5 MORPHOLINES AS SELECTIVE INHIBITORS OF CYTOCHROME P450 2A13

BACKGROUND OF THE INVENTION

In 2003. the Centers for Disease Control and Preλ ention estimated that appro \imateh 25 5 million men and 21 5 million women smoke In other words. 24 l °υ

K) of all men and 19 2% of all women are smokers There are mam reasons people start smoking, including, but not limited to. stress, life problems, peer pressure, famih histoπ . and personally tendencies There are also mam reasons people to want to quit smoking, including, but not limited to. health problems (such as lung cancer), and the smell, stained teeth, bad breath, wrinkled skin, and \ ellow nails associated with smoking Moreoλ er.

15 smoking can cause the aλ erage male to lose 13 2 \ ears of his life, and the aλ erage female to lose 14 5 \ ears of her life

People huπ e im ented mam wa\ s to quit smoking U S Pat No 6.845.777 to Pera (2005 ) emplo\ s a composition that can take a capsule, powder, or liquid form, and that satisfies a smoker's craung for nicotine U S Pat No 6.596.740 to Jones (2003 ) satisfies

20 a smoker's craung for nicotine Ma a nasal spra\ Other "quit smoking" im entions include, but are not limited to. adult pacifiers (i e . U S Pat No 6.458.159 to Peters- Combs (2002 )). cigarette aeration and filtration deuces (i e . U S Pat No 5.954.061 ( 1999)). wπstwatches (i e . U S Pat No 6.305.939 to Krstulouc (2001 )). spinal cord stimulation (i e . U S Pat No 6.233.488 to Hess (2001 )). and chemotherap\ (i e . U S

25 Pat No 6.333.357 to Eig (2001 )) There is e-\ en a cigarette pack that pla\ s an anti- smoking message each time the smoker opens it (U S Pat No 6.559.768 to Schaffner. et al (2003 ))

Despite all the creatπ e wa\ s people run e tried to quit smoking, a stud\ b\ the American Cancer Society in 2005 shows that onh 2 5% of smokers who tr\ to quit

M) smoking each \ ear actualh succeed Clearh . there is still a need for more wa\ s to quit smoking, for. not e\ eiλ method will work for each indn idual smoker

Howeλ er. there also is a need for inhibiting the phy siological effects of smoking for indn iduals who cannot quit or run e a difficult and/or prolonged program to stop smoking Thus, it can be adλ antageous to treat, inhibit, or preλ ent the adλ erse v5 phy siological effects of smoking so that the oλ erall health of a smoker is maintained or increased

- ~> _

5 SUMMARY

In one embodiment, the present im ention includes a method of inhibiting formation of cancerous metabolites in a subject Such a method can include prouding a morpholine compound that selectπ eh interacts with c\1ochrome P450 2Al 3 oλ er cy tochrome P450 2A6. administering a therapeuticalh effectπ e amount of the K) morpholine compound to the subject so as to inhibit formation of the cancerous metabolites b\ the c\1ochrome P450 2Al 3 The morpholine compound can be substantialh more selectπ e for interacting with the c\ tochrome P450 2Al 3 oλ er the c\ tochrome P450 2A6 The morpholine compound can also be substantialh non- interactn e with other phy siological components

15 In one embodiment, the present im ention can include a method of inhibiting formation of cancerous lung cells in a subject Such a method can include prouding a morpholine compound that selectπ eh interacts with c\1ochrome P450 2Al 3 oλ er c\ tochrome P450 2A6. and administering a therapeuticalh effectπ e amount of the morpholine compound to the subject so as to inhibit formation of the cancerous lung 20 cells

In one embodiment, the present im ention can include a method of inhibiting c\ tochrome P450 2Al 3 from forming carcinogen metabolites in a subject Such a method can include prouding a morpholine compound that selectπ eh interacts with c\ tochrome P450 2Al 3 oλ er c} tochrome P450 2A6. and administering a therapeuticalh 25 effectπ e amount of the morpholine compound to the subject so as to inhibit c} toclirome P450 2A13 from interacting with 4-(meth} lnitrosainino)-l -(3-p}πd} l)-l-butanone (NNK)

In one embodiment, the present im ention can include a morpholine compound that inhibits formation of cancerous metabolites in a subject Such a morpholine M) compound can include a structure that selectπ eh interacts with c}1ochrome P450 2Al 3 o\ er c\1ochrome P450 2A6

In one embodiment, the morpholine compound can be selected from Compounds I -34. more preferabh I -K)

In one embodiment, the morpholine compound can be selected from Formulas A- *5 D

In one embodiment, the morpholine compound can be included in pharmaceutical!} acceptable carrier in a therapeuticalh effectπ e amount

5 These and other embodiments and features of the present im ention w ill become more fulh apparent from the following description and appended claims, or ma\ be learned b\ the practice of the im ention as set forth hereinafter

FIGURES

To further clarift the aboλ e and other adλ antages and features of the present

K) im ention. a more particular description of the im ention w ill be rendered b\ reference to specific embodiments thereof which are illustrated in the appended drawings It is appreciated that these drawings depict onh illustrated embodiments of the im ention and are therefore not to be considered limiting of its scope The im ention w ill be described and explained with additional specifier^ and detail through the use of the accompanung

15 draw ings in w Inch

Figure IA is a graph illustrating the UV/usible spectrum of purified CYP2A protein, which shows a Soret peak at 418 nin. indicatn e of a low-spin, six-coordinate configuration of the heme iron bound to an actπ e site water molecule

Figure IB is a plot illustrating the spectrum shift of cytochrome P450 enzunes

20 that occurs as protein is titrated with increasing concentrations of a hgand and the heme- bound water is displaced The inset shows how the changes in absorbance are plotted -\ s hgand concentration (in this case coumaπn) and used to determine hgand affinity (ka)

λ alues

Figure 2 is a graph illustrating -\ ahdation of the HTS λ ersion of the spectral

25 binding assa\ in Figure I B This assa\ uses onh six waλ elengths for single concentrations of different hgands This example shows binding of PEITC. a positπ e control that binds CYP2A13. a negatπ e control (2A13-1 and 2A13-2). and two new compounds identified to bind CYP2A13 (N6-1 and N6-2)

Figure 3 A is a graph that illustrates titration with CYP2A13 reλ eals binding of 4- M) (2-chloro-6-fluorobenz\ l)morpholine to CYP2A enzunes

Figure 3B is a graph that is used to determine 2Al 3 Kd from data in 3 A Multiple titrations re\ eal an a\ erage Kd of 5 8 μM

Figure 3C is a graph that illustrates that titration with CYP2A6 reλ eals no binding

^5 Figure 4A is a graph that illustrates titration with CYP2A13 reλ eals binding of 4-

(2-meth\ lbenz\ l)morpholine to CYP2A enzunes

Figure 4B is a graph that is used to determine 2Al 3 Kd from data in 4A Multiple titrations reλ eal an a\ erage Kd of 19 3 μM

5 Figure 4C is a graph that illustrates that titration with CYP2A6 reλ eals no binding

Figure 5 A is a graph that illustrates titration w ith CYP2A13 reλ eals binding of 4- (2-chlorobenz\ l)morpholine to CYP2A enzunes

Figure 5B is a graph that is used to determine 2Al 3 Kd from data in 5 A K) Multiple titrations re\ eal an a\ erage Kd of 7 3 μM

Figure 5C is a graph that illustrates that titration with CYP2A6 reλ eals no binding

DETAILED DESCRIPTION

Lung cancer is a leading cause of human mortality and smoking is the most

15 critical factor in the elopment of 80-90° o of lung cancer (Proc Natl Acad Sa U S A

2004 July 6 101(2") 10143- 1014H) The human \enobiotic cy tochrome P450 2Al 3

(CYP2A13 ). w Inch is mainh expressed in the respiratoπ tract, efficiently catah zes metabolism of the tobacco-smoke procarcinogen 4-(meth\ lnitrosamino)-l -(3-p\ πd\ I)-I- butanone (NNK) into two toxic metabolites that intercalate DNA and cause lung cancer in

20 smokers NNK is one of the most preλ alent compounds in cigarette smoke and one of the strongest procarcinogens As such, the function of CYP2A13 has a significant role in the formation of toxic metabolites that function as carcinogens These carcinogens can induce the formation of am cancer, but are highh hkeh to be im oh ed in lung cancer

One method of inhibiting the formation of the carcinogens would be to block the actiut\

25 of CYP2A13 Howeλ er. CYP2A13 is 93 5% identical to the human lπ er dmg- metabohzing c\1ochrome P450 2A6 (CYP2A6). which onh metabolizes NNK \ er\ inefficiently As such, a molecule that inhibits CYP2A13 actiut\ ma\ also block

CYP2A6 actiut} . which is undesirable To oλ ercome the problem associated with similaπt} of the enzunes. specificit> for CYP2A13 oλ er CYP2A6 (and other P450s) can

M) be a factor in identify ing an inhibitor for CYP2A13 An inhibitor that selectπ eh inhibits human CYP2A13 o\ er other c> tochrome P450 enzy mes, but especialh CYP2A6. can be used to preλ ent in nro generation of the NNK metabolites that cause lung cancer or other cancers in smokers

I. Inhibiting CYP2A13 v5 The inhibition of CYP2A13 represents an lnnoλ atπ e approach to preλ ention of lung cancer While mam smoking inteπ ention approaches tπ to alter smoker behaλ ior. replace nicotine delπ eπ . or act at nicotinic receptors to reduce the phy siological rewards of smoking, the inhibition of CYP2A13 is an entireh new approach that inhibits the

5 formation of toxic carcinogens that cause lung cancer w ithout requiring alteration of nicotine exposure Thus, this strateg} aims to reduce the risk of eloping lung cancer for smokers or those exposed to second hand smoke

To identif} potential inhibitors of CYP2A13. a spectral hgand binding assa> w as used in a high throughput format to screen a large number of molecules Confirmation of

K) hits (e g . molecules that interact w ith CYP2A13 ) in a binding titration assa> can be used to identif} specific molecules or molecule families with similar core scaffolds This strateg} has led to the identification of at least two molecular morpholine-based scaffolds (e g . genus) that run e mam different specific scaffold derπ atπ es (e g . morpholine derπ atπ e species molecules) that bind specificalh to CYP2A13. but showed little or no

15 binding to the close homolog. CYP2A6 This high selectiut} of CYPA13 oλ er CYP2A6 is desirable, and such molecules can be used in the therapeutic protocols described herein

Preliminaπ studies indicate that some of the molecules based on these scaffolds also inhibit enzunatic metabolism of CYP2A13 substrates That is. the molecules not onh bind CYP2A13 selectπ eh . but in doing so also inhibit the general function of

20 CYP2A13 Optimal drug candidates run e specific interactions as inhibitors of CYP2A13. but not CYP2A6 The identification of selectπ e inhibitors of CYP2A13 can be used to proλ ide more effectπ e drugs w ith higher functionaht} and selectiut} . and also can be used in lung cancer chemopreλ ention Selectπ e drug candidates can target reduction in the m vivo formation of nicotine-derπ ed carcinogens w ithout requiring changes in

