(12) United States Patent (10) Patent No.: US 9,181.237 B2 Grote (45) Date of Patent: Nov

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(12) United States Patent (10) Patent No.: US 9,181.237 B2 Grote (45) Date of Patent: Nov. 10, 2015

(54) SUBSTITUTED BERBINES AND THEIR OTHER PUBLICATIONS SYNTHESIS Jiaranaikulwanitch, J. et al.: Triazolyl tryptoline derivatives as beta (71) Applicant: MALLINCKRODT LLC, Hazelwood, Secretase inhibitors. Biorg. & medicinal Chem. letters, vol. 20, pp. MO (US) 6572-6576, 2010.* Kobayashi, J. et al.: Theoneberine; The first brominated (72) Inventor: Christopher W. Grote, Webster Groves, benzyltetrahydroberberine alkaloid from the Okinawan marine MO (US) sponge TheOnella sp., vol. 57, pp. 6680-6682, 1992.* Rastrelli, L. et al.: New Protopine and (73) Assignee: Mallinckrodt LLC, Hazelwood, MI benzyltetrahydroprotoberberine alkaloids from Aristolochia (US) constricta and their activity on isolated guinea-pig ileum.J. of Natu ral Prod., vol. 60, pp. 1065-1069, 1997.* (*) Notice: Subject to any disclaimer, the term of this Grycova et al., Quaternary protoberberine alkaloids, Phytochem. patent is extended or adjusted under 35 2007, 68(2): 150-175. U.S.C. 154(b) by 0 days. Mali et al., Novel syntheses of 1-substituted-7,8-dialkoxyisochro man-3-ones and 8-substituted.-2,3,9,10-tetramethoxyberbines, Tetra (21) Appl. No.: 14/504,906 hedron (1986), 42(7), 2075-82. Memetzidis, et al., Synthesis of Aromatic Chloroberbines, Hetero (22) Filed: Oct. 2, 2014 cycles (1990), 31(2), 341-51. Memetzidis, et al., Structure-affinity relationships of berbines or (65) Prior Publication Data 5,6,13,13a-tetrahydro-8H-dibenzoa, gduinolizines at O-adrenocep tors, Eur, J. Med. Chem., 1991, 26: 605-611. US 2015/OO99772 A1 Apr. 9, 2015 Nagubandietal. The mechanism of the Bischler-Napierski reaction, J. Heterocyclic Chem., 1980, 17: 1457-1463. Related U.S. Application Data Sotomayor et al., Bischler-Napieralski Cyclization-N/C-Alkylation Sequences for the Construction of Isoquinoline Alkaloids. Synthesis (60) Provisional application No. 61/886,695, filed on Oct. of Protoberberines and Benzocphenanthridines via C-2'- 4, 2013. Functionalized 3-Arylisoquinolines, Journal of Organic Chemistry (1996), 61(12), 4062-4072. (51) Int. Cl. Valpuesta et al., Regio-and Stereoselective Stevens Rearrangement CO7D 22/8 (2006.01) of Benzyltetrahydroprotoberberinium Salts, Eur, J. Org. Chem. 2004, CO7D 22/04 (2006.01) 4313-4318. CO7D 455/03 (2006.01) Yamamoto et al., Total synthesis of 8-epi-javaberine A and javaberine (52) U.S. Cl. A, Heterocycles 2014, 88(2): 1311-1321. CPC ...... C07D455/03 (2013.01) Zhang, et al., A Novel Analgesic Isolated from a Traditional Chinese (58) Field of Classification Search Medicine, Current Biology, 2014, vol. 24(2), pp. 1-7. USPC ...... 546/73, 71 International Search Report and Written Opinion dated Jan. 14, 2015 See application file for complete search history. from related international application No. PCT/US2014/058806, 11 pg.S. (56) References Cited PubChem. Compound Summary for CID 6342, Acetonitrile, Create Date: Sep. 16, 2004. retrieved on 20.22.2014 from the Internet U.S. PATENT DOCUMENTS pubchem.ncbi.nlm.nih.gov/6342. PubChem. Compound Summary for CID 8025, Ethyl formate, Create 4.013,666 A 3, 1977 Lenz ...... 546,71 Date: Mar. 26, 2005. retrieved on Nov. 21, 2014 from the Internet 4,052,389 A 10, 1977 Monkovic pubchem.ncbi.nlm.nih.gov/8025. 6.255,317 B1 7, 2001 Kim 8,003,795 B2 8, 2011 Liu 8,163,912 B2 4/2012 Grote * cited by examiner 8,431,705 B2 4/2013 Grote 2010.0113494 A1 5, 2010 Hu Primary Examiner — Charanjit Aulakh 2010, 0120810 A1* 5, 2010 Leblond et al...... 514,280 2012,0004223 A1 1/2012 Liu 2012/0059026 A1 3/2012 LaVoie (57) ABSTRACT The present invention provides substituted berbines, pro FOREIGN PATENT DOCUMENTS cesses for the synthesis of substituted berbine compounds, as EP 0 028959 5, 1981 well as intermediates used in the synthesis of substituted WO 2009/007457 1, 2009 berbine compounds. Also provided are methods for using the WO 2010.075469 T 2010 substituted berbines to inhibit cancer cell growth. WO 2010, 128061 11, 2010 WO 2012, 1631 79 12/2012 5 Claims, No Drawings US 9, 181,237 B2 1. 2 SUBSTITUTED BERBINES AND THEIR SUMMARY OF THE INVENTION SYNTHESIS Among the various aspects of the present invention is a compound comprising Formula (V-1): CROSS-REFERENCE TO RELATED APPLICATIONS (V-1) R1 R12 R11

