylpyrrolidine

Step 1 .

To a solution of (+)-Carbobenzyloxy-D-proline (1 .5 g, 6.0 mmol), EDC (2.3 g, 12.0 mmol) and HOBt (800 mg, 12.9 mmol) in DMF (20 ml) v=vas added trietylamine (1.5 ml) and pyrrolidine (1.0 mi, 12.0 mmol). It was stirred for 18 hat rt. Sat. NaHCO; was add ed, it was extracted with CH2CL2 (three times). The organic layers were separated and dried over

Na,SOq. The solvent was removed and the residue was purified by silica gel chromatography (EtOAc) to give 1-(R)-[N-(benzyloxycarbonyl)-pyrolyl]peyrrolidine as a white solid (94%). 'H NMR (400 MHz, CDCl, all rotamers) 0 1.57—1.66 (m, 1H), 1.71-2.02 Cm, SH), 2.04-2.19 (m, 2H), 3.26-3.43 (m, 3H), 3.44-3.78 (m, 3H) 4.41 (dd, J=4.5, 7.6 HZ, 0.5H), 4.52 (dd, J=3.7, 7.6 Hz, 0.5H), 4.99 (d,J= 12.1 Hz, 0.5 H), 5.05 (d, /= 12.5 Hz, -0.5H), 5.13 (d, J=12.1 Hz, 0.5H), 5.20 (d, J= 12.5 Hz, 0.5H), 7.27-77.38 (m, 5H). ] Step 2

A mixture of 1-(R)-[N-(benzyloxycarbonyl)proly—1jpyrrolidine (2.7 g, 8.9 mrimol) and 5% Pd-C catalyst (270 mg) in methanol (15 ml) were stir=ted under a hydrogen atnmosphere for 20 h. The reaction mix ture was filtered through celite= and the solvent was remeoved yielding 2(MR)-prolylpyrrolidine as a viscouas oil (80%), which was used without further purificatiorm for the next step. 'H INMR (400 MHz, de-DMSO) 8 1.52-1.78 (m, 5H), 1.82-1.85 (m, 2H), 1.97-2.04 (m, 1H), 2.63-2.71 (m, 1H), 2.97-3.02 (m, 1H), 3.22-3.35 (m, 3H), 3.4 8-3.54 (m, 1H), 3.72 (dd, J=6.1.,8.0Hz, 1H).

Step 3 2-(R)-Prolylpyrrolidine (1.2 g, 7.1 mmol) was dissolved in THF (10 ml). The reaction mixture was cooled to 0° C and B Ha, 1M in THF (10 ml, 10 naamol) was dropwise at 0 C. The recaction mixture was refluxed fosr 16 h, 3 M HCI (4.7 ml). 2 M NaOH solution was added until. pH 10 was reached. The prodwict was extracted with 5% MeOH in CH,Cl; (three times). The organic layers were dried over Na;SO4 and the solvent wa s removed to provide the title commpound as a slightly yellow liquid (73%), which was used without further purificatior for the next step. 'H "NMR (400 MHz, d¢-DMSO) 8 1.22-1.30 (m, 1H), 1.55-1.6% (m, 6H), 1.71-1.79 (m, 1H), 2-26-2.30 (m, 1H), 2.33-2.38 (m, 1H), 2.40-2.45 (m, 4H), 2.6=5-2.71 (m, 1H), 2.78- 2.84 (m, 13H), 3.02-3.09 (m, 1H).

Promceeding as described in Examp Te 1 above, but substituting =4-(morpholin-4-yl)- . piperidine with 2-(S)-pyrrolidin-1-ylmeth=ylpyrrolidine (prepared as described above, by substitutin_g (+)-carbobenzyloxy-D-proline with carbobenzyloxy-L-pr-oline) provided (3Z)-3- : {[3,5-dimesthy}-2S-(pyrrolidin-1-ylmethyl Jpyrrolidin-1-ylcarbonyl]-1H-pyrrol-2- ylmethylidllene}-5-fluoro-1,3-dihydro-2H-dndol-2-one.

Example 33 - Synthesis of 5-[5-fluoro-2-Oxo0-1,2-dihydro-indol-(3Z) -ylidene-methyl]-2,4- dimethyl- Z1H-pyrrole-3-carboxylic acid

Step 1

Dimmethylformamide (25 mL, 3 eq -) was cooled with stirring ira an ice bath. To this was added POC; (1.1 eq., 10.8 mL). After 30 minutes, a solution of tthe 3,5-dimethyl-4- ethylester pyrrole (17.78, 105.8mmol) in DMF (2M, 40 mL) was add ed to the reaction and stirring co ntinued. After 2 hour, the reaction was diluted with water (C250 mL) and basified to , pH=11 wi th IN aqueous NaOH. The whi te solid was removed by filt-ration, rinsing with water and then hexanes and dried to afford 5-formyl-2,4-dimethyl-1/" -pyrrole-3-carboxylic: acid ethyl ester (19.75 g, 95%) as a tan solid.

'H NMR (360 MHz, DMS O-d6) § 12.11 (brs, 1H, NEI), 9.59 (s, 1H, CHO), 4.177 (q,

J = 6.7Hz, 2H, OCH,CH,), 2.44 (s, 3H, CH.) 2.40 (s, 3H, CH.3), 1.26 (d, J= 6.7Hz, 3H. «CH,CHa). =tep 2 5-Formyl-2,4-dimethyl-1F-pyrrole-3-carboxylic acid ethyl ester (2 g, 10 mmol) was added to a solution of potassium hydroxide (3 g, 53 mmol) dissolved in methanol (3 mI) and water (10 mL). The mixture was refluxed for 3 hours, cooled ®o room temperature and acidified with 6 N hydrochloric acid to pH 3. The solid was collected by filtration, washed with water and dried in a vacuum oven overnight to give 5-fommyl-2,4-dimethyl-1H-pyr_Tole- 3~carboxylic acid (1.6 g, 93%). 'H NMR (300 MHz, DMSO0-d6) 3 12.09 (s, br, 2H, NH & COOH), 9.59 (s, 1H,

CHO), 2.44 (s, 3H, CHa), 2.40 (s, 3H, CHa).

Step 3 5-Fluoroisatin (8.2 g, 49.7 mmol) was dissolved in 508 mL of hydrazine hydrate and refluxed for 1 hour. The reaction mixtures were then poured in ice water. The precipitate was then filtered, washed with water and dried under vacuuno oven to give 5-fluoro-2- oxindole (7.5 g). .

Step 4

The reaction mixture of 5- fluorooxindole (100 mg, C3.66 mmol), 5-formyl-2,4- dimethyl-1H-pyrrole-3-carboxylic acid (133 mg, 0.79 mmol), and 10 drops of piperidime in ethanol (3 mL) was stirred at 60 °C overnight and filtered. The solid was washed with. 1 M of aqueous hydrochloride solution, water, and dried to afford 5—(5-fluoro-2-oxo-1,2-dihyciro- indol-3-ylidenemethyl)-2,4-dimethyl-17-pyrrole-3-carboxyMic acid (201 mg, quantitataive) as a yellow solid. MS m/z (relative intensity, %) 299 (IM-1T", 100).

Example 4 - Synthesis of 5-(5-Fluoro-2-0xo-1,2-dihydro-#ndol-3-ylidene-methyl)-22,4- dimethyl-1H-pyrrole-3-carboxylic acid (3-diethylamino-22-hydroxy-propyl)-amid =e

Step 1

To 2-chloromethyloxirane (95 g, 1.03 mole) was added a mixture of water (3.08 g, ) 0.17 mole) and diethylamine (106.2 mL, 1.03 mole) at 30 °CC. The reaction mixture was then stirred at 28-35 °C for 6 hour and cooled to 20-25 °C to gives 1-chloro-3-diethylamino—propan- 2-ol.

Step 2

A soRution of sodium hydroxide (47.9 g, 1.2 mole) in 78 mL water =was added 1-

A chloro-3-die thylamino-propan-2-ol. The resultant was stirred at 20-25 °C for 1 hour, diluted with 178 mI __ of water and extracted with eth er twice. The combined ether solution was dried - with solid peotassium hydroxide and evaporated to give 135 g of crude procuct which was purified by &taction distillation to give pure glycidyldiethylamine (98 g, 76=%) as an oil.

Step 3

To the ice-cold solution of ammonium hydroxide (25 mL, 159 mm ole) of 25% (w/w) was added g=lycidyldiethylamine dropwise (3.2 g, 24.8 mmol) over 10 minutes. The reaction mixture wass stirred at 0 — 5 °C for 1 hour and then room temperature for 1-4 hours. The resulting re=action mixture was evaporated amd distilled (84-90 °C at 500-6 00 mT) to yield 1- amino-3-diesthylamino-propan-2-ol (3.3 g, 22%). MS m/z 147 ((M+11).