25 smoking habits, which ma} decrease disease This approach, alone or in combination with existing therapies, can help reduce the incidence of lung cancer Also, tins approach can be used for indπ idual that are tr\ ing to quite smoking

In one embodiment, the present im ention can include the use of one or more of a series of morpholine derπ atπ es for inhibiting the formation of the carcinogens, and

M) thereb} inhibiting the formation of cancer, such as lung cancer The morpholine derπ atπ es can be molecules that are aλ ailable commercialh . analogs of commercialh aλ ailable compounds, or new compounds that are suithesized de IIOλ O based on the scaffolds presented herein The morpholine derπ atπ es in accordance w ith the present im ention are selectπ e inhibitors of the enz\ me CYP2A13 oλ er CYP2A6 It is preferred v5 that the compound has high selectπ it} for CYP2A13 λ ersus CYP2A6 A selectπ it} constant that is essentialh a ratio of inhibition of CYP2A13 o\ er inhibition of CYP2A6 can be identified to characterize a potential inhibitor Thus, the selectπ it} constant proλ ides as high a ratio of 2A13/2A6 inhibition as possible

5 Preuoush known inlnbitors of c\ tochromes P450 2A enz\ mes include isothioc\ anates (e g . -N=C=S ) The morphohne scaffolds of the present e not been preuoush identified as P450 inlnbitors in general or specificalh as 2Al 3- selectπ e inhibitors Since the isothioc\ anates are highh reactπ e and potentialh toxic. the\ are not suitable daig compounds The moiphohne scaffolds are deλ oid of

K) isothioc\ anate motifs While some of the morphohne derπ atπ es ma\ be commercialh aλ ailable for other uses, the method of use of such morphohne derπ atπ es for inhibiting CYP2A13 is iκn el These morpholines lκπ e the potential to inhibit cy tochrome CYP2A13 actπ it\ in human tissue, such as lung tissue, to preλ ent the com ersion of the nicotine-derπ ed nitrosamine 4-(meth\ lnitiOsamino)-l-(3-p\ rid\ l)-l -butanone (NNK) into

15 the two carcinogenic compounds (e g . Carcinogen A and Carcinogen B) that cause the DNA damage that can initiate lung cancer The selectπ it\ of the morphohne derπ atπ e compounds for CYP2A13 oλ er the closeh related human lπ er CYP2A6 allows for normal lπ er actπ it\

Carcinogen A

+

CH, -NE :N

20 Carcinogen B

The morphohne compounds can be used as lung cancer chemopreλ entatπ es As chemopreλ entatπ es. the moiphohne compounds can be used to reduce the risk of lung cancer either in smokers that use nicotine or in people exposed to second-hand smoke Preλ ention of lung cancer is an important objectπ e because lung cancer is a leading cause

2.-) of human mortality Smoking is the most critical factor in the deλ elopment of lung cancer, but mam smokers either cannot or will not gπ e up nicotine exposure Also, while tπ ing to stop smoking, mam smokers use nicotine gum As such, inhibition of CYP2A13 can also be used along w ith nicotine gum to help maintain health during a stressful period and inhibit the formation of carcinogens Thus, the inhibition of

M) CYP2A13 can be used in smokers, and other people susceptible to the intake of cigarette toxins, such as smokers that are tπ ing to curb their nicotine addiction

5 II. Morpholines

Moipholines in accordance with the present im ention ha\ e been shown to ha\ e selectπ ih for CYP2A13 o\ er CYP2A6 The morpholines screened, as shown below, are candidates for derπ atization As such, the morpholine compounds shown in Tables 1 and 2 below can be prepared into analogues that ha\ e modulated potency . selectπ ih . and solubility

K) in order to proude useful leads for drug disco\ eπ and drug de\ elopment During optimization, new analogues can be designed considering issues of drug delπ en . metabolism. no\ elt> . and safety The information obtained from the compounds of Tables 1 and 2 was used in order to design additional moipholines that ha\ e selectπ Uλ for CYP2A13

Additionally , am of the compounds of Compounds 1 -34 can be

15 derπ atized/analogued as is well known in the art of combinatorial and medicinal chemistπ The analogs or derπ atπ es can be prepared b\ adding and/or substituting functional groups at λ aπous locations on am of compounds of Compounds 1-34 As such, the compounds of Compounds 1 -34 can be com erted into derπ atπ es/analogues using w ell known chemical synthesis procedures For example, all of the hy drogen atoms

20 or substituents can be selectπ eh modified to generate new analogues Also, the linking atoms or groups can be modified into longer or shorter linkers with carbon backbones or hetero atoms Also, the ring groups can be changed so as to run e a different number of atoms in the ring and/or to include hetero atoms Moreoλ er. aromatics can be com erted to c\ chc rings, and uce -\ ersa For example, the rings ma\ be from 5-7 atoms, and ma\

25 be homoc\cles or heteroc\cles

As used herein, the term "analog ^" , "analogue. ^" or "derπ atπ e ^" is meant to refer to a chemical compound or molecule made from a parent compound or molecule b\ one or more chemical reactions As such, an analog can be a compound with a structure similar to that of Compounds 1-34 or based on a scaffold of Compounds 1-34. but differing from

M) it in respect to certain components or structural makeup, which ma\ run e a similar or opposite action metabolicalh An analog or derπ atπ e of am of Compounds 1-34 in accordance with the present im ention can be used to selectπ eh inhibit CYP2A13

In one embodiment, the compounds of Compounds 1-34 can independenth be derπ atized/analogued b\ modifs ing h\ drogen groups independenth from each other into v5 other substituents That is. each atom on each molecule can be independenth modified with respect to the other atoms on the same molecule Am traditional modification for producing a derπ atπ e/analogue can be used For example, the atoms and substituents can be independenth comprised of h\ drogen. an alk\ 1. aliphatic, straight chain aliphatic.

aliphatic haung a chain hetero atom, branched aliphatic, substituted aliphatic. c\ chc aliphatic. heteroc} clic aliphatic haλ ing one or more hetero atoms, aromatic, heteroaromatic. poh aromatic, poh amino acids, peptides, poh peptides, combinations thereof, halogens, halo-substituted ahphatics. and the like Additionalh . am ring group on a compound can be derπ atized to increase and/or decrease ring size as w ell as change the backbone atoms to carbon atoms or hetero atoms

In one embodiment, the morphohne compounds can be described b\ the chemical structures of Formulas A-D. salt thereof, or prodrug thereof

Formula A

In Formula A X _1-6 are independent!} selected from C. N. NH. N-alk} l. O. or S so long as to form a morphohne or morpholine dem atπ e (e g . thiomorpholines and piperazines are considered to be morpholine derπ atπ es). Z is a linker such as an alk} l. amide, amine. carbo\> l. ketone, ester, inline, urea, thiourea, or thioamide functional group (either constitutional isomer, where possible), and Yi is a substituted or unsubstituted straight chain or branched aliphatic (e g . C l -C l O). an adamant} 1 (e g . 2- adamant} ! or adamantane derπ atπ e). or c} cle or heteroc} cle selected from phem l.

5 py ridine. p\ πmidine. p\ razine. 1.3.5-tπazine. 1.2.4-tπazine. quinoline. isoquinoline. acπdine. phenantlirohnes. benzoquinolines. phenathπdines. phenazines. quinoxalines. quinazolines. phthalazines. pteπdines. cinnohnes. p\rroles. imidazoles. 1.2.3-tπazoles.

1.2.4. tπazoles. tetrazoles. isoxazoles. 1.3-thiazoles. benzimidazoles. indoles, indazoles. benzothiazoles. phenols, naphthols. 2-furan. 3-furan. 2-thiophene. 3-tliiophene. 2-p\ rrole. lo 3-p\ rrole. 2-o\azole. 4-o\azole. 5-o\azole. 2-thiazole. 4-tliiazole. 5-thiazole. 2-imidazole.

4-imidazole. 5-imidazole. 3-iso\azole. 4-iso\azole. 5-iso\azole. 3-isotliiazole. 4- lsothiazole. 5-isothiazole. 4-( 1.2.3) oxadiazole. 5-( 1.2.3 ) oxadiazole. 4-( 1.2.3) tπazole. 5-

( 1.2.3 ) tπazole. or 2-( 1.3.4) thiadiazole. and n = 0. 1. 2. or 3. where the substituted c\ cle or heterocλ cle is substituted at am position w ith H. a halogen. Cl. F. CH-,. CH-,CH:. or 15 higher or lower substituted or unsubstituted straight chain or branched aliphatic The first ring of a morpholine includes X _1-6 . and Yi can be a second ring

Formula B can be substantially Formula A with R ¹ a substituent on one or more ring atoms and independenth being H. a halogen. Cl. F. Br. NCK CH-,. CH-XH ₂ . or higher or lower substituted or unsubstituted straight chain or branched aliphatic (e g . C l- 20 C lO). or c\ cle or heteroc\cle

Formula C can be substantialh Formula A or Formula B. and R ¹ ma\ be present or absent, and when present being a substituent on one or more ring atoms and independenth being H. a halogen. Cl. F. Br. NCK CH-,. CH-,CH ₂ . or higher or lower substituted or unsubstituted straight chain or branched aliphatic (e g . C l -C lO). or c\ cle 25 or heteroc\ cle Y2-7 are independenth selected from C. N. NH. N-alk\ l. O. or S. and the ring formed therefrom ma\ be c\ chc or aromatic Y7 ma\ be present or absent so as to form a 5-membered ring

Formula D can be substantialh Formula A. Formula B or Formula D. and R ¹ and

R ² ma\ be present or absent, and when present each independenth being a substituent on M) one or more ring atoms and independenth being H. a halogen. Cl. F. Br. NCK CH-,.

CH-,CH ₂ . or higher or lower substituted or unsubstituted straight chain or branched aliphatic (e g . C l -C lO). or c\ cle or heteroc\ cle Y2-7 are independenth selected from C.

N. NH. N-alk\ l. O. or S. and the ring formed therefrom ma\ be c\ chc or aromatic Y7 ma>be present or absent so as to form a 5-membered ring v5 Additionalh . Yi or R ² can be a mono-morpholine. bi-morpholine. or tπ- morpholine. wherein each morpholine of such multi-morpholines are each independenth described b\ Formulas A-D

5 As used herein, the term "analog ^" , "analogue. ^" or "derπ atπ e ^" is meant to refer to a chemical compound or molecule made from a parent compound or molecule b\ one or more chemical reactions As such, an analog can be a compound with a structure similar to that of Compounds 1 -34 or based on a scaffold of Formulas A-D. but differing from it in respect to certain components or structural makeup, which ma\ run e a similar or

K) opposite action metabolicalh Thiomorpholines and piperazines are specific examples of morpholine derπ atπ es

In one embodiment, the compounds of Formulas A-D can independenth be derπ atized/analogued b\ modif\ ing the R groups independenth from each other That is. each R group on each molecule can be independenth modified with respect to the other R

15 groups on the same molecule Am traditional modification for producing a derπ atπ e/analogue can be used For example, the R groups of the derπ atπ es/analogues can be independenth comprised of h\ drogen. an alk\ 1. aliphatic, straight chain aliphatic, aliphatic haung a chain hetero atom, branched aliphatic, substituted aliphatic. c\ chc aliphatic. heteroc\ chc aliphatic haung one or more hetero atoms, aromatic.