This application claims the priority of U.S. provisional application Ser. No. 61/886,696, filed Oct. 4, 2013, which is hereby incorporated by reference in its entirety. 10

15 FIELD OF THE INVENTION

The present invention generally relates to substituted ber wherein: bines, processes for the synthesis of substituted berbines, R. R. R., and Rindependently are hydrogen, halogen, OR',NR'N', nitro, cyano, thiol, hydrocarbyl, sub intermediate compounds used in the preparation of Substi stituted hydrocarbyl, or together RandR along with tuted berbines, and methods of using substituted berbines. the ring carbons to which they are attached form a ring comprising { }O(CH2)O{-}; R. R. R', and Rindependently are hydrogen, halogen, 25 OR', NR'N', nitro, cyano, thiol, hydrocarbyl, sub stituted hydrocarbyl, or together RandR along with the ring carbons to which they are attached form a ring comprising { }O(CH2).O—}; provided that at BACKGROUND OF THE INVENTION least two of R. R. and Rare other than methoxy; 30 R. R', R', and R' independently are hydrogen, The berbine class of heterocyclic compounds is structur hydrocarbyl, or substituted hydrocarbyl, R',R,R,R, and Rindependently are hydrogen, ally related to the plant alkaloid berberine. Berbine com halogen, OR, NR'N', nitro, cyano, thiol, hydro pounds have been reported to have numerous therapeutic carbyl, or substituted hydrocarbyl; effects. For example, they have been found to have antibac 35 R" and R' independently are hydrogen, hydrocarbyl, terial, antifungal, antiparasitic, antipyretic, antihypertensive, or substituted hydrocarbyl, m is an integer of 0 or greater; antidepressant, antiemetic, tranquilizing, and analgesic n is an integer from 1 to 3; and activities. Because of the potential therapeutic value of ber the dashed lines represent optional double bonds. Another aspect of the disclosure encompasses a process for bine compounds and derivatives thereof, there is a need for 40 preparing a compound comprising Formula (V). The process new derivatives than may be more potent and/or efficacious. comprises contacting a compound comprising Formula (II) Moreover, there is a need for efficient synthesis processes for with a cyclizing agent to form a compound comprising For mula (IV), and contacting the compound comprising Formula the preparation of pure preparations of specific enantiomers (IV) with a reducing agent to form the compound comprising of these substituted berbines. Formula (V) according to the following reaction scheme:

RI R12 R11

Cyclizing agent US 9, 181,237 B2

-continued

Reducting agent a

wherein: the ring carbons to which they are attached form a ring R is hydrogen, hydrocarbyl, or substituted hydrocarbyl, comprising { }O(CH2)O{-}; R. R. R., and Rindependently are hydrogen, halogen, R. R', R'', R', R', and Rindependently are hydro OR', NR'N', nitro, cyano, thiol, hydrocarbyl, sub gen, hydrocarbyl, or substituted hydrocarbyl; stituted hydrocarbyl, or together RandR along with n is an integer from 1 to 3; and the ring carbons to which they are attached form a ring comprising { }O(CH2)O{-}; 25 the dashed lines represent optional double bonds. R. R. R', and Rindependently are hydrogen, halogen, Other aspects and features of the invention will be in part OR', NRN nitro, cyano, thiol, hydrocarbyl, substi apparent and in part pointed out hereinafter. tuted hydrocarbyl, or together RandR along with the ring carbons to which they are attached form a ring DETAILED DESCRIPTION comprising { }O(CH), O—}; 30 R. R', R'', R', R', and R' independently are hydro The present invention provides new substituted berbine gen, hydrocarbyl, or substituted hydrocarbyl; compounds and processes for preparing Substituted berbines, n is an integer from 1 to 3: as well as intermediate compounds for use in the preparation X is halogen, { }OSOR", or {-}OCOR', wherein 35 of substituted berbines. The processes disclosed herein allow R" is hydrocarbyl or substituted hydrocarbyl; and for regiochemical and stereochemical synthesis of substituted the dashed lines represent optional double bonds. berbines. For example, Syn diastereomers may be prepared A further aspect of the present disclosure provides a using the processes disclosed herein. Furthermore, the pro method for inhibiting growth of a cancer cell. The method cesses disclosed herein are more efficient, more specific, and comprises contacting the cancer cell with an effective amount 40 provide greater yields than currently available synthesis pro of a compound comprising Formula (V): cesses. Additionally, it has been discovered that substituted berbine compounds inhibit cancer cell growth. (V) For ease of discussion, the ring atoms of berbine com

RI pounds are numbered as diagrammed below. 45

wherein: Substituted berbine compounds may have at least two chiral R is hydrogen, hydrocarbyl, or substituted hydrocarbyl, carbons, namely, C-14 and C-8, as indicated above with aster R. R. R. and Rindependently are hydrogen, halogen, 60 isks. OR', NR'N', nitro, cyano, thiol, hydrocarbyl, sub stituted hydrocarbyl, or together RandR along with (I) Compounds the ring carbons to which they are attached form a ring (a) Compounds Comprising Formula (III) comprising { }O(CH2)O{-}; One aspect of the present disclosure encompasses com R. R. R', and Rindependently are hydrogen, halogen, 65 pounds that may be used as intermediates in the preparation of OR', NRN nitro, cyano, thiol, hydrocarbyl, substi Substituted berbine compounds. In general, the intermediate tuted hydrocarbyl, or together RandR along with compounds comprise Formula (III): US 9, 181,237 B2 6 In one alternative of this embodiment, the compound com

(III) prising Formula (III) may be a compound comprising For mula (IIIa):

(IIIa)