Step 4

To =the solution of 5-formyl-2,4-dim ethyl-1H-pyrrole-3-carboxylic- acid (100 mg, 0.43 mmol), ED=C (122.7 mg, 0.64 mmol) and H OBt (86.5 meg, 0.64 mmol) in 1.0 mL of DMF was added 1-ancino-3-diethylamino-propan-2-oX (93.2 mg, 0.64 mmol). The reessulting reaction solution was stirred at room temperature overnight and evaporated. The residue was suspended in 10 mL of water and filtered. “The solid was washed with sat-urated sodium bicarbonate and water and dried in a high v-acuum oven overnight to give crude product : which wass purified on column chromatography eluting with 6% methano1-dichlormethane containings, triethylamine (2 drops/ 100mL ©f 6% methanol-dichloromethane) to give 5-(5- fluoro-2-o x0-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrro le-3-carboxylic acid (3-diethyl=amino-2-hydroxy-propyl)-amide (62 mg, 34%) as a yellow soli d. 'H- NMR (400 MHz, DMSO0-d6) & 13.70 (s, 1H, NH-1°), 10.90 (s— 1H, NH-1), 7.76 (dd, J=2..38, 9.33 Hz, 1H, H-4),7.72 (s, 1H, vinyl-H), 7.60 (m, br., 1H,

CONHCH,CH(OH)-CH,N(C,Hs),-4°), 6.93 (dt, J = 2.38, 8.99 Hz, 1H, F1-5), 6.85 (dd, J = 4.55, 8.999 Hz, 1H, H-6), 3.83 (m, br, 1H, OH), 3.33 (m, 4H), 2.67 (m, br, 5H), 2.46 (s, 3H,

CH3), 2.4-4 (s, 3H, CH3), 1.04 (m, br, 6H, «CH;x2). MS m/z (relative intemnsity, %) 427 ((M+1]",. 100). :

Example 5 - Synthesis of 5-[5-Fluoro-2-oxo-1,2-dihyd mro-indol~(3Z)-ylidene-mmethyl]-2,4- dimethyl-1 H-pyrrole-3-carboxylic acid (2-hydroxy-3- morpholin-4-yl-propyl)—amide (R), (S) and (R/S) (Compowmnds 4, 5 and 6) . Step 1

A mixture of morpholine (2.6 mL, 30 mmol) and epichlorohydrin (2.35 m 1, 30 mmol) in ethanol (50 mL) was stirred at 70 °C overnight. After removing the solvent, thes residue was diluted with methylene chloride (50 mL). The clear solid precipitated was collected by vacuum filtration to givse 1-chloro-3-morpholin-4-yl-progpan-2-ol (2.0g, 37%). 'H NMR (DMSO-d) 8 3.49 (t, J=4.8 Hz, 2H), 3.60 (t, J=4.6Hz, 27H), 3.75 (m, 4H, 2xCH>), 4.20 (dd,

J=5.2, 12 Hz, 2H), 4.54% (m, 2H), 4.62 (m, 1H, CH), 6.64% (d, J=6.4 Hz, 1H, OH). IMS (m/z) 180.2 (M+1).

Step 2 1-Chloro-3-mozxpholin-4-yl-propan-2-ol (2.0g, 1 1 mmol) was treated with_ the solution of NH; in methanol (225% by weight, 20 mL) at room termperature. Nitrogen was “bubbled into the reaction mixture to remove the ammonia. Evaporation of solvent gave the hydrogen chloride salt of 1-amin_o-3-morpholin-4-yi-propan-2-ol (C2.0g, 91%). '"H NMR (E»MSO-dg) 8 2.30 (d, J=6.0Hz, 2H), 2.36 (m, 4H, NCH), 2.65 (dd, J==18.4, 12.8Hz, 1H), 2.91 (dd, J=3.6, 12.8Hz, 1H), 3.52 (mx 4H, OCHy), 3.87 (m, 1H, CH), 5 .32 (s, 1H, OH), 8.02 (brss,, 3H,

NH;"). MS (m/z) 161. 1 (M+1).

Step 3 5-(5-Fluoro-2-=0x0-1,2-dihydro-indol-3-ylidenenethyl)-2,4-dimethyl-1H—pyrrole-3- carboxylic acid (120 mag, 0.4 mmol) was condensed witTh 1-amino-3-morpholin-£3-yl-propan- 2-0l(74 mg, 0.48 mmol) to precipitate 5-[5-fluoro-2-0x™-1,2-dihydro-indol-(3Z)— ylidenemethyl}-2,4-di-methyl-1H-pyrrole-3-carboxylic acid (2-hydroxy-3-morpheolin-4-yl- propyl)-amide (65 mgs, 36%). The mother liquid was ev-aporated to dryness and t=he residue was purified by flash «chromatography to give additiona_l 2N (70 mg, 39%). '"H NeMR (DMSO- de) 82.28 (m, 1H), 2.332 (m, 1H), 2.40 (m, 4H), 2.40, 2. 42 (2xs, 6H, 2xCH3), 3.1 5(s, 1H), 3.31 (m, 1H), 3.55 (zm, 4H), 3.78 (m, 1H), 4.73 (brs, 11H, OH), 6.82 (dd, J=4.5, 8 .4Hz, 1H), 6.90 (td, 21=2.8, 33=10).0Hz, 1H), 7.53 (m, 1H), 7.70 (s, 1H), 7.74 (dd, J=2.0, 9.6=Hz, 1H) } (aromatic and vinyl), 10.87 (s, 1H, CONH), 13.66 (s, 17H, NH). LC-MS (m/z) 4481.4 (M-1).

Svnthesis o f2-hydroxy-7-oxa-4-azoni aspiro[3.5|nonane chlo=xide ~ No _1 ethanol a n>" . oO C= —_— :

Lo~ HN] 2) acetone oJ ’ To a 1L 3-neck r-ound bottom flask, fitted wi th a thermocouple, nitrogZen inlet and a 250ml addition funnel, vas charged morpholine (91 .5g, 91.5 ml, 1.05 mole, 1.0 eq.) and 100ml of ethanol. The solution was stirred rapidly ‘while adding epichlorohy~drin (100g, 84.5 ml, 1.08 mole, 1.03 eq.) from the addition funnel over about 30 minutes. Th etemperature was monitored and whem the pot temperature reached 27°C, the reaction was cooled with an ice water bath. The clear solution was stirred for 1& hours. The reaction wass assayed by GC (dilute 5 drops of reaction mixture into 1 ml of ethamnol and inject onto a 1501 DB-5 capillary

GC column with the fol lowing run parameters, Injector 250°C, detector 250=C, initial oven temperature 28°C warming to 250°C at 10°C per minute.) The reaction was complete with less than 3% morpholine remaining. The reaction v-vas concentrated on the r- otoevaporated at 50°C with full house va_cuum until no more distillate could be condensed. T he resulting oil was stored at room tempperature for 24-48 hours or wintil a significant mass o ferystals was observed (seeded will speed up the process). The sJurry was diluted with 250ml of acetone and filtered. The solidss were dried in the vacuum omven at 60°C for 18-24 hows. This provided 84g of crystal line product. The mother licquors could be concentrated and the crystallization process mrepeated in increase recovery. '"H NMR (400 MHz, IDMSO-dg) 6.55 (d, 1 H), 4.64 (m, 1 H), 4.53 (m, 2H), 4.18 (m, 2 A), 3.74 (m, 4 H), 3.60 (rm, 2 H), 3.48 (m, 2

H). >*C NMR (100 MHa z, DMSO-ds) 6 70.9, 61.39, 61.04,60.25, 58.54, 57.280.

Synthesi sof 1-amino-3-(4-morphok inyl)-2-propanol (Racemmic)

Ca CL EE oo on oo no HCI

Hy NHg, MeOH (ONY NE 3 Co oo oJ OH

To a 3L 1-neck= round bottom flask with a rnagnetic stir bas was chaarged 2-hydroxy-7- oxa-4-azoniaspiro[3.5 ]nonane chloride (150g, 83 5-mmole) followed by 23 wt. % anhydrous ammonia in methanol (2120ml). The flask was stoppered and the resulting clear solution was stirred at 20-23°C for 18 hours. GC under the con ditions above showed no remaining starting material. The stopper was removed and the ammownia allowed to bubble oumt of the solution for 30 minutes. The flask was then transferred to a rotoevaporated and concemntrated to a white solid with 45°C bath and full house vacuum. 'H NMR (400 MHz, DMSS0-ds) 3.57 (dd, 2HD), 3.3-3.5 (m, 6 H), 2.59 (m, 2 H), 2.2-2.4 (m, 6 H); °C NMR (100 MHz DMSO-ds) 8 ’ 70.8, 67.1, 60.1, 53.8, 48.1. . Following the procedure described in Example 3 above but substituting 2-(RS)-1- amino-3-morpholin-4-yl-propan-2-ol with 2-(S)-1 -amino-3-morpholin-4-yl-p-ropan-2-ol prepare«d as described below the desired compound 5-[5-fluoro-2-oxo-1 ,2-dih_ydro-indol- (3Z)-yladenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-(S)-hydro- xy-3- morphoslin-4-yl-propyl)-amide was obtained.