20 heteroaromatic. poh aromatic, poh amino acids, peptides, poh peptides, combinations thereof, halogens, halo-substituted aliphatics. and the like

Additionalh . the X groups of the morpholine can be independenth selected from C. O. N. S. P. and like atoms, such as other hetero atoms

As used herein, the term "hetero atoms ^" is meant to refer to atoms other than

25 carbon atoms such as ox\gen. nitrogen, sulfur, phosphoais. and the like Usualh . a heteroatom is multπ alent so as to form at least tw o αn alent bonds, which can be used in a linking group or other moier\

As used herein, the term "aliphatic ^" is meant to refer to a h\ drocarb\ l moiet\ . such as an alk\ l group, that can be straight or branched, saturated or unsaturated, and/or

M) substituted or unsubstituted. which has twent\ or less carbons in the backbone An aliphatic group ma\ comprise moieties that are linear, branched. c\ chc and/or heteroc\ clic. and contain functional groups such as ethers, ketones, aldehy des, carbox} lates. and the like Exemplar} aliphatic groups include but are not limited to substituted and/or unsubstituted groups of meth\ l. eth\ l. prop\ l. but\ l. pent\ l. hex\ l. v5 hepr\ l. oct\ l. nom l. dec> l. undec> l. dodec> l. tπdec> l. tetradec> l. pentadec> l. hexadec> l. heptadec> l. octadec> l. nonadec> l. eicos> l. alk\ l groups of higher number of carbons and the like, as well as 2 -meth\ lprop\ l. 2-metbι 1-4-etlτs lbut> 1. lprop> l. 3- prop> l. 2.8-dibut\ ldec> l. 6.6-dimeth> loct> l. 6-prop> l. 2-meth> lbut> l. 2-

5 meth\ lpent\ 1. 3-meth\ lpent\ l. 2-eth\ lhe\\ 1. and the like The terms aliphatic or alk\ l also encompasses alkem l groups, such as un\ l. alh l. aralk\ l and alk\ n\ l groups

Substitutions within an aliphatic group can include am atom or group that can be tolerated in the aliphatic moier\ . including but not limited to halogens, sulfurs. thiols, thioethers. thioesters. amines (pπmaπ . secondary . or tertian ), amides, ethers, esters. io alcohols. ox\ gen. and the like The aliphatic groups can b\ wa\ of example also comprise modifications such as azo groups, keto groups, aldehy de groups, carbom l groups. carbox\ 1 groups, nitro. mtroso or nitπle groups. heteroc\ cles such as imidazole. h\ drazino or h\dro\\ lamino groups. isoc\ anate or c\ anate groups, and sulfur containing groups such as sulfoxide, sulfone. sulfide, and disulfide Additionally the substitutions

15 can be Ma single, double, or triple bonds, when releλ ant or possible

Further, aliphatic groups ma\ also contain hetero substitutions, which are substitutions of carbon atoms. b\ hetero atoms such as. for example, nitrogen. ox\gen. phosphorous, or sulfur As such, a linker comprised of a substituted aliphatic can e a backbone comprised of carbon, nitrogen. ox\ gen. sulfur, phosphorous, and/or the like

20 Heteroc} clic substitutions refer to alk\ 1 rings haλ ing one or more hetero atoms Examples of heterocy clic moieties include but are not limited to morpholino. imidazole, and p\ rrohdino

Also, am c\ cloalk\ l groups shown in Formulas A-D can be substituted with a aromatic group haλ ing about the same number of members in the ring so long as the

25 molecule is still a morphohne

As used herein, the term "aromatic ^" is meant to refer to molecule is one in which electrons are free to c\ cle around circular or c\ chc arrangements of atoms, which are alternateh singh and doubh bonded to one another More properh . these bonds ma\ be seen as a h\ bπd of a single bond and a double bond, each bond in the ring being identical

M) to e\ er\ other Examples of aromatic compounds that can be present include benzene. benz\ l. toluene. x\ lene. and the like The aromatic compound can include hetero atoms so as to be a hetero aromatic such as py ridine, furan. tetrah\drofuran. and the like Also, an aromatic can be a poh c> clic aromatic such as naphthalene, anthracene, phenanthrene. poh c> clic aromatic hy drocarbons, indole, quinoline. isoquinoline. and the like v5 As used herein, the term "amine" is meant to refer to moieties that can be derπ ed directh or indirecth from ammonia b> replacing one. two. or three hy drogen atoms b> other groups, such as. for example. alk\ l groups Pπmaπ amines haλ e the general structures RNH: and secondaπ amines haλ e the general structure R:NH The term amine

5 includes, but is not limited to meth\ lamine. eth\ lamine. prop\ lamine. isoprop\ lamine. aniline. c\ clohex\ lamine. benz\ lamine. pol\ c\ clic amines, heteroatom substituted aπ l and alk\ lamines. dimeth\ lamine. dieth\ lamine. diisoprop\ lamine. dibur\ lamine. meth\ lprop\ lamine. meth\ lhex\ lamine. meth\ lc\ cloprop\ lamine. eth\ lc\ lohex\ lamine. meth\ lbenz\ lamine. meth\ c\ clohex\ lmeth\ lamine. bur\ lc\ clohex\ lamine. morpholine.

K) thiomoiphohne. py rrolidine, pipeπdine. 2.6-dimeth\ lpipeπdine. piperazine. and heteroatom substituted alk\ 1 or an 1 secondaπ amines

As used herein, the term "halo ^" means fluoro. chloro. bromo. or iodo. preferabh fluoro and chloro

As used herein, the term "poh (amino acid) ^" or "poh peptide ^" is a poh amide

15 formed from amino acids Poh(amino acid)s will generalh range from about 200-2.000 molecular weight or greater than about 2.000 molecular weight, or haung no upper molecular weight limit, and normalh being less than 10.000.000 and usualh not more than about 600.000 daltons

As used herein, the term "peptide ^" is meant to refer to am compound formed b>

20 the linkage of two or more amino acids b> amide (peptide) bonds, usualh a poh mer of α- amino acids in which α-amino group of each amino acid residue (except the NH ₂ terminus) is linked to the l group of the next residue in a linear chain The terms "peptide. ^" "pohpeptide. ^" and "poh (amino acid) ^" are used suiom moush herein to refer to this class of compounds without restriction as to size The largest members of this

25 class are referred to as proteins

Additionalh . some of the compounds of the present im ention can be prepared as racemic mixtures of isomers, mixtures of isomers, or opticalh isolated isomers Compounds that haλ e the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space are termed "isomers ^"

M) Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers ^"

Stereoisomers that are not mirror images of one another are termed

"diastereomers ^" and those that are non-supeπmposable mirror images of each other are termed "enantiomers ^" When a compound has an asunmetπc center, for example, it is bonded to four different groups, a pair of enantiomers is possible An enantiomer can be v5 characterized b> the absolute configuration of its asunmetπc center and is described b> the R- and S-sequencing rules of Calm and Prelog. or b> the manner in which the molecule rotates the plane of polarized light and designated as dextrorotatoπ or

5 orotatoπ (i e . as ( + ) or (- (-isomers respectπ eh ) A chiral compound can exist as either indiλ idual enantiomer or as a mixture thereof A mixture containing equal proportions of the enantiomers is called a "racemic mixture ^"

The compounds of this im ention ma\ possess one or more as\ mmetric centers Unless indicated otherw ise, the description or naming of a particular compound in the io specification and claims is intended to include both indiudual enantiomers and mixtures, racemic or otherw ise, thereof The methods for the determination of stereochemistry and the separation of stereoisomers are w ell-known in the art (see discussion in Chapter 4 of "Adλ anced Organic Chemistπ ^" . 4 ^M edition J March. John Wile\ and Sons. New York. 1992 )

15 III. Therapeutic Methods

The compounds of the present im ention can be used for the treatment, inhibition, and/or preλ ention of cancer in a subject This can include lung cancer or other cancers The abiht} of a compound of the present im ention to inhibit CYP2A13 ma\ proude for new therapeutic methods for cancer

20 As used herein, the term "treating ^" or "treatment ^" of a disease, such as cancer, includes (a) preλ enting the disease. / e causing the clinical s\ mptoms of the disease not to deλ elop in a mammal that ma\ be exposed to or predisposed to the disease but does not \ et experience or displa\ s\ mptoms of the disease, (b) inhibiting the disease. i e . arresting or reducing the deλ elopment of the disease or its clinical s\ mptoms. or (c)

25 reheλ ing the disease. / e . causing regression of the disease or its clinical s\ mptoms Inhibiting cancer can also include inhibiting cancer propagation Preλ ention of cancer can include total preλ ention as w ell as a temporaπ preλ ention so as to dela\ onset Inhibition and preλ ention can be useful for subjects that haλ e been identified to be susceptible to cancer, such as a smoker

M) In one embodiment, a compound of the present im ention can be administered to a subject that is susceptible to or has cancer As such, the treatment, inhibition, and/or preλ ention of cancers can be performed b\ administering to a subject in need thereof an effectπ e amount of a compound as described herein Optionalh . the compound can be administered in combination w ith a pharmaceutical!} acceptable additπ e. earner or v5 excipient

In one embodiment, a therapeutic method can include a method for inhibiting and/or preλ enting the growth of cancers Such a method can include identif} ing a subject to haλ e a malignant tumor or cancer (e g . lung cancer), and then administering a

5 compound of the present im ention in an inhibitor} or therapeuticalh effectπ e amount or concentration

The therapeutic methods can be used with one or more of the compounds described herein Also, the compounds can be coadministered together or with other therapeutic compounds, such as other compounds that can be used in managing cancer lo As such, the compounds of the present im ention can be administered alone, in combination w ith each other, or the} can be used in combination with other known compounds For instance, the compounds can be used in conjunctπ e therap} with other known anti-angiogenic chemotherapeutic or antineoplastic agents (e g . unca alkaloids, antibiotics, antimetabolites, platinum coordination complexes, etc ) For instance, the

15 compounds can be used in conjunctπ e therap\ w ith a unca alkaloid compound, such as unblastine. uncπstine. taxol. etc . an antibiotic, such as adπam\ cin (doxoαibicin). dactinom\ cm (actinom\ cin D). daunorubicin (daunom\cin. rubidom\ cin). bleomy cin. phcam\ cin (mithram\ cin) and mitomy cin (mitom\ cin C). etc . an antimetabolite, such as methotrexate. c> tarabine (AraC). azauπdine. azaπbine. fluorodeox} undine.