15 wherein: R is hydrogen, hydrocarbyl, or substituted hydrocarbyl, R. R. R., and Rindependently are hydrogen, halogen, OR',NR'N', nitro, cyano, thiol, hydrocarbyl, sub wherein: stituted hydrocarbyl, or together RandR along with R is hydrogen, alkyl, Substituted alkyl, heterocyclo, Sub the ring carbons to which they are attached form a ring stituted heterocyclo, aryl, substituted aryl, heteroaryl, comprising { }O(CH), O—}; or substituted heteroaryl; R. R. R. and Rindependently are hydrogen, halogen, R’ is hydroxy oralkyoxy and R is hydrogen, or together OR', NRN nitro, cyano, thiol, hydrocarbyl, substi 25 tuted hydrocarbyl, or together RandR along with R° and R form {-}O CH-O{-} the ring carbons to which they are attached form a ring R and R independently are hydrogen, halogen, comprising { }O(CH2)O{-}; hydroxy, alkoxy, or alkyl; R. R', R'', R', R', and R' independently are hydro R and R' independently hydrogen, halogen, hydroxy, gen, hydrocarbyl, or substituted hydrocarbyl; 30 alkyoxy, alkyl aryloxy, Substituted aryloxy, nitro, n is an integer from 1 to 3: amino, amine, or amide; and X is halogen, { }OSOR', or {-}OCOR', wherein X is halogen, { }OSOR', or {-}OCOR', wherein R" is hydrocarbyl or substituted hydrocarbyl; and R" is hydrocarbyl or substituted hydrocarbyl. In another alternative of this embodiment, the compound the dashed lines represent optional double bonds. 35 comprising Formula (III) may be a compound comprising In some embodiments, R may be hydrogen, alkyl, hetero Formula (IIIb): cylic, aryl, heteroaryl, substituted alkyl, substituted hetero cyclic, substituted aryl, or substituted heteroaryl. In various

iterations, R may be lower alkyl, which is defined herein as (IIIb) C-C, and may be linear or cyclic. In otheriterations, R may 40 be morpholinyl, piperizinyl, phenyl, benzyl, pyridyl, pyridazinyl, pyranyl, oxazinyl, piperonyl, etc. Any of the foregoing may be substituted with at least one alkyl, alkenyl, alkynyl, aryl, halogen, Oxo, keto, hydroxy, acyl, acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, nitro, amino, amine, 45 amide, thiol, cyano, ketal, acetal, ester, or ether. In various embodiments R* and R independently may be hydrogen, halogen, hydroxy, alkyoxy, alkyl or together R and R may form { }O CH, O{ }. In other embodi 50 ments, RandR independently may be hydrogen, halogen, hydroxy, alkoxy, or alkyl. In further embodiments, RandR independently may be hydrogen, halogen, hydroxy, alkoxy, alkyl, aryloxy, Substituted aryloxy, nitro, amino, amine, or wherein: amide. In other embodiments, each of R. R', R'', and R' 55 R is hydrogen, alkyl, Substituted alkyl, heterocyclo, Sub may be hydrogen. In various embodiments, the ring contain stituted heterocyclo, aryl, substituted aryl, heteroaryl, ing the dashed lines may have one, two, or three double or substituted heteroaryl; bonds. In certain embodiments, X may be chloride, bromide, R’ is hydroxy oralkyoxy and R is hydrogen, or together { }OSO-trifluoromethane, { }OSO-methane, or R° and R form {-}O CH-O{-} { }OSO-toluene. The configuration of C-14 may be R or S. 60 R and R independently are hydrogen, halogen, In specific embodiments, R may be heterocyclo, substi hydroxy, alkoxy, or alkyl; tuted heterocyclo, aryl, substituted aryl, heteroaryl, or substi tuted heteroaryl. In some embodiments, R may be phenyl, R and R' independently hydrogen, halogen, hydroxy, substituted phenyl, benzyl, or substituted benzyl. For alkyoxy, alkyl aryloxy, Substituted aryloxy, nitro, example, the substituted phenyl or substituted benzyl may 65 amino, amine, or amide; and have at least one Substituent chosen from halogen, hydroxy, X is halogen, { }OSOR', or {-}OCOR', wherein alkoxy, alkyl, nitro, amino, or amine. R" is hydrocarbyl or substituted hydrocarbyl. US 9, 181,237 B2 7 8 (b) Compounds Comprising Formula (IV) In one alternative of this embodiment, the compound com Another aspect of the present disclosure provides a com prising Formula (IV) may be a compound comprising For pound comprising Formula (IV): mula (IVa):

(IV)