Synthesis of 1-amino-3-(4-morpholinyl)-2-propanol (Non-Racemic) o}

To 1L 3-neck round bottom flask, fitted with mechanical stirring, thermocouple and additiom funnel, was charged morpholine (91.5g, 91.5 ml, 1.05 mole, 1.0 eq.) and 45 ml of t- butanol. The solution was stirred rapidly while adding R-epichlorohydrin (100g, 84.5 ml, 1.08 meole. 1.03 eq.) from the addition funnel over about 30 minutes. The termperature was monitored and when the pot temperature reached 27°C, the reaction was cool ed with an ice water beath. The clear solution was stirred for 18 hours. The reaction was assayed by GC (dilute 5 drops of reaction mixture into 1 ml of ethanol and inject onto a 15m DB-5 capillary

GC col umn with the following run parameters, Injector 250°C, detector 250° C, initial oven temper ature 28°C warming to 250°C at 10°C per minute). The reaction was =complete with less thzan 3% morpholine remaining. The solution was cooled to 10°C and a Z20 wt% solution of pota_ssium t-butoxide in THF (576g) was added dropwise keeping the temperature less than 15°C. The resulting white slurry was stirred at 10-15°C for 2 hours and chec=ked by GC using the above conditions. None of the chlorohydrin could be observed. The mix ture was concermtrated on the rotoevaporated using 50°C bath and full house vacuum. The resulting mixture was diluted with water (500ml) and methylene chloride. The phases- were separated and thes aqueous phase washed with methylene chloride (500ml). The combined organic layers were dried over sodium sulfate and concentrated to a clear, colorless owil. This provid ed 145g, 97% yield of the epoxide. 'H NMR (400 MH,, DMSO-d) 8 3.3 (dd, 4 H),

3.1 (m, 1 H), 2.6 (dd, 1 HX), 2.5 (dd, 1 H), 2.4 (m, 4 H), 2.2 (dd, 2 H); BC NMR (1 00 MH,

IDOMSO0- dg) 8 65.4, 60.1, 53.1,48.9, 43.4.

The above crude espoxide was charged to a 3L 1-n eck round bottom flask with a magnetic stir bar. Anhycrous ammonia in methanol (24% w/w 2.5L) was added, the flask } wvas stoppered and the mixture stirred at room temperatur e for 24 hours. GC under the conditions above showed no remaining starting material. The stopper was removeed and the

Ammonia allowed to bub ble out of the solution for 30 mirutes. The flask was them transferred to a rotoevapeorated and concentrated to a clear colorless oil with 45°C bath and

Full house vacuum. This provided 124g of product. '"H NIMR (400 MH,, DMSO—ds) 8 3.57

Cad, 2H), 3.3-3.5 (m, 6 FH), 2.59 (m, 2 H), 2.2-2.4 (m, 6 HD); °C NMR (100 MH,, BDMSO- de)

S70.8,67.1, 60.1, 53.8, 48.1.

Synthesis of 1-amino-3-(4-morpholin—vyl)-2-(S)-propanol

To 1L 3-neck rorand bottom flask, fitted with mec hanical stirring, thermocouple and addition funnel, was cha-rged morpholine (91.5g, 91.5 ml , 1.05 mole, 1.0 eq.) and 200 ml of 1ethanol. The solution —was stirred rapidly while adding R-epichlorohydrin (100s, 84.5 ml, 1.08 mole, 1.03 eq.) frorm the addition funnel over about 30 minutes. The temperature was monitored and when the pot temperature reached 27°C, the reaction was cooled v=vith an ice water bath. The clear soslution was stirred for 18 hours. 'E he reaction was assayed by GC (dilute 5 drops of reaction mixture intol ml of ethanol amd inject onto a 15m DB—S5 capillary

GC column with the following run parameters, Injector 250°C, detector 250°C, iritial oven temperature 28°C warm ing to 250°C at 10°C per minute_) The reaction was com_plete with less than 3% morpholines remaining. The solution was co-oled to 10°C and a 25 w~t. %esolution of sodium methoxide in methanol (233g, 1.08 mole, 247 ml) was added dropwise keeping the temperature less than 15°C. The resulting white slurry was stirred at 10-15°C fom 2 hours and checked by GC using thae above conditions. None of the chlorohydrin could be o=bserved.

The mixture was concertrated on the rotoevaporator usirig 50°C bath and full ho=use vacuum.

The resulting mixture was diluted with water (500ml) an.d methylene chloride. T he phases were separated and the - aqueous phase washed with metlaylene chloride (500ml). The combined organic layer—s were dried over sodium sulfate and concentrated to a cl-ear, colorless oil. This provided 145g, 97% yield of 1,2-epoxy-3-morpoholin-4-ylpropane. 'H MMR (400

MHz, DMSO-ds) 83.3 (dd, 4 H), 3.1 (m, 1 H), 2.6 (dd, I H), 2.5 (dd, 1 H), 2.4 (mm, 4 H), 2.2 (dd, 2 Hy; *C NMR (100 MHz, DMSO-de) 8 65.4, 60.1, 53.1, 48.9,43.4.

wy0 2004/075775 PCT/US2004/005283

The above crude 1,2-epoxy-3-moxpholin-4-ylpropane was charged toa 3L 1-neck round bottom flask with a magnetic stir bar. Anhydrous ammonia in methzanol (24% w/w 2.51) was added, the flask was stoppered and the mixture stirred at room tesmperature for 24 : hours. GC under the conditions above showed no remaining starting mater—ial. The stopper wass removed and the ammonia allowed to bubble out of the solution for 30 minutes. The flassk was then transferred to a rotoevaporated and concentrated to a clear c=olorless oil with 45°C bath and full house vacuum. This provided 124g of 1-amino-3-(4-meorpholinyl)-2-(S)- pro-panol. 'H NMR (400 MHz, DMSO-d; 8 3.57 (dd,2H), 3.3-3.5 (m, 6 H), 2.59 (m, 2 H), 2.2- 2.4 (m, 6H); '*C NMR (100 MHz, DMS O-ds) § 70.8, 67.1, 60.1, 53.8, 48. 1. ~ 0

HN N

3 AL oN TN

NSN NH OH ©

H IN —— —» °F / f

N S-fluorooxindole °

EtsN, THF co" : y

H

Imidazole amide (7.0 g, 32.3 mmol), amine (15.0 g, 64.6 mmol), S—fluorooxindole (4.93 g, 32.6 mmol), triethylamine (9.79 g, 96.9 mmol), and THF (88 ml) —were mixed and heaated to 60°C. A brown solution formed. After stirring for 24 h at 60°C, the yellow slurry was cooled to rt (room temperature) and filtered. The cake was washed with 80 ml THF and dri_ed overnight at 50°C under house vacuum. A brown solid (23.2 g) was obtained. The solid was slurried in 350 ml water for 5 h at rt and filtered. The cake was —washed with 100 ml water and dried at 50°C under house vacuum overnight. 8.31 g were ototained with 56% chemical yield.

F Te fe) x

TL + 0 IN NS + HN YT NY

N z L_o 8 HO fo) »GER®!

N HO

F / o

N

A 0.25L flask fitted with am thermometer, condenser, nmagnetic stirring, and nitrogen irajet was charged with 4.92g 5-Fl-uorooxindole, 7.0g Imidazosle amide, 15.5g (R)-11 -Amino-3- (4-morpholinyl)-2-propanol, 9.78 _g Triethylamine and 88ml Tetrahydrofuran. The= mixture : was heated to 60° C for 16.5 hour—s. The reaction is cooled tos ambient temperature= and filtered. The solids obtained are sslurried (3) three successive times in acetonitrile sat 11ml/g, dried in vacuo for 3.6g (25.25%). [HPLC, Hypersil BDS, C- 18, 5p, (6:4), AcetonTitrile:0.1M

Ammonium Chloride, PHA-5714 37 = 4.05 min.] H'NMR (IOMSO): § 10.86 (1H, bs); 7.75 (1H,d); 7.70 (1H,s); 7.50 (1H,m); 6.88 (2H,m); 4.72 (1H,bs); 3.78 (1H,bs); 3.56 (4H, m); 3.32 (6H,m); 3.15 (1H,m); 2.43 (8H,brm). :

Example 6 - Synthesis of 2,4-dirnethyl-5-[2-0x0-1,2-dihydmro-indol-(3Z)-yliden ~emethyl]- 1_H-pyrrole-3-carboxylic acid (2=-hydroxy-3-morpholin-4-=y]-propyl)-amide 5-(2-Ox0-1,2-dihydro-ind- ol-3-ylidenemethyl)-2,4-dirmethyl-1 H-pyrro le-3-acarboxylic acid (113 mg, 0.4 mmol) was con_densed with 1-amino-3-mompholin-4-yl-propan-2-ol (74 mg, 0.48 mmol) to precipitate 2,4-dinmethyl-5-[2-oxo-1,2-dihydro -indol-(3Z)-ylidenermethyl]-14- pyrrole-3-carboxylic acid (2-hydr—oxy-3-morpholin-4-yl-prop yl)-amide (77 mg, 45 .3%). 'H NMR (DMSO-d) 8 2.227 (m, 1H), 2.32 (m, 1H), 2 .40 (m, 4H), 2.40, 2.482 (2xs, 6H, 2xCHa), 3.15 (s, 1H), 3.32 (m, 11), 3.55 (m, 4H), 3.77 (m, 1H), 4.74 (d, J=4.8Hz, 1H, OH), 6.86 (d, J=7.6Hz, 1H), 6.96 (t, J=-7.2 Hz, 1H), 7.10 (t, J=7.6Fz, 1H), 7.49 (t, J=5.6= Hz, 1H), 7.61 (s, 1H), 7.77 (d, J=8.0 Hz, 1H) (aromatic and vinyl), 10-88 (s, 1H, CONH), 1 3.62 (s, 1H,

INH). LC-MS (m/z) 425.4 (M+1).