20 deox> coforni} cm. mercaptopuπne. etc . or a platinum coordination complex, such as cisplatin (cis-DDP). carboplatin. etc In addition, the compounds can be used in conjunctπ e therap} with other known anti-angiogenic chemotherapeutic or antineoplastic compounds

As used herein, the term "coadministration ^" or "combination therap} ^" is used to

25 describe a therap} in which at least two actπ e compounds in effectπ e amounts are used to treat lung tumors Although the term coadministration preferabh includes the administration of two actπ e compounds to the patient at the same time, it is not necessaπ that the compounds be administered to the patient at the same time, although effectπ e amounts of the indn idual compounds will be present in the patient at the same time

M) IV. Pharmaceutical Compositions

Compounds according to the present im ention ma> be used in pharmaceutical compositions ing biological/pharmacological actπ it} for the treatment of cancers b> selectπ eh inhibiting CYP2A13 These compositions comprise an effectπ e amount of am one or more of the compounds disclosed herein, optionalh in combination w ith a v5 pharmaceutical!} acceptable additπ e. carrier, or excipient Also, the compounds can be combined and/or prepared into pharmaceutical!} acceptable salts The compounds ma} also be co-administered with other therapeutic agents The effectπ e amount can be a

5 therapeutically effectπ e amount of the compound sufficient for use in treating, inhibiting, and/or preλ enting cancer, such as lung cancers, as well as other cancers

As used herein, the terms "an effectπ e amount ^" , "therapeutic effectπ e amount ^" , or "therapeuticalh effectπ e amount ^" shall mean an amount or concentration of a compound according to the present im ention which is effectπ e within the context of its

K) administration or use Thus, the term "effectπ e amount ^" is used throughout the specification to describe concentrations or amounts of compounds according to the present im ention which ma> be used to produce a faλ orable change in the disease or condition treated, whether that change is a remission, a decrease in growth or size of cancer or a tumor, a faλ orable phy siological result, a reduction in the growth or

15 elaboration of a microbe, or the like, depending upon the disease or condition treated

As used herein, the term "pharmaceutical!} acceptable excipient ^" means an excipient that is useful in preparing a pharmaceutical composition that is generalh safe, non-toxic and neither biologicalh nor otherwise undesirable, and includes an excipient that is acceptable for λ eteπnar} use as well as human pharmaceutical use A

20 "pharmaceutical!} acceptable excipient ^" as used in the specification and claims includes both one and more than one such excipient

As used herein, the term "pharmaceutical!} acceptable acid addition salts ^" refers to those salts which retain the biological effectπ eness and properties of the free bases, which are not biologicalh or otherwise undesirable, and which are formed with inorganic

25 acids such as h}drochloπc acid, h} drobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, gh colic acid, puuuc acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid. he acid, and the like

M) Groups which form pharmaceutical!} acceptable acid addition salts include amines, h} drazines. amidines. guanidines. substituted an 1/heteroan 1 and substituted alk} 1 groups that earn at least a nitrogen bearing substituent such as amino, uamdine. amidino. uamdine and the like

The compounds of the present im ention can be formulated into a v5 pharmaceutical!} acceptable formulation Such a composition can be useful to preλ ent. o\ aπan cancers, and thereb} can be used as an inhibitor, proph} lactic, or treatment for breast and/or o\ an an cancers

5 In embodiments of the present lm ention. the pharmaceutical composition comprises an actπ e component and inactn e components The actπ e components are compounds described herein and their derπ atπ es/analogues The inactn e components are selected from the group consisting of excipients. carriers, soh ents. diluents, stabilizers, enliancers. additπ es. adhesπ es. and combinations thereof

K) The amount of the compound in a formulation can \ an w ithin the full range emplo\ ed b\ those skilled in the art T\ picalh . the formulation w ill contain, on a weight percent basis, from about 0 01-99 99 weight percent of the compounds of the present im ention based on the total formulation, with the balance being one or more suitable pharmaceutical excipients Preferabh . the compounds are present at a leλ el of about 1-80

15 weight percent

Pharmaceutical preparations include sterile aqueous or non-aqueous solutions, suspensions and emulsions Examples of non-aqueous soh ents are prop\ lene gh col. poh ethy lene gh col. -\ egetable oil such as olπ e oil. injectable organic esters such as eth\ loliate Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or

20 suspensions, including saline and buffered media Parenteral -\ eludes include sodium chloride solution. Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils Intraλ enous -\ eludes include fluid and nutrient replenishers. electroMe replenishes, (such as those based on Ringer's dextrose), and the like Preseπ atπ es and other additπ es ma\ also be present such as. for example, antimicrobials, antioxidants.

25 chelating agents and inert gases and the like Those of skill in the art can readih determine the -\ anous parameters for preparing these pharmaceutical compositions without resort to undue experimentation

Pharmacological compositions ma\ be prepared from water-insoluble compounds, or salts thereof, such as aqueous base emulsions In such embodiments, the

M) pharmacological composition w ill t\picalh contain a sufficient amount of pharmaceutical!} acceptable emulsifung agent to emulsift the desired amount of the pharmacological agent Useful emulsifung agents include, but are not limited to. phosphatid} 1 cholines, lecithin, and the like

Additionalh . the compositions ma\ contain other additπ es. such as pH-adjusting v5 additπ es In particular, useful pH-adjusting agents include acids, such as hy drochloric acid, bases or buffers, such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, or sodium gluconate

5 Furthermore, pharmacological agent compositions ma\ . though not alwa\ s. contain microbial preseπ atπ es Microbial preseπ atπ es that ma\ be emplo\ ed include, but are not limited to. meth\ lparaben. prop\lparaben. and benz\ l alcohol The microbial preseπ atπ e ma\ be emplo\ ed when the pharmacological agent formulation is placed in a MaI designed for multi-dose use Pharmacological agent compositions for use in lo practicing the subject methods ma\ be hophihzed using techniques well known in the art

The compositions ma\ also include components, such as c\ clodextπns. to enhance the solubility of one or more other components included in the compositions

C\ clodextπns are wideh known in the literature to increase the solubility of poorh water-soluble pharmaceuticals or drags and/or enhance pharmaceutical/drug stabilit\

15 and/or reduce unwanted side effects of pharmaceuticals/daigs For example, steroids, which are hy drophobic, often exhibit an increase in water solubility of one order of magnitude or more in the presence of c\ clodextπns Am suitable c\ clodextπn component ma\ be emplo\ ed in accordance with the present im ention The useful c\ clodextπn components include, but are not limited to. those materials which are effectπ e in

20 increasing the apparent solubilit\ . preferabh water solubilit\ . of poorh soluble actπ e components and/or enhance the stabilit\ of the actπ e components and/or reduce unwanted side effects of the actπ e components Examples of useful c\ clodextπn components include, but are not limited to β-c\ clodextπn. dem atπ es of β-c\ clodextπn. carbox} meth\ l-β-c> clodextπn. carbox> l-eth\ l-β-c> clodextπn. 1-β-

25 c> clodextπn. dimeth> l-β-c> clodextπn. meth> l-β-c>clodextπn. random meth> l-β- c> clodextπn. glucos> l-β-c> clodextπn. maltos> l-β-c> clodextπn. drox> 1-β- c> clodextπn. diOx> prop> l-β-c> clodextπn. sulfobutλ lether-β-c> clodextπn. and the like and mixtures thereof

The specific c> clodextπn component selected should huπ e properties acceptable

M) for the desired application The c> clodextπn component should haλ e or exhibit reduced toxicit} . particularh if the composition is to be exposed to sensitπ e bod> tissue, for example. e> e tissue, etc Veπ useful β-c> clodextπn components include β-c> clodextπn. derπ atπ es of β-c> clodextπn and mixtures thereof Particularh useful c> clodextπn components include sulfobur> lether β-c> clodextπn. h>diOx>prop> l c> clodextπn and v5 mixtures thereof Sulfobutλ lether β-c> clodextπn is especialh useful, for example, because of its substantialh reduced toxicit>

The pharmaceutical^ acceptable excipients. such as -\ ehicles. adjiπ ants. earners or diluents, are readih aλ ailable to the public Examples of suitable excipients can

5 include, but are not limited to. the following acidulents. such as lactic acid, hy drochloric acid, and tartaric acid, solubihzing components, such as non-ionic, catiomc. and anionic surfactants, absorbents, such as bentonite. cellulose, and kaolin, alkalizing components, such as diethanolamine. potassium citrate, and sodium bicarbonate, anticaking components, such as calcium phosphate tπbasic. magnesium tπsilicate. and talc.

K) antimicrobial components, such as benzoic acid, sorbic acid. benz\ l alcohol, benzethonium chloride, bronopol. alk\ l parabens. cetπmide. phenol, phem lmercuπc acetate, thimerosol. and phenox\ ethanol. antioxidants, such as ascorbic acid, alpha tocopherol. prop\ l gallate. and sodium metabisulfite. binders, such as acacia, alginic acid. carbox\ meth\ 1 cellulose. h\drox\ eth\ l cellulose, dextrin, gelatin, guar gum. magnesium

15 aluminum silicate, maltodextπn. poudone. starch, λ egetable oil. and zein. buffering components, such as sodium phosphate, malic acid, and potassium citrate, chelating components, such as EDTA. malic acid, and maltol. coating components, such as adjunct sugar. cer\ l alcohol, poh um l alcohol, carnauba wax. lactose maltitol. titanium dioxide, controlled release λ eludes, such as microcπ stalline w ax. white wax. and \ ellow wax.

20 desiccants. such as calcium sulfate, detergents, such as sodium lauπ l sulfate, diluents, such as calcium phosphate, sorbitol, starch, talc, lactitol. poh methacπ lates. sodium chloride, and gh ceπ l palmitostearate. disintegrants. such as colloidal silicon dioxide, croscarmellose sodium, magnesium aluminum silicate, potassium polacπlin. and sodium starch gh colate. dispersing components, such as poloxamer 386. and poh ox\ ethy lene

25 farh esters (poh sorbates). emollients, such as ceteaπ l alcohol, lanolin, mineral oil. petrolatum, cholesterol. isoprop\ l m\ πstate. and lecithin. emulsifung components, such as anionic emulsifung wax. monoethanolamine. and medium chain trigly cerides, flaλ oπng components, such as eth\ 1 maltol. eth\ 1 λ anilhn. fumaric acid, malic acid, maltol. and menthol, humectants. such as gh ceπn. prop\ lene gh col. sorbitol, and

M) tπacetin. lubricants, such as calcium stearate. canola oil. gh ceπ l palmitostearate. magnesium oxide, poloxuner. sodium benzoate. stearic acid, and zinc stearate. soh ents. such as alcohols. benz\ l pheny l formate, 1 oleate. gh cerol. gh cofurol. for indigo carmine, poh ethy lene gh col. for sunset > ellow . for tartazine. tπacetin. stabilizing components, such as c> clodextπns. albumin, xanthan gum. v5 and tonicit} components, such as gh cerol. dextrose, potassium chloride, and sodium chloride, and mixture thereof Excipients include those that alter the rate of absorption. ailabihtλ . or other pharmacokinetic properties of pharmaceuticals, dietaπ supplements, alternatπ e medicines, or nutraceuticals

5 Other examples of suitable excipients. binders and fillers are listed in Remington's

Pharmaceutical Sciences. 18th Edition, ed Alfonso Gennaro. Mack Publishing Co Easton. Pa . 1995 and Handbook of Pharmaceutical Excipients. 3rd Edition, ed Arthur H Kibbe. American Pharmaceutical Association. Washington D C 2000. both of which are incorporated herein b> reference lo In some embodiments, the compounds in the compositions ma> be present as a pharmaceutical^ acceptable salt The term "pharmaceutical!} acceptable salts ^" includes salts of the composition, prepared, for example, with acids or bases, depending on the particular substituents found within the composition and the treatment modalit} desired Pharmaceutical!} acceptable salts can be prepared as alkaline metal salts, such as lithium.