RI (IVa) 10

wherein: R is hydrogen, hydrocarbyl, or substituted hydrocarbyl, R. R. R. and Rindependently are hydrogen, halogen, OR', NR'N', nitro, cyano, thiol, hydrocarbyl, sub wherein: stituted hydrocarbyl, or together RandR along with 25 R is hydrogen, alkyl, Substituted alkyl, heterocyclo, Sub the ring carbons to which they are attached form a ring stituted heterocyclo, aryl, substituted aryl, heteroaryl, comprising { }O(CH2)O{-}; or substituted heteroaryl; R. R. R. and Rindependently are hydrogen, halogen, OR', NRN nitro, cyano, thiol, hydrocarbyl, substi R’ is hydroxy oralkyoxy and R is hydrogen, or together tuted hydrocarbyl, or together Rand R7 along with 30 R° and R form {-}O CH-O{-} the ring carbons to which they are attached form a ring R and R independently are hydrogen, halogen, comprising { }O(CH), O—}; hydroxy, alkoxy, or alkyl, and R. R', R'', R'', R', and R' independently are hydro R and R7 independently hydrogen, halogen, hydroxy, gen, hydrocarbyl, or substituted hydrocarbyl; 35 alkyoxy, alkyl aryloxy, Substituted aryloxy, nitro, n is an integer from 1 to 3; and amino, amine, or amide. the dashed lines represent optional double bonds. In another alternative of this embodiment, the compound In some embodiments, R may be hydrogen, alkyl, hetero comprising Formula (IV) may be a compound comprising cylic, aryl, heteroaryl, substituted alkyl, substituted hetero Formula (IVb): cyclic, substituted aryl, or substituted heteroaryl. In various 40 iterations, R may be lower alkyl, which is defined herein as C-C, and may be linear or cyclic. In otheriterations, R may (IVb) be morpholinyl, piperizinyl, phenyl, benzyl, pyridyl, pyridazinyl, pyranyl, oxazinyl, piperonyl, etc. Any of the foregoing may be substituted with at least one alkyl, alkenyl, 45 alkynyl, aryl, halogen, Oxo, keto, hydroxy, acyl, acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, nitro, amino, amine, amide, thiol, cyano, ketal, acetal, ester, or ether. In various embodiments R and R independently may be hydrogen, halogen, hydroxy, alkyoxy, alkyl or together R 50 and R may form { }O CH, O{ }. In other embodi ments, RandR independently may be hydrogen, halogen, hydroxy, alkoxy, or alkyl. In further embodiments, RandR independently may be hydrogen, halogen, hydroxy, alkoxy, 55 alkyl, aryloxy, Substituted aryloxy, nitro, amino, amine, or wherein: amide. In other embodiments, each of R. R', R', and R' R is hydrogen, alkyl, Substituted alkyl, heterocyclo, Sub may be hydrogen. In various embodiments, the ring contain stituted heterocyclo, aryl, substituted aryl, heteroaryl, ing the dashed lines may have one, two, or three double or substituted heteroaryl; bonds. The configuration of C-14 may be R or S. 60 R’ is hydroxy oralkyoxy and R is hydrogen, or together In specific embodiments, R may be heterocyclo, substi R° and R form {-}O CH-O{-} tuted heterocyclo, aryl, substituted aryl, heteroaryl, or substi tuted heteroaryl. In some embodiments, R may be phenyl, R and R independently are hydrogen, halogen, substituted phenyl, benzyl, or substituted benzyl. For hydroxy, alkoxy, or alkyl, and example, the substituted phenyl or substituted benzyl may 65 R and R7 independently hydrogen, halogen, hydroxy, have at least one Substituent chosen from halogen, hydroxy, alkyoxy, alkyl aryloxy, Substituted aryloxy, nitro, alkoxy, alkyl, nitro, amino, or amine. amino, amine, or amide. US 9, 181,237 B2 9 10 (c) Compounds Comprising Formula (V-1) In some embodiments, the compound comprising Formula Still another aspect of the present disclosure provides a (V-1) may be a compound comprising Formula (V-1a): compound comprising Formula (V-1):