Example 7 - Synthesis of S-[S-cHhloro-2-oxo-1,2-dihydro-iradol-(3Z)-ylidene-me=thyl}-2,4- dimethyl-1H-pyrrole-3-carboxy~lic acid (2-hydroxy-3-mor—pholin-4-yl-propyl)—amide (Compound 7) 5-(5-Chloro-2-oxo-1,2-dilhydro-indol-3-ylidenemethy~1)-2,4-dimethyl-1 H-poyrrole-3- carboxylic acid (126.6 mg, 0.4 m_mol) was condensed with 1—amino-3-morpholin--4-yl- propan-2-ol (74 mg, 0.48 mmol) to precipitate 5-[S-Chloro-2--oxo-1,2-dihydro-indLol-(3Z)- ylidenemethyl]-2,4-dimethyl-1H—pyrrole-3-carboxylic acid (2-hydroxy-3-morpho in-4-yl- propyl)-amide (107 mg, 58%). "H NMR (DMSO-dg) 8 2-29 (m, 1H); 2.33 (m, 1H), 22.39(m, 4H), 2.40, 2.4-2 (2xs, 6H, 2xCHa), 3.15 (s, 1H), 3.37 (m, 1E1), 3.55 (m, 4H), 3.77 (m, 1 H), 4.74 (d, J=4.8Hz,. 1H, OH), 6.85 (d, J=8.4Hz, 1H), 7.11 (dd, J=2.0, 8.0Hz, 1H), 7.53 (t, J =5.6Hz, 1H), 7.75 (s~ 1H), 7.97 (d, 1=2.0Hz, 1H) (aromatic and vinyl), 10.99 (s, 1H, CONH) , 13.62 (s, 1H, NH). IL.C-MS (w/z) 457.4 (M-1).

Example 8 - Synthesis of 5- 5-bromo-2-oxo-1,2-dibydro-i ndol-(3Z)-ylidene-mettmyl]-2,4- dimethyl-1H-pyrrole-3-carBooxylic acid (2-hydroxy-3-mompholin-4-yl-propyl)-ammide 5-(5-Bromo-2-oxo0-1, =-dihydro-indol-3-ylidenemeth-yl)-2,4-dimethyl-1H-pyrmrole-3- ‘ carboxylic acid (72.2 mg, 0.2 mmol) was condensed with 1—amino-3-morpholin-4-yl -propan- 2-01 (38mg, 0.24 mmol) to pmecipitate 5-[5-Bromo-2-0x0-1 ,2-dihydro-indol-(3Z)- ylidenemethyl}-2,4-dimethyl -1H-pyrrole-3-carboxylic acid &2-hydroxy-3-morpholin—4-yl- propyl)-amide (55 mg, 55%) .

IH NMR (DMSO-de) 62.27 (m, 1H), 2.32 (m, 1H), =2.39(m, 4H), 2.41, 2.42 ((2xs, 64, 2xCHs), 3.13 (s, 1H), 3.35 (xm, 1H), 3.55 (m, 4H), 3.77 (m, JH), 4.74 (d, J=4.4Hz, 1E], OH), 6.80 (d, J=8.4Hz, 1H), 7.24 «dd, J=2.0, 8.0Hz, 1H), 7.51 (t, .J=5.6Hz, 1H), 7.76 (s, 1 EJ), 8.09 (d, J=2.0Hz, 1H) (aromatic &and vinyl), 10.99 (s, 1H, CONE), 13.62 (s, 1H, NH). LC=-MS (m/z) 503.4 (M-1).

Example 9 - Synthesis of 2 _4-dimethyl-5-[2-0xo-1,2-dihyedro-indol-(3Z)-ylidene- mmethyl]- 1H-pyrrole-3-carboxylic acid (2-hydroxy-3-[1,2,3]triazoM-1-yl-propyl)-amide

Step 1

A mixture of 3-[1,2,3]triazole (2.0 g, 29 mmol), epicchlorohydrin (3.4 ml, 43. 5 mmol) and N, N-diisopropyl-ethylamine (2.6 mL, 15 mmol) in eth-anol (50 mL) was stirred at room temperature overnight. Afte removing the solvents, the res—idue was purified by flash chromatography (CHCl,/CH;0H=100/ 1-100/2-100/4) to g=ive 1-chloro-3-(1,2,3)-triz azol-2- ylpropan-2-ol (2.1 g, 45%). '"H NMR (CDCl3) & 3.52 (m, 2=H, OH and CH>), 3.60 (3d, J=5.2, 11.2 Hz, 1H), 4.36 (m, 1H, CH), 4.68 (m, 2H), 7.67 (s, 2H)». MS (m/z) 162.1 M+1) and 1- chloro-3-(1,2,3)triazol-1-ylporopan-2-ol (2.3 g, 49%). 'H NMR (CDCl;) 6 3.56 (s, 1_H), 3.57 (s, 1H), 4.35 (m, 1H), 4.53 «dd, J=7.2, 14 Hz, 1H), 4.67 (d3, J=3.8, 14Hz, 1H), 7.67 (s, 1H), 7.71 (s, 1H). MS (m/z) 162.1 (M+1). :

Step 2 : 1-Chloro-3(1,2,3)tri_azol-1-ylpropan-2-ol (2.3g, 13 mmmol) was treated with t-he solution of NH; in methanol (25% by weight, 20 mL) at 60 °C overnight in a sealed pressure vessel. After cooling to room temperature, nitrogen was bumlbbed into the reaction mixture to remove the ammonia. Evaporation of solvent gave the hydrogen chloride salt of 1-ammino-3- (1,2,3)triazol-1-ylpropan-2 ~ol (2.57g, 100%).

'H NMR (DMSO-d) & 2.68 (dd, J=8.8, 12.8Hz, 1H), 2.97 (dd, J=3.6, 12.8Hz, 1H), 4.15 (m, 1H), 4.44 (dd, J=6.4, 14Hz, 1H), 4.57 (dd, J=4.6, 14H=, 1H), 5.95 (d, J=5.2Hz, 1H,

OH), 7.77 (s, 1H), 8.01 (brs., 3H, NH;"), 8.12 (5, 1H). MS (m/z) 143.1 (M+1).

Step 3 . 5-(2-Oxo-1,2-dihydro—indol-3-ylidenemethyl)-2,4-dimewsthyl-1H-pyrrole-3 -carboxylic acid (113 mg, 0.4 mmol) was condensed with 1-amino-3(1,2,3) triazole-1-yl-propan-2-ol 85 mg, 0. 48mmol) to precipitate 2,4-dimethyl-5-[2-0x0-1,2-dihyd To-indol-(3Z)-ylidenemethyl ll - 1H-pyrrole-3-carboxylic acid (2-hydroxy-3-[1,2,3]triazol-1-yl-poropyl)-amide (70 mg, 41%). "H NMR (DMSO-dg) 32.45, 2.48 (2xs, 6H, 2xCHj), 3.35 (m, 2H), 4.02 (m, 1H), 4.32 (dd, J=7.6, 14 Hz,1H), 4.53 (dd, J=3.4, 14 Hz,1H), 5.43 (d, ]=5 .6Hz, 1H, OH), 6.91 (d,

J=7.6Hz, 1H), 7.01 (t, J=7.6 Hz, 1H), 7.15 (t, ]=8.0Hz, 1H), 7.66 (s, 1H), 7.12 (t, 3=5.6 Hz, 1H), 7.74 (s, 1H), 7.77 (d, J=7.6 Hz, 1H), 8.11 (s, 1H), 10.93 (s-, 1H, CONH), 13.68 (s, 1H,

NH). LC-MS (w/z) 405.4 (M1).

Example 10 - Synthesis of S—[5-fluoro-2-o0xo-1,2-dihydro-incAol-(3Z)-ylidene-methyl}-2,4&- dimethyl-1H-pyrrole-3-carboxylic acid (2-hydroxy-3-[1,2,3] triazol-1-yl-propyl)-amide 5-(5-Fluoro-2-oxo-1 ,2-dihydro-indol-3-ylidenemethyl)—2,4-dimethyl- 1H-pyrrole-3- carboxylic acid (120 mg, 0.4 mmol) was condensed with 1-ami_no-3(1,2,3)triazol-1-yl- propan-2-ol (85 mg, 0. 48mmol) to precipitate 5-[5-fluoro-2-0x=0-1,2-dihydro-indol-(3Z)- ylidenemethyl]-2,4-dimethyl—1H-pyrrole-3-carboxylic acid (2-knydroxy-3-[1,2,3]triazol-1-yl— propyl)-amide (100 mg, 62%). 'H NMR (DMSO-dg) 82.42, 2.44 (2xs, 6H, 2xCHa), 3.227 (m, 2H), 3.98 (m, 1H), 4.27 (dd, J=7.6, 14 Hz,1H), 4.50 (dd, J=3.4, 13.6 Hz,1H), 5.38 (d, J==5.6Hz, 1H, OH), 6.82 (dd,

J=4.4, 8.4Hz, 1H), 6.91 (id, 2J=2.4, *J=9.0Hz, 1H), 7.70 (m, 33), 7.75 (dd, J=2.4, 9.2Hz, 1H), 8.11 (s. 1H), 10.93 (s, 1H, CONH), 13.73 (s, 1H, NH). LC-MS (m/z) 423.4 (M-1).