15 sodium, or potassium salts, or as alkaline earth salts, such as ben Ilium, magnesium or calcium salts Examples of suitable bases that ma} be used to form salts include ammonium, or mineral bases such as sodium h} dioxide, lithium h} dioxide, potassium h} dioxide, calcium h} dioxide, magnesium h} dioxide, and the like Examples of suitable acids that ma} be used to form salts include inorganic or mineral acids such as

20 h} drochloπc. h} drobromic. h} droiodic. hy drofluoric. nitric. carbonic, monoh} drogencarbomc. phosphoric, monoh} drogenphosphoπc. dih} drogenphosphoπc. sulfuric, monoh} drogensulfuπc. phosphorous acids and the like Other suitable acids include organic acids, for example, acetic, propionic, isoburuic maleic. malonic. benzoic, succinic, suberic, fumaπc. mandelic. phthalic. benzenesulfonic. p-toh lsulfonic.

25 citric, tartaric, methanesulfonic. glucuronic, galactunoπc. salic} lic. formic, naphthalene- 2-sulfonic. and the like Still other suitable acids include amino acids such as arginate. aspartate, glutamate. and the like

In general, pharmaceutical!} acceptable carriers for are well-known to those of ordinaπ skill in the art This carrier can be a solid or liquid and the t}pe is generalh

M) chosen based on the pe of administration being used Suitable pharmaceutical carriers are. in particular, fillers, such as sugars, for example lactose, sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tπcalcium phosphate or calcium h}drogen phosphate, furthermore, binders such as starch paste, using, for example, com. wheat, rice or potato starch, gelatin, tragacanth. meth} lcellulose v5 and/or poh um lpurolidone. if desired, disintegrants. such as the aboλ ementioned starches, furthermore carbox} meth} 1 starch, crosslinked poh um lpurohdone. agar, alginic acid or a salt thereof, such as sodium alginate, auxiliaries are primanh ghdants. flow -regulators and lubricants, for example silicic acid. talc, stearic acid or salts thereof.

5 such as magnesium or calcium stearate. and/or poh eth\ lene gh col Sugar-coated tablet cores are prouded with suitable coatings which, if desired, are resistant to gastric juice, using, inter alia, concentrated sugar solutions which, if desired, contain gum arable, talc, poh un\ lp\ rrohdone. poh ethy lene gh col and/or titanium dioxide, coating solutions in suitable organic soh ents or soh ent mixtures or. for the preparation of gastric juice- io resistant coatings, solutions of suitable cellulose preparations, such as acet\ lcellulose phthalate or h\ dro\\ prop\ lmeth\ lcellulose phthalate Colorants or pigments, for example, to identift or to indicate different doses of actπ e ingredient. ma\ be added to the tablets or sugar-coated tablet coatings

Additional pharmaceutical^ acceptable carriers that ma\ be used in these

15 pharmaceutical compositions include, but are not limited to. ion exchangers, alumina, aluminum stearate. lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, gh cine, sorbic acid, potassium sorbate. partial gh ceride mixtures of saturated -\ egetable fatr\ acids, water, salts or electrohies. such as prolamine sulfate, disodium h\ drogen phosphate, potassium h\ drogen phosphate, sodium chloride.

20 zinc salts, colloidal silica, magnesium tπsilicate. poh um l p> rrohdone. cellulose-based substances, poh ethy lene gh col. sodium carbox\ meth\ lcellulose. poh acπ lates. waxes. poh eth\ lene-poh ox\prop\ lene-block poh mers. poh ethy lene gh col and wool fat

Additional formulations for use in the present im ention can be found in Remington's Pharmaceutical Sciences (Mack Publishing Compam . Philadelphia. Pa .

25 17th ed ( 1985 )). which is incorporated herein b\ reference Moreoλ er. for a brief reuew of methods for daig delπ eπ . see. Langer. Science 249 1527-1533 ( 1990). winch is incorporated herein b\ reference The pharmaceutical compositions described herein can be manufactured in a manner that is known to those of skill in the art. / e . b\ means of com entional mixing, dissolung. granulating, dragee-making, leugating. emulsifung.

M) encapsulating, entrapping or hophihzing processes Other examples of suitable pharmaceuticals are listed in 2000 Med Ad News 19 56-60 and The Phy sicians Desk Reference. 53rd edition. 792-796. Medical Economics Compam ( 1999). both of which are incorporated herein b\ reference

In general, compounds of this im ention can be administered as pharmaceutical v5 compositions b\ am one of the following routes oral. s\ stemic (e g transdermal, intranasal or b\ suppositoπ ). or parenteral (e g . intramuscular, intraλ enous or subcutaneous) administration One manner of administration is oral using a com enient daih dosage regimen which can be adjusted according to the degree of affliction

5 Compositions can take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or am other appropriate compositions Another manner for administering compounds of this im ention is inhalation

Suitable preparations for parenteral administration are primaπh aqueous solutions io of an actπ e ingredient in water-soluble form, for example a water-soluble salt, and furthermore suspensions of the actπ e ingredient, such as appropriate oih injection suspensions, using suitable lipophilic soh ents or λ ehicles. such as fatt\ oils, for example sesame oil. or s\ nthetic fatt\ acid esters, for example eth\ l oleate or trigly cerides, or aqueous injection suspensions which contain uscosit\ -increasing substances, for example

15 sodium carbox\ meth\ lcellulose. sorbitol and/or dextran. and. if necessaπ . also stabilizers

Suitable rectal h utihzable pharmaceutical preparations are. for example, suppositories, which consist of a combination of the actπ e ingredient w ith a suppositoπ base Suitable suppositoπ bases are. for example, natural or s\ nthetic trigly cerides.

20 paraffin hy drocarbons, poh ethy lene gh cols or higher alkanols Furthermore, gelatin rectal capsules which contain a combination of the actπ e ingredient with a base substance ma\ also be used Suitable base substances are. for example, liquid trigly cerides, poh eth\ lene gl\ cols or paraffin h\ drocarbons

Recenth . pharmaceutical formulations lκπ e been eloped especialh for daigs

25 that show poor ailabilit\ based upon the principle that ailabiht\ can be increased b\ increasing the surface area / e . decreasing particle size For example. U S Pat No 4.107.288 (herein incorporated b\ reference) describes a pharmaceutical formulation haung particles in the size range from K) to 1.000 ran in which the actπ e material is supported on a crosslinked matrix of macromolecules U S Pat No 5.145.684

M) (herein incorporated b\ reference) describes the production of a pharmaceutical formulation in which the drug substance is puh eπzed to nanoparticles (aλ erage particle size of 400 ran) in the presence of a surface modifier and then dispersed in a liquid medium to gπ e a pharmaceutical formulation that exhibits remarkabh high bioaλ ailabilitλ v5 According to the methods of the present lm ention. the compositions of the im ention can be administered b\ injection b\ gradual infusion o\ er time or b> am other medicalh acceptable mode Am medicalh acceptable method ma> be used to administer the composition to the patient The particular mode selected will depend of course, upon

5 factors such as the particular daig selected, the se-\ eπt\ of the state of the subject being treated, or the dosage required for therapeutic efficac\ The methods of this im ention. generalh speaking. ma\ be practiced using am mode of administration that is medicalh acceptable, meaning am mode that produces effectπ e els of the actπ e composition without causing clinicalh unacceptable adλ erse effects io The administration ma\ be localized (/ e . to a particular region, phy siological s\ stem. tissue, organ, or cell t\ pe) or s\ stemic For example, the composition ma\ be administered through parental injection, implantation, oralh . λ aginalh . rectalh . buccalh . pulmonaπ . topicalh . nasalh . transdermal^ . surgical administration, or am other method of administration where access to the target b\ the composition is achieλ ed Examples of

15 parental modalities that can be used with the im ention include intraλ enous. intradermal, subcutaneous, intracaut\ . intramuscular, intraperitoneal, epidural, or intrathecal Examples of implantation modalities include am implantable or injectable daig delπ eπ s\ stem Oral administration ma\ be used for some treatments because of the com enience to the patient as well as the dosing schedule Compositions suitable for oral

20 administration ma\ be presented as discrete units such as capsules, pills, cachettes. tables, or lozenges, each containing a predetermined amount of the actπ e compound Other oral compositions include suspensions in aqueous or non-aqueous liquids such as s\ rup. an elixir, or an emulsion

For injection, the compounds can be formulated into preparations b\ dissoh ing.

25 suspending or emulsifung them in an aqueous or nonaqueous soh ent. such as -\ egetable or other similar oils. s\nthetic aliphatic acid gh ceπdes. esters of higher aliphatic acids or prop\ lene gh col. and if desired, with com entional additπ es such as solubihzers. isotonic agents, suspending agents, emulsifung agents, stabilizers and preseπ atπ es Preferabh . the compounds can be formulated in aqueous solutions, preferabh in ph> siologicalh

M) compatible buffers such as Hanks's solution. Ringer's solution, or phy siological saline buffer For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation Such penetrants are generalh known in the art

For oral administration, the compounds can be formulated readih b> combining with pharmaceutical^ acceptable carriers that are well known in the art Such earners v5 enable the compounds to be formulated as tablets, pills, dragees. capsules, emulsions, lipophilic and h\ drophihc suspensions, liquids, gels. s> rups. slurries, suspensions and the like, for oral ingestion b> a patient to be treated Pharmaceutical preparations for oral use can be obtained b> mixing the compounds with a solid excipient. optional!} grinding a

5 resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores Suitable excipients are. in particular, fillers such as sugars, including lactose, sucrose, mannitol. or sorbitol, cellulose preparations such as. for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth. meth\ l cellulose. h\dro\\prop\ lmeth\ l cellulose, sodium lo carbox\ meth\ lcellulose. and/or pol\ un\ lp\ rrolidone (PVP) If desired, disintegrating agents ma\ be added, such as the cross-linked poh um l p\ rrolidone. agar, or alginic acid or a salt thereof such as sodium alginate

Pharmaceutical preparations which can be used oralh include push-fit capsules made of gelatin, as w ell as soft, sealed capsules made of gelatin and a plasticizer. such as

15 gh cerol or sorbitol The push-fit capsules can contain the actπ e ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and. optionalh . stabilizers In soft capsules, the actπ e compounds ma\ be dissoh ed or suspended in suitable liquids, such as fatt\ oils, liquid paraffin, or liquid poh ethy lene gh cols In addition, stabilizers ma\ be added All formulations for

20 oral administration should be in dosages suitable for such administration

For buccal administration, the compositions ma\ take the form of tablets or lozenges formulated in com entional manner

For administration b\ inhalation, the compounds for use according to the present im ention are com enienth delπ ered in the form of an aerosol spra\ presentation from

25 pressurized packs or a nebulizer, with the use of a suitable propellant. e g . dichlorodifluoromethane. trichlorofluoromethane. dichlorotetrafluoroethane. carbon dioxide or other suitable gas. or from propellant-free. dπ -powder inhalers In the case of a pressurized aerosol the dosage unit ma\ be determined b\ proλ iding a λ ah e to delπ er a metered amount Capsules and cartridges of. e g . gelatin for use in an inhaler or

M) insufflator ma\ be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch

Additionalh . the morpholines or morpholine derπ atπ es (e g . thiomorpholines) can be formulated into cigarettes, cigars, or the like This can allow the compound of the im ention to be administered to a smoker so that the actπ e compound is delπ ered while v5 smoking The compounds of the im ention can be incorporated into tobacco products similar to the methods of incorporating other compounds into tobacco products

The compounds can be formulated for parenteral administration b\ injection, e g . b\ bolus injection or continuous infusion Formulations for injection ma\ be presented in

5 unit dosage form, e g . in ampules or in multidose containers, w ith an added preseπ atπ e The compositions ma} take such forms as suspensions, solutions or emulsions in oil} or aqueous λ ehicles. and ma> contain formulator agents such as suspending, stabilizing and/or dispersing agents

In addition to the formulations described preλ ioush . the compounds ma> also be

K) formulated as a depot preparation Such long acting formulations ma> be administered b> implantation (for example subcutaneoush or intramuscularh ) or b> intramuscular injection Thus, for example, the compounds ma> be formulated with suitable poh meπc or hy drophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as spaπngh soluble derπ atπ es. for example, as a spaπngh soluble

15 salt

The compounds can be encapsulated in a -\ ehicle such as liposomes that facilitates transfer of the bioactπ e molecules into the targeted tissue, as described, for example, in U S Pat No 5.879.713 to Roth et til and Woodle. et til . U S Pat No 5.013.556. the contents of which are hereb\ incorporated b> reference The compounds can be targeted

20 b> selecting an encapsulating medium of an appropriate size such that the medium delπ ers the molecules to a particular target For example, encapsulating the compounds within microparticles. preferabh biocompatible and/or biodegradable microparticles. which are appropriate sized to infiltrate, but remain trapped within, the capillar} beds and ah eoh of the lungs can be used for targeted delπ en to these regions of the bod}

25 following administration to a patient b} infusion or injection

Microparticles can be fabricated from different poh mers using a -\ anet} of different methods known to those skilled in the art The soh ent eλ aporation technique is described, for example, in E Mathiow itz. et al . J Scanning Microscop} . 4. 329 ( 1990). L R Beck, et al . Fertil Steπl . 31. 545 ( 1979). and S Benita. et al . J Pharm Sci . 73.

M) 1721 ( 1984) The hot-melt microencapsulation technique is described b} E Mathiowitz. et al . Reactπ e Poh mers. 6. 275 ( 1987) The spra} dπ ing technique is also well known to those of skill in the art Spra} dπ ing im oh es dissolung a suitable poh mer in an appropriate soh ent A known amount of the compound is suspended (insoluble drags) or co-dissoh ed (soluble drags) in the poh mer solution The solution or the dispersion is v5 then spra} -dried Microparticles ranging between 1 -10 microns are obtained with a morpholog} which depends on the t\ pe of poh mer used

Microparticles made of gel-t} pe poh mers. such as alginate, can be produced through traditional ionic gelation techniques The poh mers are first dissoh ed in an

5 aqueous solution, mixed with barium sulfate or some bioactπ e agent, and then extruded through a microdroplet forming deuce, which in some instances employ s a flow of nitrogen gas to break off the droplet A slowh stirred (approximate^ 100-170 RPM) ionic hardening bath is positioned below the extruding deuce to catch the forming microdroplets The microparticles are left to incubate in the bath to allow sufficient time

K) for gelation to occur Microparticle particle size is controlled b> using λ aπous size extruders or -\ an ing either the nitrogen gas or poh mer solution flow rates

Particle size can be selected according to the method of delπ en which is to be used. t\picall\ size IV injection, and where appropriate, entrapment at the site where release is desired

15 In one embodiment, the liposome or microparticle has a diameter which is selected to lodge in particular regions of the bod} For example, a microparticle selected to lodge in a capillar} w ill t\ picall\ e a diameter of between 10 and 100. more preferabh between 10 and 25. and most preferabh . between 15 and 20 microns Numerous methods are known for preparing liposomes and microparticles of an}

20 particular size range S}iithetic methods for forming gel microparticles. or for forming microparticles from molten materials, are known, and include poh meπzation in emulsion, in spra} ed drops, and in separated phases For solid materials or preformed gels, known methods include wet or dn milling or grinding, puh eπzation. classification b} air jet or sie-\ e. and the like

25 Embodiments ma} also include administration of at least one pharmacological agent using a pharmacological delπ en deuce such as. but not limited to. pumps (implantable or external deuces), epidural injectors. s}πnges or other injection apparatus, catheter and/or reseπ oir operatπ eh associated with a catheter, injection etc For example, in certain embodiments a delπ en deuce emplo} ed to delπ er at least one

M) pharmacological agent to a subject ma} be a pump. s} ringe. catheter or reseπ oir operabh associated w ith a connecting deuce such as a catheter, tubing, or the like Containers suitable for delπ en of at least one pharmacological agent to a pharmacological agent administration ice include instruments of containment that ma} be used to delπ er. place, attach, and/or insert at least one pharmacological agent into the delπ er} deuce for v5 administration of the pharmacological agent to a subject and include, but are not limited to. uals. ampules, tubes, capsules, bottles. s}ringes and bags

Sterile injectable forms of the compositions of this im ention ma} be aqueous or a substantial!} aliphatic suspension These suspensions ma} be formulated according to

5 techniques known in the art using suitable dispersing or wetting agents and suspending agents The sterile injectable preparation ma\ also be a sterile injectable solution or suspension in a non-to\ic parenteralh -acceptable diluent or soh ent. for example as a solution in 1.3-butanediol Among the acceptable -\ ehicles and soh ents that ma\ be emplo\ ed are water. Ringer's solution and isotonic sodium chloride solution In addition.

K) sterile, fixed oils are com entionalh employ ed as a soh ent or suspending medium For this purpose, am bland fixed oil ma\ be emplo\ ed including s\nthetic mono- or di- gh ceπdes Fatr\ acids, such as oleic acid and its gh ceπde derπ atπ es are useful in the preparation of injectables. as are natural pharmaceutical^ -acceptable oils, such as olπ e oil or castor oil. especialh in their poh ox\ eth\ lated λ ersions These oil solutions or

15 suspensions ma\ also contain a long-chain alcohol diluent or dispersant

The pharmaceutical compositions of this im ention ma\ also be administered topicalh . especialh when the target of treatment includes areas or organs readih accessible b\ topical application, including diseases of the e\ e. the skin, or the lower intestinal tract Suitable topical formulations are readih prepared for each of these areas

20 or organs

Pharmacological agents ma\ be delπ ered transdermal^ . b\ a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols For example, embodiments ma\ include a pharmacological agent formulation in the form of a discrete patch or film or

25 plaster or the like adapted to remain in intimate contact with the epidermis of the recipient for a period of time For example, such transdermal patches ma\ include a base or matrix la\ er. e g . poh meπc la\ er. in which one or more pharmacological agent(s) are retained The base or matrix la\ er ma\ be operatπ eh associated w ith a support or backing Pharmacological agent formulations suitable for transdermal administration ma\ also be

M) delπ ered b\ iontophoresis and ma\ take the form of an optionalh buffered aqueous solution of the pharmacological agent compound Suitable formulations ma> include citrate or bis/tπs buffer (pH 6) or ethanol/w ater and contain a suitable amount of actπ e ingredient Topical application for the lower intestinal tract can be effected in a rectal suppositoπ formulation (see e) or in a suitable enema formulation Topicalh - v5 transdermal patches ma> also be used

For other topical applications, the pharmaceutical compositions ma> be formulated in a suitable ointment containing the actπ e component suspended or dissoh ed in one or more earners Carriers for topical administration of the compounds of this

5 im ention include, but are not limited to. mineral oil. liquid petrolatum, white petrolatum, propy lene gh col. poh ox\ eth\ lene. poh ox} prop} lene compound. emulsifung wax and water Altematπ eh . the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the actπ e components suspended or dissoh ed in one or more pharmaceutical^ acceptable carriers Suitable carriers include, but are not limited to.

K) mineral oil. sorbitan monostearate. poh sorbate 60. cer\ l esters wax. ceteaπ l alcohol. 2- oct} ldodecanol. benz} l alcohol and water

For ophthalmic use. the pharmaceutical compositions ma} be formulated as micromzed suspensions in isotonic. pH adjusted sterile saline, or. preferabh . as solutions in isotonic. pH adjusted sterile saline, either with or without a preseπ atπ e such as

15 benz} lalkonium chloride Altematπ eh . for ophthalmic uses, the pharmaceutical compositions ma} be formulated in an ointment such as petrolatum

The pharmaceutical compositions of this im ention ma} also be administered b} nasal aerosol or inhalation Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and ma} be prepared as solutions in

20 saline, emploung benz} l alcohol or other suitable preseπ atπ es. absorption promoters to enhance bioaλ ailabiht> . fluorocarbons. and/or other com entional solubihzing or dispersing agents

Depending on the mode of administration, the pharmaceutical composition will preferabh comprise from 0 05 to 99% b} weight, more preferabh from 0 1 to 70% b}

25 weight of the actπ e ingredient, and. from 1 to 99 95% b} weight, more preferabh from 30 to 99 9% b} weight of a pharmaceutical!} acceptable carrier, all percentages being based on the total composition

The compositions of the present im ention ma} be gπ en in dosages, generalh at the maximum amount while aλ oiding or minimizing am potentialh detrimental side

M) effects The compositions can be administered in effectπ e amounts, alone or in a cocktail with other compounds, for example, other compounds that can be used to treat and/or preλ ent breast and/or oλ aπan cancer An effectπ e amount is generalh an amount sufficient to inhibit breast and/or oλ aπan cancer within the subject

In one embodiment of the present im ention. therapeuticalh effectπ e amounts of v5 compounds of the present im ention ma} range from approximate!} 0 05 to 50 mg per kilogram bod} weight of the recipient per da} , preferabh about 0 01-25 mg/kg/da} . more preferabh from about 0 5 to 10 mg/kg/da} Thus, for administration to a 70 kg person, the dosage range would most preferabh be about 35-70 mg per da}