Example 11 - Synthesis of S-[5-chloro-2-0x0-1,2-dihydro-in dol-(3Z)-ylidene-methyl}-2,<4- dimethyl-1H-pyrrole-3-carboxylic acid (2-hydroxy-3-[1,2,3 ]triazol-1-yl-propyl)-amide 5-(5-Chloro-2-oxo-1, 2-dihydro-indol-3-ylidenemethyl) -2,4-dimethyl-1H-pyrrole-3- carboxylic acid (126.6 mg, 0.4 mmol) was condensed with 1-a—mino-3(1,2,3)triazole-1-yl- propan-2-ol (85 mg, 0. 48mimol) to precipitate 5-[5-Chloro-2-c>x0-1,2-dihydro-indol-(3Z)- ylidenemethyl]-2 [A-dimethyk-1H-pyrrole-3-carboxylic acid (2-_hydroxy-3-[1,2,3]triazol-1-yl - propyl)-amide (48 mg, 27%).

TH NMR (DMSO-dg) § 2.42, 2.44 (2xs, 6H, 2xCHs), 3.27 (m, 2H) , 3.99 (m, 1H), 4.28 (dd, 1=7.8, 14 Hz,1H), 4.51 (dd, J=3.2, 14 Hz,1H), 5.39 (4, J=6.0Hz, 1H, OH), 6.85 (d,

J =8.4Hz, 1H), 7.12 (dd, J=2.0, 8.2Hz, 1H), 7.70 (m, 2H), 7.74 (s, 1H), 7.97 (d, J=2.0Hz, 1H), 8.07 (s, 1H), 10.99 (s, 1H, CONH), 13.65 (s, 1H, NH). LC-MS (vz) 439 _.4 (M-1). } Example 12 - Synthesis of 5-[5-bromo-2-oxo-1,2-dihydro-indol-(3Z)-y-lidene-methyl]-2,4- climethyl-1H-pyrrole-3-carboxylic acid (2-hydroxy-3-[1,2,3]triazol-1-=y}-propyl)-amide 5-(5 -Bromo-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimetBhyl-1 H-pyrrole-3- carboxylic acid (144.4 mg, 0.4 mmol) was condensed with 1-amino-3(1,2=,3)triazole-1-yl- propan-2-ol (85 mg, 0.48mmol) to precipitate 5-[5-bromo-2-ox0-1,2-dihy=dro-indol-(3Z)- wlidenemethyl]-2,4-dimethyl- 1 H-pyrrole-3-carboxylic acid (2-hydroxy-3—[1,2,3]triazol-1-yl- propyl)-amide (130 mg, 67%). 'H NMR (DMSO-dg) 8 2.41, 2.44 (2xs, 6H, 2xCHs), 3.27 (m, 2H)m, 3.99 (m, 1H), 4.28 (dd, I=7.6, 14 Hz,1H), 4.50 (dd, J=3.6, 14 Hz,1H), 5.40 (d, J=5.6Hz, 1H, OH), 6.81 (4d,

J=8.4Hz, 1H), 7.24 (dd, J=2.0, 8.0Hz, 1H), 7.70 (m, 2H), 7.77 (s, 1H), 8.07 (s. 1H), 8.10 (d,

J=1.6Hz, 1H), 11.0 (s, 1H, CONH), 13.64 (s, 1H, NH). LC-MS (m/z) 485.4 (M-1).

Example 13 - Synthesis of 5-(5-Fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl-2,4- dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl)amide (Compound 1) 5-Fluoro-1,3-dihydroindol-2-one (0.54 g, 3.8 mmol) was condens ed with 5-formyl- 2.4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylaminoethy!)amide t=o give 0.83 g (55%) of the title compound as a yellow green solid.

Alternative synthesis of 5-(5-Fluoro-2-oxo-1,2-dihydroindol-3-yliden semethyl)-2,4- dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl)amide

Hydrazine hydrate (55 %, 3000 mL) and 5-fluoro-isatin (300 g) vere heated to 100 °C. An additional 5-fluoro-isatin (500 g) was added in portions (100 g) over 120 minutes with stirring. The mixture was heated to 110 °C and stirred for 4 hours. The r=nixture was cooled 1o room temperature and the solids collected by vacuum filtration to give= crude (2-amino-5- fluoro-phenyl)-acetic acid hydrazide (748 g). The hydrazide was suspeneded in water (700 mL) and the pH of the mixture adjusted to < pH 3 with 12 N hydrochlori- c acid. The mixture was stirred for 12 hours at room temperature. The solids were collected by vacuum filtration and washed twice with water. The product was dried under vacuum to give 5-fluoro-1,3- dihydro-indol-2-one (600 g, 73 % yield) as a brown powder. "H-NMR (climethylsulfoxide-

dg) 83.46 (5, 2H, CH»), 6.75, 6.95, 7.05 (3 xm, 3H, arormnatic), 10.35 (s, 1H, NH)—- MS m/z 152 [M+1]. 3,5-Dimethyl-1H-pyrrole-2,4-dicarboxylic acid 2=-tert-buty] ester 4-ethyl e-ster (2600 g) and ethanol (7800 mL) wesre stirred vigorously while 10 N hydrochloric acid (3650 mL) . was slowly added. The temperature increased from 25 °CC to 35 °C and gas evolution began.

The mixture was warmed to _54 °C and stirred with furtheer heating for one hour at which time the temperature was 67 °C. "The mixture was cooled to S °Cand 32 L of ice and vovater were slowly added with stirring. Whe solid was collected by vacuum filtration and washed three times with water. The solid was air dried to constant we=ight to give of 2,4-dimet hyl-1H- pyrrole-3-carboxylic acid etlayl ester (1418 g, 87 % yielA) as a pinkish solid. "H-NMR (dimethylsulfoxide-ds) & 2.1.0, 2.35 (2xs, 2x3H, 2xCH3)w, 4.13 (q, 2H, CH>), 6.37 (s, 1H,

CH), 10.85 (s, 1H, NH). MS m/z 167 [M+1].

Dimethylformamide (322 g) and dichloromethan_e (3700 mL) were cooled in an ice bath to 4 °C and phosphorus oxychloride (684 g) was added with stirring. Solid 24- dimethyl-1H-pyrrole-3-carboxylic acid ethyl ester (670 =) was slowly added in al iquots over minutes. The maximum temperature reached was 18 °C. The mixture was heated to reflux for one hour, cooled te 10 °C in an ice bath and 1 .6 L of ice water was rapidly added with vigorous stirring. The ®emperature increased to 15 °C. 10N Hydrochloric amcid (1.6 L) was added with vigorous stimring. The temperature increased to 22 °C. The mixture was allowed to stand for 30 minutes and the layers allowed t o separate. The temperature reached a maximum of 40 °C. The aqueous layer was adjusted to pH 12-13 with 10 N potassium hydroxide (3.8 L) at a rate that allowed the temperature to reach and remain at 55 °C during the addition. After the addition was complete the mixtu_re was cooled to 10 °C ard stirred for 1 hour. The solid was collescted by vacuum filtration arid washed four times witln water to give 5-formyl-2,4-dimethyl—1H-pyrrole-3-carboxylic ac id ethyl ester (778 g, 100% yield) as a yellow solid. "H-NMR (IDMSO-de) 8 1.25 (1, 3H, CHE), 2.44, 2.48 (2xs, 2x3H , 2xCHj), 4.16 (q, 2H, CH), 9.59 (s, LH, CHO), 12.15 (br s, 1H, INH). MS m/z 195 [M+1_]. 5-Formyl-2,4-dimetEayl- 1H-pyrrole-3-carboxylic acid ethyl ester (806 g), potassium hydroxide (548 g), water (2-400 mL ) and methanol (300 mL) were refluxed for two hours with stirring and then coole«d to 8 °C. The mixture was extracted twice with dichloromethane. The aqueeous layer was adjusted to p H 4 with 1000 mL of 10 WN hydrochloric acid keeping tlhe temperature under 15 °C. Water was added to facilitate stirring. The solid was collected by vacuum filtration, washed three times vith water and dried under vacuum at 50 °C to give 5-formyl-2,4-dimethyl-1H-pyrrole-3 -c=arboxylic (645 g, 93.5 % yield) acid as a yellow solid. "H-NMR (IDMSO-ds) 6 2.40, 2.43 (2=xs, 2x3H, 2xCH), 9.57 (s, 1H , CHO), 12.07 (br s, 2H, NH+COOH) . MS m/z 168 [M+1]. : 5-F-ormyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (1204 g) and «6020 mL of dimethylformamide were stirred at room temperature while 1-(3-dimethyl-=:aminopropyl-3- ethylcarbocliimide hydrochloride (2071 g), hydro xybenzotriazole (1460 g), triethylamine (2016 mL) and diethylethylenediamine (1215 mI) were added. The mixtumre was stirred for hours at: room temperature. The mixture was diluted with 3000 mL of water, 2000 mL of brine and 3000 mL of saturated sodium bicarbon ate solution and the pH ad_justed to greater than 10 with 10 N sodium hydroxide. The mixture was extracted twice with 5000 mL each time of 10 % methanol in dichloromethane and the extracts combined, drie d over anhydrous magnesium sulfate and rotary evaporated to dryness. The mixture was wish diluted with 1950 mL cof toluene and rotary evaporated again to dryness. The residue was triturated with 3:1 hexane=:diethyl ether (4000 mL). The solids were collected by vacuun~ filtration, washed twice with. 400 mL of ethyl acetate and dried under vacuum at 34 °C for 2 hours to give 5- formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl)—amide (819 g, 43 % yield) a.s a light brown solid. "H-NMR (dimethylsulfoxide-ds) & 0.96 (t, 6H, 2xCH3), 2.31, 2.38 (2xs, 2x CHa), 2.51 (m, 6H 3xCH,), 3.28 (xm, 2H, CH, ), 7.34 (m, 1H, amide NH), 9.56 (s, 1H, CEH0), 11.86 (s, 1H, pyrrole NH). MS m/z 266 [M+1]. 5-Formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (2-diethylam—inoethyl)-amide (809 g), 5—fluoro-1,3-dihydro-indol-2-one (438 g), ethanol (8000 mL) and pyrrolidine (13 mL) were heated at 78 °C for 3 hours. The mixture was cooled to room te—mperature and the solids colL ected by vacuum filtration and washed with ethanol. The solidss were stirred with ethanol (5 900 mL) at 72 °C for 30 minutes. The mixture was cooled to romom temperature.