5 In another embodiment of the present im ention. dosages ma> be estimated based on the results of experimental models, optionalh in combination w ith the results of assa> s of the present im ention Generalh . daih oral doses of actπ e compounds will be from about 0 01 mg/kg per da> to 2000 mg/kg per da> Oral doses in the range of 10 to 500 mg/kg. in one or seλ eral administrations per da> . ma> \ ield suitable results In the e\ ent io that the response of a particular subject is insufficient at such doses. e-\ en higher doses (or effectπ e higher doses b> a different, more localized delπ eπ route) ma> be emplo\ ed to the extent that patient tolerance permits Multiple doses per da> are also contemplated in some cases to achieλ e appropriate s> stemic leλ els of the composition Dosage amount and inteπ al ma> be adjusted indn idualh to proλ ide plasma els of the actπ e

15 compound which are sufficient to maintain therapeutic effect Preferabh . therapeuticalh effectπ e serum els w ill be achieλ ed b> administering multiple doses each da> In cases of local administration or selectπ e uptake, the effectπ e local concentration of the drug ma> not be related to plasma concentration One haung skill in the art w ill be able to optimize therapeuticalh effectπ e local dosages w ithout undue experimentation

20 Although the exact dosage w ill be -\ aπ dependent upon the percent composition of the dosage of compounds of the present im ention. in most cases some generalizations regarding the dosage can be made The daih dosage regimen for an adult human patient ma> be. for example, an oral dose of betw een 0 1 mg and 2000 mg of each actπ e ingredient, preferabh betw een 1 mg and 500 mg. e g 5 to 200 mg In other embodiments.

25 an intraλ enous. subcutaneous, or intramuscular dose of each actπ e ingredient of between 0 01 mg and 100 mg. preferabh betw een 0 1 mg and 60 mg. e g 1 to 40 mg is used In cases of administration of a pharmaceutical!} acceptable salt, dosages ma} be calculated as the free base In some embodiments, the composition is administered 1 to 4 times per da} Alternatπ el} the compositions of the im ention ma} be administered b} continuous

M) intraλ enous infusion, preferabh at a dose of each actπ e ingredient up to 1000 mg per da} As w ill be understood b} those of skill in the art. in certain situations it ma} be necessaπ to administer the compounds disclosed herein in amounts that exceed, or e\ en far exceed, the e-stated dosage range in order to effectπ el} and aggressπ eh treat particularh aggressπ e diseases or infections In some embodiments, the compounds w ill v5 be administered for a period of continuous therap} . for example for a week or more, or for months or } ears

Dosage amount and inteπ al ma} be adjusted indπ idualh to proλ ide plasma els of the actπ e moiet} w hich are sufficient to maintain the modulating effects, or minimal

5 effectπ e concentration (MEC ) The MEC will -\ aπ for each compound but can be estimated from in vitro and in vivo data Dosages necessaπ to achieλ e the MEC w ill depend on indiudual characteristics and route of administration How eλ er. HPLC assa> s or bioassa} s can be used to determine plasma concentrations

Use of a long-term release implant ma> be particularh suitable in some cases

K) "Long-term release." as used herein, means that the implant is constructed and arranged to delπ er therapeutic els of the composition for at least 30 or 45 da> s. and preferabh at least 60 or 90 da> s. or e-\ en longer in some cases Long-term release implants are w ell known to those of ordinal} skill in the art. and include some of the release s> stems described aboλ e

15 Am suitable dosage ma> be administered The compound, the carrier, and the amount will -\ aπ w ideh depending on bod} w eight, the se-\ eπt> of the condition being treated and other factors that can be readih eλ aluated b> those of skill in the art Generalh a dosage of betw een about 1 mg per kg of bod} w eight and about 100 mg per kg of bod> w eight is suitable

20 In pharmaceutical dosage forms, agents ma> be administered alone or with an appropriate association, as w ell as in combination, w ith other pharmaceutical!} actπ e compounds As used herein, "administered w ith ^" means that at least one pharmacological agent and at least one other adjm ant (including one or more other pharmacological agents) are administered at times sufficient!} close that the results obseπ ed are

25 indistinguishable from those achieλ ed when one pharmacological agent and at least one other adjm ant (including one or more other pharmacological agents) are administered at the same point in time The pharmacological agent and at least one other adjm ant ma} be administered simultaneous!} (/ e . concurrent!} ) or sequentialh Simultaneous administration ma} be carried out b} mixing at least one pharmacological agent and at

M) least one other adjm ant prior to administration, or b} administering the pharmacological agent and at least one other adjm ant at the same point in time Such administration ma} be at different anatomic sites or using different routes of administration The phrases "concurrent administration. ^" "administration in combination. ^" "simultaneous administration ^" or "administered simultaneous!} ^" ma} also be used interchangeabh and v5 mean that at least one pharmacological agent and at least one other adjm ant are administered at the same point in time or immediateh follow ing one another In the latter case, the at least one pharmacological agent and at least one other adjm ant are administered at times sufficient!} close that the results produced are s} iiergistic and/or are

5 indistinguishable from those achieλ ed when the at least one pharmacological agent and at least one other adjm ant are administered at the same point in time Altematπ eh . a pharmacological agent ma\ be administered separateh from the administration of an adjm ant. which ma\ result in a s\ nergistic effect or a separate effect The methods and excipients described herein are mereh exemplar} and are in no wa\ limiting io Therapeuticalh effectπ e dosages for the compounds described herein can be estimated initialh from cell culture assa\ s For example, a dose can be formulated in animal models to achieλ e a circulating concentration range that includes the ICNo as determined in cell culture (/ e . the concentration of test compound that is lethal to 50 ° ₀ of a cell culture), or the ICioo as determined in cell culture (ι e . the concentration of

15 compound that is lethal to 100% of a cell culture) Such information can be used to more accurateh determine useful doses in humans Initial dosages can also be estimated from in vivo data

Moreoλ er. toxicity and therapeutic efficac\ of the compounds described herein can be determined b\ standard pharmaceutical procedures in cell cultures or experimental

20 animals, e g . b\ determining the LD _^0 . (the dose lethal to 50 ° ₀ of the population), the ED _^ o (the dose therapeuticalh effectπ e in 50 ° ₀ of the population), and EC _^0 (the excitatoπ concentration effectπ e in 50 ° ₀ of the population) The dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as the ratio between LDso and EDso Compounds which exhibit high therapeutic indices are

25 candidates for further deλ elopment The data obtained from these cell culture assa\ s and animal studies can be used in formulating a dosage range that is not toxic for use in human The dosage of such compounds lies preferabh w ithin a range of circulating concentrations that include the EDso with little or no toxicit\ The dosage ma\ -\ aπ within this range depending upon the dosage form empkn ed and the route of

M) administration utilized The exact formulation, route of administration and dosage can be chosen b\ the indπ idual phy sician in Mew of the patient's condition (See. e g . Fingl et al . 1975. In "The Pharmacological Basis of Therapeutics ^" . Ch 1. p 1 ) Additionalh . the EC^o can be important to measure

In one embodiment, a catheter is used to direct the composition directh to the v5 location of the targeted tumor As will be readih apparent to one skilled in the art. the useful in v/vo dosage to be administered and the particular mode of administration w ill ■\ aπ depending upon the age. weight and mammalian species treated, the particular compounds emplo> ed. and the specific use for which these compounds are emplo> ed

5 The determination of effectπ e dosage els. that is the dosage els necessaπ to achieλ e the desired result, can be accomplished b\ one skilled in the art using routine pharmacological methods T\ picalh . human clinical applications of products are commenced at lower dosage els. with dosage leλ el being increased until the desired effect is achieλ ed Alternatπ eh . acceptable in vitro studies can be used to establish useful io doses and routes of administration of the compositions identified b\ the present methods using established pharmacological methods

The exact formulation, route of administration and dosage for the pharmaceutical compositions of the present im ention can be chosen b\ the indn idual ph\ sician in λ lew of the patient's condition (See e g et ul 1975. in "The Pharmacological Basis of

15 Therapeutics ^" , which is hereb\ incorporated herein b\ reference in its entirety . with particular reference to Ch 1. p 1 ) T\picalh . the dose range of the composition administered to the patient can be from about 0 5 to 1000 mg/kg of the patient's bod\ weight The dosage ma\ be a single one or a series of two or more gπ en in the course of one or more da\ s. as is needed b\ the patient In instances where human dosages for

20 compounds e been established for at least some condition, the present im ention w ill use those same dosages, or dosages that are between about 0 l °υ and 50OV more preferabh between about 25% and 250% of the established human dosage Where no human dosage is established, as will be the case for new h -discoλ ered pharmaceutical compounds, a suitable human dosage can be inferred from EC _^0 . ED^, or ID^ ₀ -\ alues. or

25 other appropriate -\ alues derπ ed from in vitro or in vivo studies, as qualified b\ toxicity studies and efficac\ studies in animals

It should be noted that the attending phy sician w ould know how to and when to terminate, interrupt, or adjust administration due to toxicity or organ d\ sfunctions Com erseh . the attending phy sician would also know to adjust treatment to higher els

M) if the clinical response were not adequate (precluding toxicity ) The magnitude of an administrated dose in the management of the disorder of interest will -\ aπ w ith the seλ eπty of the condition to be treated and to the route of administration The se-\ eπt> of the condition ma> . for example, be eλ aluated. in part. b> standard prognostic eλ aluation methods Further, the dose and perhaps dose frequenc\ . w ill also λ an according to the v5 age. bod> weight, and response of the indiudual patient A program comparable to that discussed aboλ e ma> be used in λ eteπnaπ medicine

Compounds disclosed herein can be eλ aluated for efficac} and to\icit> using known methods For example, the to\icolog> of a particular compound, or of a subset of

5 the compounds, sharing certain chemical moieties. ma\ be established b\ determining in vitro toxicity towards a cell line, such as a mammalian, and preferabh human, cell line The results of such studies are often predictπ e of toxicity in animals, such as mammals, or more specificalh . humans Alternatπ eh . the toxicit\ of particular compounds in an animal model, such as mice. rats, rabbits, or monke\ s. ma\ be determined using known

K) methods The efficac\ of a particular compound ma\ be established using seλ eral recognized methods, such as in vitro methods, animal models, or human clinical trials Recognized in vitro models exist for nearh e-\ er\ class of condition, including but not limited to cancer, cardioλ ascular disease, and λ aπous immune ch sfunction Similarh . acceptable animal models ma\ be used to establish efficac\ of chemicals to treat such

15 conditions When selecting a model to determine efficac\ . the skilled artisan can be guided b\ the state of the art to choose an appropriate model, dose, and route of administration, and regime Of course, human clinical trials can also be used to determine the efficac} of a compound in humans

EXPERIMENTAL

20 1.