The solidss were collected by vacuum filtration, washed with ethanol and diried under vacuum at 54 °C for 130 hours to give 5-[5-fluoro-2-0xo -1,2-dihydro-indol-(3Z)-y lidenemethyl]-2,4- dimethyl- 1H-pyrrole-3-carboxylic acid (2-diethylamino-ethyl)-amide (10R 3 g, 88 % yield) as an orange= solid. l11-NMR (dimethylsulfoxide-ci6) & 0.98 (t, 6H, 2xCH3), 2.43, 2.44 (2xs, 6H, 2xCHI3), 2.50 (m, 6H, 3xCH2), 3.28 (q, 2H, CH2), 6.84, 6.92, 7.42,7_.71, 7.50 (5xm, 5H, aromatic, vinyl, CONH), 10.88 (s, 1H, CONH), 13.68 (s, 1H, pyrrole NH)». MS m/z 397 [M- 11.

The malic salt ©f 5-(5-Fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4 -dimethyl- 1H-pyrrole-3-carboxyliic acid (2-diethylamino-ethyl)annide can be prepared accordiing to the disclosure of U.S. Pate-mt Application Serial No. 10/281 ,985, filed August 13, 2002 , which . claims priority to U.S. Patent Provisional Application TNo. 60/312,353, filed August 15, 2001, which is incorporated toy reference in its entirety.

Synthesis of 5-e(5-bromo-2-o0xo-1,2-dihydro-inclol-3-ylidenemethyl)-2,4-dirmethyl-14- pyrrole-3-carboxylic acid, 5-(5-chloro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-=2,4- dimethyl-1H-pyrrole-3 -carboxylic acid, 5-(2-oxo0-1,2-Aihydro-indol-3-ylidenemeth_yt)-2,4- dimethyl-1H-pyrrole- 3 -carboxylic acid is described in Serial No. 09/783,264 filed on

February 14%, 2001, titled “PYRROLE SUBSTITUTED 2-INDOLINONE --PRO_TEIN

KINASE INHIBITOR_S”, the disclosure of which is incorporated herein in its entirety.

Example 14 - Synthessis of 5-(5-Fluoro-2-oxo-1,2-dikaydro-indol-3-ylidenemeth Zyl)-2,4- dimethyl-1H-pyrrole=-3-carboxylic acid (2-pyrrolidin-1-yl-ethyl)-amide (Compmwound 2) 5-Fluoro-1,3-dihydro-indolin-2-one was conde=nsed with 5 -formyl-2,4-dimeethyl-1H- pyrrole-3-carboxylic =xcid (2-pyrrolidin-1 -yl-ethyl)-amaide to yield the title compound.

MS + ve APC 397 [M+1].

Exmaple 15 - Synthe=sis of 5-(5-Fluoro-2-0xo-1,2-d&hydro-indol-(3Z)-ylidenenmethyl)-2,4- dimethyl-1H-pyrrole-3-carboxylic acid (2-ethylami no-ethyl)-amide (Compourad 8) 5-Formyl-2,4—dimethyl-1H-pyrrole-3-carboxykic acid (2-ethylamino-ethyl) -amide (99g), ethanol (400 nm), 5-fluoro-2-oxindole (32 g) amd pyrrolidine (1.5 g) were re=fluxed for 3 hours with stirring. The mixture was cooled to roorm temperature and the solids collected by vacuum filtration. The solids were stirred in ethan ol at 60°C, cooled to room temperature and collected by vacuaum filtration. The product was «ried under vacuum to give S-(5-

Fluoro-2-oxo-1 2-dihaydro-indol-(3Z)-ylidenemethyl)—2,4-dimethyl- 1H-pyrrole-3-ccarboxylic acid (2-ethylamino-e-thyl)-amide (75g, 95% yield). 'EHH-NMR (dimethylsulfoxide-«ds) § 1.03 (t, 3H, CHa), 2.42, 2. 44 (2xs, 6H, 2xCHa), 2.56 (q, 24, CHy), 2.70, 3.30 (2xt, 4H, 2xCH,), 6.85, 6.92, 7.58, 7.72=,7.76 (5xm, 5H, aromatic, vinyl , and CONH), 10.90 (br s, 136, CONH), 13.65 (brs, 1H, pyrreole NH).

MS m/z 369 [M-1].

Example 146 - Synthesis of 5-(5-Fluoro-2-o=xo-1,2-dihydro-indol-3—ylidenemethyl)-2,3- dimethyl-1 H-pyrrole-3-carboxylic acid (2—morpholin-4-yl-ethyl)-=amide (Compound 3) 5-FRuoro-1,3-dihydro-indolin-2-one was condensed with 5-foormyl-2,4-dimethyl-_1H- ) pyrrole-3-c-arboxylic acid (2-morpholin-1-yl —ethyl)-amide to yield thee title compound. . Biological Examples

Example 1 7- 5-(5-Fluoro-2-0xo-1,2-dihydiro-indol-3-ylidenemetha yl)-2,4-dimethyl-1 _H- pyrrole-3-ecarboxylic acid (2-diethylaminae-ethyl)-amide (compou nd 1) inhibits phosphory=lation of CSF1R : 3T=2-huCSF1R cells were starved oveemight (RPMI 1640/0.1%24 FBS), and then resuspende=d in fresh RPMI 1640 containing 0.1% FBS + compound 1, using 20 million cells per conditi-on in 6-well plates. Cells were tr eated at 37 °C for 2 hour s with compound 1... then stimulated for 10 mins with human M-CSF =at 100 ng/ml (R&D Systems, Minneapolis, MIN).

Cells were lysed immediately after stimulation and lysates spun at 4 °C for 20 mins.

Supernatart was transferred to new microfu ge tubes. For each samp le, 500 pg of total protein wa_s immunoprecipitated overnight vith a rabbit polyclonal amntibody to bead- conjugated human CSF1R (Santa Cruz Biotechnology, CA). A p-CSSF1R Western blot ~was then perfomrmed, using anti-phospho-CSFIR_ (Tyr723) antibody (Cell. Signaling Technoleogy,

Beverly, NA) at 1:1000 dilution. Antibody to actin was used as a control for total prote=in, as the antibody to CSF1R does not recognize o-CSFIR well.

Thue results, shown in Figure 1, dem-onstrate that compound 1 inhibits CSFIR phosphorylation in a dose-dependent manner. As the positive control lane shows, absert inhibition_, M-CSF stimulates CSF1R phosphorylation. However, with increasing dosess of compouncd 1, CSF1R phosphorylation decli ned. The IC50 for inhibmtion of CSF1R whe=n expressed. by NIH3T3 cells by compound 1 was 50-100 nM.

Example 18 - 5-(5-Fluoro-2-oxo-1 ,2-dihy =dro-indol-3-ylidenemethhyl)-2,4-dimethyl-T1 H- pyrrole-3-carboxylic acid (2-diethylamin_ ¢-ethyl)-amide (compowund 1) inhibits muxine osteoclast development in vitro

Bone marrow cells were isolated from female Balb/c mice and cultured either im medium zlone or with addition of 10 ng/mL murine M-CSF and 100 ng/ml! murine RANIK ligand (R_ANKL) from day 0 to induce oste=oclast development. To cultures with cytokines, different «concentrations of compound 1 we=re added, from 1 nM to M0 uM. Osteoclast development on day 7 was assessed by colorimetric quantitation of tartrate resistant aci_d phosphat=ase activity (TRAP) as well as counting TRAP positive cells with > 3 nuclei. As

Figure 2a shows, compound 1 inhibits thee development of osteoclasts at 10-100 nM concentrations. : . A similar study was performed, adding cytokines at day 0 but —varying the time of addition of compound 1(D0, D2 or D4). TRAP staining and quantitat_ion were performed at : day 6. This study demonstrated that the mechanism of action was inimibition of M-CSF effect= (early phase development), and not RANKL effect (late phase development. Figure 2b summariz=es the data, and shows that at concentrations of both 100 nM and 10 nM, the later precursor cells are exposed to compound 1, the less inhibitory effect the compound exhibits.

Examples 19 - 5-(5-Fluoro-2-0xo-1,2-dmhydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H- pyrrole-3-carboxylic acid (2-diethylarmino-ethyl)-amide (compoumnd 1) inhibits breast cancer g rowth in vivo

T~he MDA-MB-435-HAL-luc bresast cancer xenograft model vas used to confirm significant inhibition of the growth of breast cancer bone metastases. The presence of live tumor ce=lls was monitored by bioluminescence imaging with the Xerogen IVIS™ system.

Mice treated with compound 1 showed =a significant reduction in live- tumor.

Cell Lines

Ihe human breast carcinoma cel line 435/HAL was obtainec® (Pharmacia Corp., St.

Louis, MO). This line was isolated usixag an in vivo selection procediure to identify a derivatiwe of MDA-MB-435 human brezast carcinoma cell line that exhibited increased primary- tumor growth rate and increased pulmonary metastasis in vi-vo (30). Stable transfec tion of 435/HAL cells with luciferase (light emitting enzyme of firefly Photinus pyralis) was then performed. The cells were cotransfected with pGL_3-control (Promega,

Madison, WI) and pTK-Hygro (Clontech, Palo Alto, CA) at a 1:4 ratio using Lipofectamine 2000 (Imvitrogen, Carlsbad, CA). Cell=s were maintained in Hygronmycin (200 pg/ml) (Invitroegen) and resistant colonies isolaated by ring cloning. Hygro-mresistant colonies were screene=d for luciferase expression usin_g Promega brite-glo reagent, normalized as RLU/ug protein . A subclone with the highest lwiciferase activity was selected, which we refer to as ‘435/H_AL-luc’’

These cells were cultured in REPMI 1640 supplemented with. 10% fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruv-ate (Life Technologies Inc., Gaithersburg, MD), and maintained routinely in a humidified cchamber at 37 °C and 5% carbon dioxide. Cells were harvested from culture flasks during exponential growth, washed orace with sterile phospha_te-

buffered saline (PBS)®, counted, and resuspended in PBS to 2 suitable concentration prior to implantation. ~ Mice

Female athymic #u/nu mice were obtained (Charles River Laboratoriess, Wilmington, - MA). The Mice weree housed under pathogen-free conditions in microisolator cages with sterile rodent chow amd water available ad libitum. All xenograft animal studi es were performed in an AAALAC, International accredited vivarium and in accordanece with the

Institute of Laboratory Animal Research Guide for the «Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD). Mice w «re approximately 8 weeks old when cells were implanted. via the left ventricle of the heart to evaluate growth in bawne, and 10-11 weeks old when tumor pieces were implanted into the #2 mammary fat pad to evaluate orthotopic growth.

Breast Cancesr Detection in Mice using the IVIS™ Imaging system

Mice were irjected intraperitoneally with 150 mg/kg of luciferin (Xen ogen Corp.,

Alameda, CA), followed by anesthetization with Ketarmine/Xylazine five min utes later. After another five minutes, mice were imaged using an inten sified charge-coupled device (ICCD) camera in the Xeno gen vis™ imaging system (Xenoggen Corp.) to evaluate ®he bioluminescence of cancer in the mouse. Briefly, mice were placed on the tex-mperature- controlled bed of th_e imaging chamber and a gray-scal e whole body image of the ventral side of the mice was captured, followed by an overlay of a bioluminescence map representing the spatial distribution of photons detected from cleaved lxaciferin in the cancer cells expressing luciferase. A final image at day 46 was taken of the dorsal side of the mice tc monitor growth of tumor in the spine. The bioluminescent sigral was quantified by c=ounting the pixels within the area drawn around each site of photon emission, using a customized version of the IGOR Pro version 4.0 Software (WaveMetrics, Inc., Lake Oswego, OIR) called Living

Image version 2.11. (Xenogen Corporation, Alameda, CA). The sum of all dlletected photon counts within a regzion of interest containing the entires cancer lesion was detesrmined. For evaluation of tumeor growth inhibition, the Student’s z test was used to assess differences in photon emission rezadings between treated and control groups (p < 0.05 was considered significant).

Example 20 - 5-(5-Fuoro-2-o0xo-1,2-dihydro-indo-1}-3-ylidenemethyl)-2,4-dmethyl-1H- pyrrole-3-carboxyli: acid (2-diethylamino-ethyl)— amide (compound 1) inhibits osteolysis induced by breast cancer metastasis in vivo

Compound 1 treatment in an experimental bone metastasis model

Athymic mices were inoculated with 3 x 10° =435/HAL-luc cells into the= left ventricle } of the heart on day 0 Twenty days later, mice were imaged using the VIS™ imaging system and placed into two matched groups of 16 mmice based on photon emisssion, a measure of bioluminescence Of the cancer. The next day, mi ce bearing established 435 /HAL-luc tumor in bone were sadministered 80 or 40 mg per kag of compound 1 or CMC vehicle once daily by gavage to ttme end of the study (21 days). Mice were imaged approxirmately once a week. By 41 days affter implantation, mice from the= control group became cachectic and exhibited signs of hid limb paralysis triggering the end of the study. The fen—wur, mandible and spines were coll-ected from mice treated with either compound 1 or its velicle, and fixed in Streck’s Tissue Fiixative prior to bone density scamnning and histological analysis. Serum was also collected for measurement of collagen bre=akdown product pyridinoli ne (PYD) in the circulation.

Compound 1 inhibited growth of 435/HAL~ luc osseous metastasis

This experinment confirmed that compound 1 could significantly inhibmt growth of breast cancer metas@ases in bone. During therapy, =ventral bioluminescence whole body images were acquired to analyze tumor growth in long bones and mandibles. Compound 1 greatly decreased the photon count emission from t-he mouse bones at both doses (Figure 3).

Photon emission from the thoracic region is likely to be due to tumor cell dep-osition into the pericardial or pleural cavities at the time of intracar—diac injection. Prior to ter_mination, both ventral and dorsal immages were taken to also analyze spinal metastases. Regi=ons of Interest (ROI) that captured tumor growth in different sites were analyzed separately. Combined data for the change in photon emission from long bones= (femur and tibia) and mardibles over time is shown in Figure :3. Tumor growth in bone was significantly inhibited by ceompound 1 (day 41: 89% inhibitiorm, p=0.001).

Serum PYTD ELISA: The parental breast carcinoma line MDA-MB-4-35, is very well characterized as ha ving osteolytic activity (32). Measurement of serum levelks of the collagen breakdown product pyridinoline (PYD) is an estab lished assay for osteolytic activity that correlates significa_ntly with the volume of bone m_etastasis in a rat model (293, 31, 33). Serum samples were collected, aliquoted and frozen at —8-0 °C until analysis. Serum PYD was measured sing a competitive enzyme immunoassay kit following the ma nufacturer’s protocol (Serum PYD, Quidel 8019, San Diego, CA). Samples were mea. sured in duplicate.

Results from 12 vehicle treated and 14 compourad 1 treated mice showed that serum PYD levels were significantly reduced by 30% in mic=e receiving compound 1 &reatment as compared to vehicle treated mice (p = 0.047). The mean value was 1.8 +./- 0.21 ng/ml in vehicle treated versus 1.3 +/- 0.16 ng/ml in mice treated with 80 mg per k=g compound 1.

Figure 4 shhows the distribution of data points for this experiment. % ok sk 3k

It will be apparent to those skilled in thes art that various modifications and variations can be made in the methods and compositions osf the present invention without departing from the spoirit or scope of the invention. It is irmtended that the present in_vention covers such modifications and variations, provided they conme within the scope of the appended claims and their equivalents.

References

Thee disclosure of each of the following references, cited above, is incorporated herein by referen. ce. 1. Mundy, Clin. Orthop. 324: 24-23 (1996). 2. Mundy, J. Bone Miner. Res., 8: 5505-10 (1993). 3. Tanaka ef al., J. Clin. Invest., 91 :257-263 (1993). 4. Yasuda, H., Proc. Natl. Acad. Sci. USA., 95: 3597-3602 (C1998). 5. Friedman et al., Science, 150: 1465-67 (1965). 6. Takahashi et al., J Clin Invest, =5:167-171 (1995). 7. Fisher et al., Endocrinology, 1322:1411-1413 (1993). 8. Davies et al., J Cell Biol, 109:1817-1826 (1989). 9. Hentunen et al., J Bone Miner Res, 6:1091-1097 (1991). 10. Weber et al., J. Bone Miner Ress, 5:401-410 (1990). 11. Coleman et al, Cancer Treatmert Rev, 19:79-103 (1993). 12. Hall et al., Calcif. Tiss. Intl., 57 :463-465 (1995).