To date, λ irtualh all attempts to characterize full-length mammalian c\ tochromes P450 e been restricted b\ the production of onh small (nanomolar) amounts of protein Howeλ er. truncation of the non-catahiic N-terminal transmembrane helix and addition of a C -terminal 4x histidine tag allow us to recombinant^ produce tens of

25 milligrams of highh purified modified CYP2A13 and CYP2A6 proteins per liter of £ coh culture These engineered λ ersions of the proteins (CYP2Al3dH and CYP2A6dH are not shown) are fulh functional in both binding hgands and metabolism of substrates Protein expression in E coh is initiated with the addition of IPTG to media supplemented with the heme precursor ammoleuilinic acid After 48-72 hours this expression s\ stem

M) \ ields approximate^ 500-1300 nmol (27-71 mg) of CYP2A13dH or CYP2A6dH per liter of bacterial culture Bπefh . P450 proteins are liberated from E coh membranes using a specialized detergent and high salt conditions and subsequenth purified using metal- affinit} and carbox> meth> 1-sepharose (CM-sepharose) affinit> chromatograph> Oλ erall purification > ield is up to 14 mg of highh purified protein per liter of E coh culture v5 Cy tochrome P4502A13dH was expressed and purified as preuoush described (J

Biol Chem (2007). 2<V2(23 )17306-17313 ) Bπefh . CYP2A13 was solubihzed from the membrane using 4 8 mM Cunal-5 detergent (Anatrace. Maumee. OH) and 0 3M NaCl followed b> ultracentπfugation at 30.000 rpm for 60 minutes The solubihzed protein was

5 applied to Nr ⁺ -agarose resin (Qiagen. Valencia. CA) and after extensπ e washing with Buffer A (0 I M potassium phosphate. pH 7 4. 20% gh cerol. 0 2M sodium chloride. 4 8 mM c\ mal. 8 inM histidine). the protein was eluted using Buffer B (Buffer A containing 2 mM EDTA and 80 mM histidine) The fractions containing protein w ith absorbance at 415 ran were pooled, diluted 5-fold with Buffer C (5mM potassium phosphate. pH 7 4.

K) 20°υ gh cerol. 4 8 mM C\mal-5 ). and loaded onto HiTrap CM-FF column The column was washed extensπ eh w ith Buffer C without the detergent Purified CYP2A13 was eluted using Buffer C with 50 mM potassium phosphate and 500 mM NaCl An aλ erage \ ield of 14 mgs of CYP2A13 was obtained per litre of E coll culture C\1ochrome P4502A6dH was similarh prepared

15 2.

The UVλ isible spectra of purified CYP2A13dH and CYP2A6dH run e a Soret peak at 418 nm. indicatn e of a low -spin, six-coordinate configuration of the heme iron bound to an actπ e site water molecule (Figure IA) When ligands bind in the actπ e site the\ frequenth displace the water from the iron, resulting in a shift to fπ e-coordinate.

20 high spin iron with a maximal absorbance -395 nm For c\1ochrome P450 enzunes. one often records the spectrum shift that occurs as protein is titrated with hgand to determine hgand affinit\ (k _< i λ alues) (Figure I B)

In order to com ert this assa\ to HTS mode, absorbance was measured at six waλ elengths spanning the waλ elength range of the spectral shift to -\ erif> that the same

25 shifts were obseπ ed upon hgand binding in the HTS assa\ enuronment (Figure 2) Because these initial experiments did exhibit the spectral shift as expected and because reading six waλ elengths for each well is incredibh time-intensπ e. the assa\ was optimized to read onh absorbance at the maximum (395 nm) and the minimum (418 nm) for each compound and compare the resulting difference in absorbance to that obseπ ed

M) for PEITC binding (positπ e control) Compounds are screened in 384 well plates using a plate absorbance reader

A spectral shift assa\ reflecting interactions of compounds with CYP2A13 was adapted to HTS format for screening compounds Each 384-well screening plate contained 320 compounds (20 uM in 0 7 l°υ DMSO) and a set of standards comprising 32 v5 wells 16-wells each of 0 71 % DMSO (Negatπ e control), and 16-wells of Positπ e control (30 μM pheneth\ hsothioc\ anate or PEITC) Purified CYP2A13 (3uM) was added to all the wells of the assa\ plates and the protein/compound mixtures were allowed to

5 equilibrate at room temperature for 5 minutes The plates were read on SpectraMax (Molecular Deuces. CA) at 385 nm and 420 nm The raw data were directh uploaded into spreadsheets that enabled hit identification based on the difference in raw absorbance λ alues at 385 nm and 420 nm

In an initial suπ e\ of 1760 compounds, we had a hit rate of -2 5°υ This hit rate is

K) \ er} high for most HTS assay s, but ma\ reflect the nature of the target C\1ochrome P450 enz\ mes are promiscuous b\ design and might be expected to bind a higher number of hbraπ compounds than most proteins Howeλ er. suπ e\ of the same 1760 compounds with CYP2A6 uelded a hit rate of onl\ 0 28% This suggested a substantial number of compounds that are selectπ e for CYP2A13 binding

15 The compounds that induced spectral shifts close to that of the positπ e control

(PEITC) were -\ ahdated in the spectral shift assa\ using the low throughput ciπ ette method CYP2A13 protein samples were diluted to a concentration of 1 μM in 100 niM potassium phosphate buffer (pH 7 4) and were diuded equalh between reference and sample 1 inL quartz ciπ ettes Concentrated compound stocks were prepared in K)O ⁰ O

20 DMSO and the sample baseline was taken from 300 - 500 nm before beginning the titration Substrate was added to the sample ciπ ette. mixed, and allowed to equilibrate for 2 minutes An equal amount of ethanol was added to the reference ciπ ette for e-\ er\ addition of compound stock to the sample The total amount of ethanol was kept less than 2°o for each titration After each coumaπn addition, difference spectra were recorded

25 from 300 - 500 nm and the change maximum ( - 420 nm) to the minimum ( - 385 nm) was determined Graphpad Prism 4 (Graphpad Software. San Diego. CA) was used to determine the K _s and λA _max w ith nonlinear least-squares regression fitting to the equation

X4 = SU ₁ . [p _{+ S + K} _ ^ _{ p _{+ S + A} - y _ ₄ p _λ . j

3.

M) In order to -\ erif> that the t\\ o-\\ aλ elength HTS assa\ is reliable for detecting true hits for CYP2A13 binding and not false positπ es. a set of 10 compounds identified as hits from the initial 176( (-compound suπ e\ w ere further im estigated CYP2A13 was titrated with these compounds one at a time in a full spectral assa> Most of these compounds did bind to CYP2A13. but seλ eral had intrinsic absorbance in the 395 nm range that v5 precluded definitπ e spectral anah sis Of the ten. 50% were -\ ahdated as true hgands of CYP2A13 One of these compounds. 4-(2-chloro-6-fluorobenz\ l)morphohne (shown

below), has a k _t i of approximate^ 58 μM with CYP2A13. but caused absoluteh no spectral shift with CYP2A6 (Figures 3A-3C)

4-(2-chloiO-6-fuorobenz\ 1 )morpholine

4.

Identification of one CYP2A13-selectπe scaffold and subsequent analog studies Subsequent anahsis of our small compound suπe\ reλealed that two additional morpholine structures (eg. 4-(2-meth\lbenz\l)morpholine and 4-(2- chlorobenz>l)moipholine) were also identified as hits These compounds were further characterized for their binding to CYP2A13 and CYP2A6 These two additional morpholines bound to CYP2A13 with initial Kd of 4 μM and 18 μM. but ga-\e no spectral shift with CYP2A6 Using multiple titrations including that shown in Figures 4A-4C.4-(2-meth\lbenz\l)morpholine was determined to rune an aλerage Kd of 193. and no substantialh binding for CYP2A6dH Using multiple titrations including that shown in Figures 5A-5C. 4-(2-chlorobenz\l)morpholine was determined to rune an aλerage Kd for CYP2A13 of 73 μM. and no substantialh binding for CYP2A6

4-(2-meth\ 1 )morphohne

4-(2-chlorobenz> 1 )morpholine Further of 4-(2-clilorobenz>l)moipholine. 4-(2-chloro-6- fluoiObenz}l)morpholine. 4-(2-meth>lbenz>l)moipholine. and other moipholine dematπes shown in Table 1 were performed The proMded the aλerage Kd

for CYP2A13. CYP2A6. and selectπ ity by (CYP2A6 Kd)/(CYP2A13 Kd). For selectπ ity. higher numbers are more selectπ e.

Table 1

Additional moipholine analogs that may be more selective for CYP2A13 over CYP2A6 in accordance with the present invention are shown in Table 2. These compounds are currently being synthesized and e\ aluated to explore the effects of further scaffold λ ariation on the affinity and selectivity for human CYP2A enzymes.

Table 2

6.

Enz\ me inhibition studies can be performed to further λ ahdate morpholine derπ atπ es Assa\ s were focused on hgand binding because catalytic actiut\ requires not onh the cy tochrome P450 protein, but also two additional accessor} proteins. NADPH cλ tochrome P450 reductase and c> toclirome b5 To eλ aluate the abilit\ of selectπ e hgand-binding hits to selectπ eh inlnbit enzunatic tumoλ er. a coumaπn h\ dro\\ lation assa\ can be used for both CYP2A13 and CYP2A6 This relatπ eh simple assa\ produces a fluorescent metabolite. 7-h\ dro\\ coumarm. that is easih detected Bπefl> . the reconstituted s\ stem contains purified P450 protein. NADPH-c>1ochiOme P450 reductase. c> tochrome h5. coumaπn. and NADPH in a potassium buffer After incubation at 37°C for K) mm. the reaction is stopped b> the addition of trichloroacetic acid The final 7-h\ dro\\ coumaπn metabolite is detected b> fluorescence using an excitation waλ elength of 368 nm and an emission waλ elength of 453 nm Tins assa> can be adapted to a high throughput assa> for 384-well plates using a Bio-tek FL600 for fluorescent metabolite detection Initial!} , compounds can be selected b> their abiht> to reduce 7-

h\ dro\\ coumarin generation from coumarin o\ er time, compared to assa\ s w ith no inhibitor Known inhibitors can be used as controls To determine KI λ alues. formation of the metabolites can be measured in the presence of increasing concentrations of the inhibitor The substrate concentrations can range from 1/2 Am to twice the Am At each substrate concentration, metabolite formation can be monitored in the absence and presence of the inhibitors Al λ alues can be generated b\ Di\on plot anah sis and used to select the best selectπ e inhibitors of CYP2A13 o\ er CYP2A6

The present im ention ma\ be embodied in other specific forms without departing from its spirit or essential characteristics The described embodiments are to be considered in all respects onh as illustratn e and not restπctπ e The scope of the im ention is. therefore, indicated b\ the appended claims rather than b\ the foregoing description All changes which come w ithin the meaning and range of eqιm alenc\ of the claims are to be embraced within their scope All references (e g . journal articles, published patent applications, patents, and the like) identified herein are incorporated herein b\ specific reference in their entirety