13. Brandi et aZ., Proc Natl Acad Sci USA, 92:72954-2958 (1995). 14. Yoneda et cal., Cancer Res, 55:1989-1993 (1995). . 15. Jilka et al.

Science, 257:88-91, (1992). 16. O'Neill et cal, Bone, 13:23-27 (1992). 17. Quinn et aZ.,]. Pathology, 184: 31-36 (199 8). 18. Clohisy et «l., Clinical Orthopaedics and R_elated Res., 373: 104-14 (2 000). 19. Mancino e# al., J. of Surgical Res., 100: 18<-24 (2001). 20. Pederson et al., Cancer Res., 59: 5849 (1999) 21. Hunt et al. , British J.

Cancer, 85(1): 78-84 (2001). 22. Yee et al., Anticancer Res, 20(6B): 4379 &2000). 23. Kacinski e=t al., Oncogene, 6: 941 (1991). 24. Flick et al -, Oncogene, 14: 2553 (1997). 25. Maher et czl., Clin.

Cancer Res., 4: 1851 (1.998). 26. Kacinski, Ann.

Med. 27: 79 (1995). 27. Kacinski, Mol.

Reprod.

Dev., 46: 71 (19977). 28. Toy et al.

Gynecologic Oncology, 80: 194-200 (2001) 29. Demers e# al.

Cancer Supplement 88:291S (2000). 30. Schmidt et al., Clin. & Exper.

Metastasis, 17: 537-44 (1 999). 31. Robins er al., Clin.

Chem., 42: 1621-26 (1.996). 32. Hunt et ad., Br.

J.

Cancer 85: 78-84 (2001 ). 33. Tamura et al., Breast Cancer 6(1): 23-28 &(1999). 34. Tanget al, J.

Cellular Biochem., 50: 350-56 (1992).

Claims (16)

WHAT IS CLAIMED IS:

1. Use of a compound of formula I in the manufaacture of a medicament for treating excessive osteolysis in a patient, X CT SE — / H [ X.. N AN Pa (Rib ZT) (D, wherein R is indep endently H, OH, alkyl, aryl, cycRoalkyl, heteroaryl, alkoxc y, heterocyclic and amino; eachR, is independently selected from the group consisting of alkyl, halo, aryl, alkoxy, haloalkyl, haloalkoxy, cycloalkyl, heteroaryl, heterocyclic, hydroxy, -C(O)-Rs, -NRgR,g, -NReC(O)-R2 and —-CE€0)NRyR¢; eachR; is independently selected from the group consisting of alky), aryl, heteroaryl, —-C(0)-Rs -and SO,R’’, where R” is alkyl, auryl, heteroaryl, NRgN;q <r alkoxy; each Rs is independently selected from the group consisting of hydrogen, alkyl, aryl, haloalkyl, cycloalkyl, heteroaryl, heterocye lic, hydroxy, -C(O)-Rg and (CHR),R;;; XisOor S; pis 0-3; qis 0-2; ris 0-3; Rg is selected from the group consisting of —OH, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl and heterocyclic; Rs and Ri» are independently selected from the group consisting of MH, alkyl, aryl, aminoalky 1, heteroaryl, cycloalkyl and hetemrocyclic, or Rg and Rg together with N may form zaring, where the ring atoms are s elected from the group c onsisting of C, N, OandS§;

Ry is selected from the group consisting of -OH, amino, meonosubstituted amino, disubstituted amino, alkyl, ary], heteroaryl, alkoxy, cycloalicyl and heterocyclic; Riz is selected from the group consisting of alkyl, aryl, hetemroaryl, alkoxy, cycloalkyl and heterocyclic; Z is OH, O-alkyl, or -NR3Rs4, “Where R; and Ry are independ_ently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclic, o= Rj and Ry may combine with Ito form a ring where the ring atoms are selected frorm the group consisting of CH,;, NI, 0 and Sor 1 (Yn R RrR3 VC ONLY \ A C N ~~ 4 Ne | R R'/m wherein Y is independently CH, O, Nor S, QisCorN; n is independently 0-4; and mis 0-3; or a salt thereof.

2. The use of claim 1, wherein R, is hal oand pis I.

3. The use of claim 2, where Z is —NR 3R4, wherein R; and Ry form a morpholine ring. ™ R 3 n R — _ ONL Ny a C N— Yd R* R'/m wherein each Y is CH,, each nis 2, mis 0 and R; and R4 form -a morpholine ring.

4. The use of claim 1, wherein Z is:

5. The use of any of claims 1-3, wherein R; is methyl and q is 2, wheresin the methyls are bonded at thwe 3 and 5 positions.

6. The use o fclaim 1, wherein the compo und administered is a commpound of Formula II: (Te H Rag N H N (Rap H @.

7. The use of claim 6, wherein Rsis H.

8. The use of claim 6, wherein R; is methy 1,q is 2, wherein the meth yls are bonded at the 3 and positions.

9. The use of claim 6, wherein the patient has cancer that has metastasized to bone.

10. The use of claim 6, wherein the patient Fas a cancer that secretes MvI-CSF.

11. The use of claim 6, wherein the patient las osteoporosis.

12. The use of ~claim 6, wherein the patient i=s post-menopausal.

i WO 2004/07577° 5 PCT™/US2004/005283

13. The u=se of claim 1, wherein the compoun d administered is selected fr-om the group consissting of 0 Is o 5s SNE Hy n ee ~N N CH, 4 ) cm, x / H x i pH

0 . I =~ H N © : lo} J) I N Hy Joe a OH CH, N CH, X / N x / H yo yo = o : ” Pu an N = x H A ) N

:

14. The vse of claim 1, wherein the compomund of formula I is selected from the group consistingg of: o Is 0 HC { NTN HyC. NN N ca b CH,

F. H i [1 lo] . H 3 © Compound | a. Compound 2 0 Hs \ J He I / on on ; n . N 0 by : z Compound 3 Compound § 0 0 Ha N wT "= N 0 SLs SRS: . N o ’ lo] . 0 . N N H H Compound 6 Compound 7 0 HC ~~) Hy N \ _° \ PS . - OH . on ‘a F H . OF H _ 0 (o} H] N Compound 4 Compound § lo) He ~~" / cx, OH \_° " N and (o} " Compound §

15. Use of a compound of formula I in the manufacture of a medicament for inhibiti ng phosphoryiatiorm of CSFIR in a patient, X WT SE / H [ X,, H EN ~ (Rq)p = A a wherein

Ris independently H, OH, alkyl, aryl, cycloalkyl, het eroaryl, alkoxy, heterocycl ic and amino;

each R) is independently selected from the group consisting of alkyl, halo, aryl,

alkoxy, haloalkyTl", haloalkoxy, cycloalkyl, heteroaryl, heterocyclic, hydroxy,

-C(O)-Rs, -NRoR_19, -NRsC(0)-R;, and —~C(O)NRsR 1p;

each R; is indepemndently selected from the group consisting of alkyl, aryl, heteroaryl, —C(0)-Rg and SO=,R™’, where R”’ is alkyl, aryl, heteroaryl, NRaNjp or alkoxy;

each Ry is independently selected from the group consa sting of hydrogen, alkyl, amryl, haloalkyl, cycloalkyl, heteroaryl, heterocyclic, hydroxy, -C(0O)-Rs and (CHR),R;, ; XisOorS;

pis 0-3;

qis 0-2;

ris 0-3;

Rg is selected from the group consisting of ~OH, alkyl, aryl, heteroaryl, alkoxy, cycloalkyl and heteerocyclic; Ry and R,¢ are inde=pendently selected from the group consisting of H, alkyl, aryl, aminoalkyl, hetero aryl, cycloalkyl and heterocyclic, or Ry and Ryo together with N may form a ring, where the ring atoms are selected from the group consisting of C, N, Oand S; ; Ry is selected froma the group consisting of ~OH, amino, monosubstituted amino, 66 Co di substituted amino, alkyl, aryl , heteroaryl, alkoxy, cycloalky® and heterocyclic R y;is selected from the group consisting of alkyl, aryl, hetero zaryl, alkoxy, cycloalkyl ard heterocyclic; Z is OH, O-alkyl, or -NR3Ry, vwhere R; and R;; are independently selected from the group consisting of hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, and heterocyclic, or R 5 and Ry may combine with NE to form a ring where the ring atoms are selected from thee group consisting of CHy, N, O and S or rR! (Yon R3 EN A I \ A C—NC__ i Rr! R'/m whherein Y is independently CH 5, 0, Nor S, Q isCorN n #&s independently 0-4; and m is 0-3.

16. Use of a compound of formula I according to claims 1 or 15, subsstantially as herein describ ed and e=xemplified.