(V-1a) (V-1) R1 10

wherein: 25 R’ is hydroxy oralkyoxy and R is hydrogen, or together wherein: R° and R form {-}O CH-O{-} R. R. R. and Rindependently are hydrogen, halogen, R and R independently are hydrogen, halogen, OR', NR'N', nitro, cyano, thiol, hydrocarbyl, sub hydroxy, alkoxy, or alkyl; stituted hydrocarbyl, or together RandR along with 30 R and R' independently hydrogen, halogen, hydroxy, the ring carbons to which they are attached form a ring alkyoxy, alkyl aryloxy, Substituted aryloxy, nitro, comprising { }O(CH), O—}; amino, amine, or amide; R. R. R. and Rindependently are hydrogen, halogen, R" is hydrogen, halogen, hydroxy, alkoxy, alkyl, nitro, OR',NR'N', nitro, cyano, thiol, hydrocarbyl, sub 35 stituted hydrocarbyl, or together RandR along with amino, or amine; and the ring carbons to which they are attached form a ring m is 0 or 1. comprising { }O(CH2), O—}; provided that at In other embodiments, the compound comprising Formula least two of R. R', and Rare other than methoxy; (V-1) may be a compound comprising Formula (V-1b): R. R', R', and R' independently are hydrogen, 40 hydrocarbyl, or substituted hydrocarbyl, (V-1b) R. R. R. R', and Rindependently are hydrogen, halogen, OR, NR'N', nitro, cyano, thiol, hydro carbyl, or substituted hydrocarbyl; 45 R" and R' independently are hydrogen, hydrocarbyl, or substituted hydrocarbyl, m is an integer of 0 or greater, n is an integer from 1 to 3; and 50 the dashed lines represent optional double bonds. In some instances, RandR independently may be hydro gen, halogen, hydroxy, alkyOxy, alkyl or together R and R' may form { }O CH O{ }. In other embodiments, R 55 wherein: and R independently may be hydrogen, halogen, hydroxy, alkoxy, or alkyl. In further embodiments, RandR indepen R’ is hydroxy oralkyoxy and R is hydrogen, or together dently may be hydrogen, halogen, hydroxy, alkoxy, alkyl, R° and R form {-}O CH-O{-} aryloxy, Substituted aryloxy, nitro, amino, amine, oramide. In R and R independently are hydrogen, halogen, still other embodiments, R,R,R,R, and Rindepen 60 hydroxy, alkoxy, or alkyl; dently may be hydrogen, halogen, hydroxy, alkoxy, alkyl, R and R' independently hydrogen, halogen, hydroxy, nitro, amino, or amine. In additional embodiments, each of alkyoxy, alkyl aryloxy, Substituted aryloxy, nitro, R. R', R', and R' may be hydrogen. In various embodi amino, amine, or amide; ments, the ring containing the dashed lines may have one, R" is hydrogen, halogen, hydroxy, alkoxy, alkyl, nitro, two, or three double bonds. The configuration of each of C-14 65 and C-8 may be R or S. In exemplary embodiments, C-14 and amino, or amine; and C-8 have a syn stereochemistry. m is 0 or 1. US 9, 181,237 B2 11 12 Exemplary compounds comprising Formula (V-1a) are -continued presented below:

Y 10

halogen

OR 15 15 R2

R3 O N SXr Y

halogen 25

Y 30

OR 15 35 R2

R3 O N rSX Y 40

Y 50

NH2 OR 15 55

R3 O N SXr 60 Y

65 US 9, 181,237 B2 13 14

-continued 2 -continued R 21

N 5 R3 SX Y OR 15 O

alkyl, optionally Substituted 10 N N A4 Z

15 wherein: R’ is hydroxy oralkyoxy and R is hydrogen, or together R° and R form {-}O CH-O{-} R'' is hydrogen or C-C alkyl R" is hydrogen or C-C alkyl; and 2O Yand Zindependently are hydrogen, halogen, hydroxy, alkoxy, alkyl, nitro, amino, or amine. Those skilled in the art understand that the phenyl group at alkyl, optionally Substituted C-8 may be replaced with a benzyl group. Moreover, com pounds comprising Formula (V-1 b) have similar exemplary 2s compounds. (II) Processes for Preparing Compounds Comprising For R2 21 mula (V) Another aspect of the present disclosure provides pro N S cesses for the preparation of Substituted berbine compounds. R3 X In general, the process entails formation of a new ring from an asymmetric compound. The process comprises contacting a compound comprising Formula (II) with a cyclizing agent to O forma compound comprising Formula (III), which undergoes cyclization to form the compound comprising Formula (IV). N N The process further comprises contacting the compound H 35 comprising Formula (IV) with a reducing agent to form the OR 15 A 2 berbine compound comprising Formula (V). For the purposes Z of illustration, Reaction Scheme 1 depicts the synthesis of the compound comprising Formula (V) in accordance with this aspect of the disclosure:

Reaction Scheme 1:

R R12 RII R R12 RII

Step A Cyclizing agent US 9, 181,237 B2 15 16

-continued R R12 RII

Step B Reducting agent a

wherein: R. R', R', and R' independently are hydrogen, R is hydrogen, hydrocarbyl, or substituted hydrocarbyl, hydrocarbyl, or substituted hydrocarbyl, R. R. R. and Rindependently are hydrogen, halogen, n is an integer from 1 to 3: OR',NR'N', nitro, cyano, thiol, hydrocarbyl, sub- X is halogen, { }OSOR", or {-}OCOR', wherein stituted hydrocarbyl, or together RandR along with R" is hydrocarbyl or substituted hydrocarbyl; and the ring carbons to which they are attached form a ring the dashed lines represent optional double bonds. comprising { }O(CH2)O{-}; In some embodiments, a compound comprising Formula R. R. R. and Rindependently are hydrogen, halogen, (Va) may be prepared by the process depicted in Reaction OR',NR'N', nitro, cyano, thiol, hydrocarbyl, sub- Scheme 1a:

Reaction Scheme 1a:

R R12 RII

Step A Cyclizing agent Hip

Step B Reducting agent -e-

stituted hydrocarbyl, or together RandR along with wherein the variables are as defined above. 65. In another embodiment, a compound comprising Formula the ring carbons to which they are attached form a ring (Vb) may be prepared by the process depicted in Reaction comprising { }O(CH2)O{-}; Scheme 1b: US 9, 181,237 B2 17 18

Reaction Scheme 1b:

R12 R11

Step A Cyclizing agent

Step B Reducting agent

35 wherein the variables are as defined above. (i) Cyclizing Agent In some embodiments, R may be hydrogen, alkyl, hetero The reaction mixture further comprises a cyclizing agent. cylic, aryl, heteroaryl, substituted alkyl, substituted hetero The cyclizing agent may be a phosphorous oxyhalide or an cyclic, substituted aryl, or substituted heteroaryl. In various acid anhydride. The phosphorous oxyhalide may be phospho iterations, R may be lower alkyl, which is defined herein as 40 rous oxychloride (POCl), phosphorous oxybromide C1-C, and may be linear or cyclic. In otheriterations, R may (POBr.), or phosphorous oxyfluoride (POF). The cyclizing be morpholinyl, piperizinyl, phenyl, benzyl, pyridyl, agent may be an inorganic acid anhydride, for example sulfur pyridaZinyl, pyranyl, oxazinyl, piperonyl, etc. Any of the trioxide, solutions in Sulfuric acid (i.e., fuming sulfuric acid foregoing may be substituted with at least one alkyl, alkenyl, or oleums), phosphorous pentoxide or mixtures of phospho alkynyl, aryl, halogen, oxo, keto, hydroxy, acyl, acyloxy, 45 alkoxy, alkenoxy, alkynoxy, aryloxy, nitro, amino, amine, rous pentoxide in phosphoric acid (i.e., polyphosphoric acid). amide, thiol, cyano, ketal, acetal, ester, or ether. The acid anhydride may also be an alkyl anhydride or an aryl In various embodiments R and R independently may be anhydride. Non-limiting examples of suitable acid anhy hydrogen, halogen, hydroxy, alkyoxy, alkyl or together R drides include trifluoromethanesulfonic anhydride, methane and R may form { }O CH, O{ }. In other embodi 50 Sulfonic anhydride, p-toluenesulfonic anhydride, trifluoro ments, RandR independently may be hydrogen, halogen, acetic anhydride, acetic anhydride, acetic formic anhydride, hydroxy, alkoxy, or alkyl. In further embodiments, Rand R7 benzoic anhydride, butyric anhydride, chlorophthalic anhy independently may be hydrogen, halogen, hydroxy, alkoxy, dride, cyclopropylcarboxylic anhydride, cyclobutylcarboxy alkyl, aryloxy, substituted aryloxy, nitro, amino, amine, or lic anhydride, ethylenetetracarboxylic anhydride, formic amide. In other embodiments, each of R. R', R', and R' 55 anhydride, 2-furonic anhydride, gloxylic anhydride, maleic may be hydrogen. In various embodiments, the ring contain anhydride, malonic anhydride, methacrylic anhydride, nico ing the dashed lines may have one, two, or three double tinic anhydride, oxalic anhydride, phthalic anhydride, propi bonds. In certain embodiments, X may be chloride, bromide, onic anhydride, succinic anhydride, toluic anhydride, and {-}OSO-trifluoromethane, { }OSO-methane, or combinations thereof. In one embodiment, the acid anhydride {-}OSO-toluene. The configuration of C-14 may be R or S. 60 may be trifluoromethanesulfonic anhydride. (a) Step A Reaction Mixture The amount of the cyclizing agent added to the reaction Step A of the process comprises contacting a compound mixture can and will vary. In general, the mole to mole ratio comprising Formula (II) with a cyclizing agent to form a of the compound comprising Formula (II) to the cyclizing compound comprising Formula (IV). This step of the process agent may range from about 1:0.5 to about 1:3. In various commences with formation of a reaction mixture. The reac 65 embodiments, the mole to mole ratio of the compound com tion mixture comprises a compound comprising Formula (II), prising Formula (II) to the cyclizing agent may range about as detailed above. 1:0.5 to about 1:1, from about 1:1 to about 1:1.5, from about US 9, 181,237 B2 19 20 1:1.5 to about 1:2, from about 1:2 to about 1:2.5, or from about 12 hours to about 18 hours, or from about 18 hours to about 1:2.5 to about 1:3. In exemplary embodiments, the about 24 hours. In exemplary embodiments, the reaction may mole to mole ratio of the compound comprising Formula (II) be allowed to proceed for about 10 hours to about 20 hours. to the cyclizing agent may be from about 1:1 to about 1:2. In general, the compound comprising Formula (IV) is not (ii) Solvent isolated from the reaction mixture. Accordingly, step (b) of The reaction mixture generally further comprises a solvent. the process may proceed in the same reaction pot or reactor. In The solvent may be an aprotic polar solvent, a protic polar Some embodiments, however, the compound comprising For Solvent, a non-polar solvent, or combinations thereof. Suit mula (IV) may be isolated from the reaction mixture using able aprotic solvents include, without limit, acetonitrile, techniques known to those of skill in the art. Non-limiting diethoxymethane, N,N-dimethylformamide (DMF), dim 10 examples of suitable techniques include precipitation, extrac ethyl sulfoxide (DMSO), N,N-dimethylpropionamide, 1,3- dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), tion, evaporation, distillation, chromatography, and crystalli 1,3-dimethyl-2-imidazolidinone (DMI), 1,2-dimethoxy Zation. ethane (DME), dimethoxymethane, bis(2-methoxyethyl) The yield of the compound comprising Formula (IV) can ether, N,N-dimethylacetamide (DMAC), N-methyl-2-pyrro and will vary. Typically, the yield of the compound compris lidinone (NMP), 1,4-dioxane, ethyl acetate, 15 ing Formula (IV) will be at least about 40% by weight. In hexamethylphosphoramide, methyl acetate, methylene chlo certain embodiments, the yield of the compound comprising ride, methoxyethane, nitrobenzene, nitromethane, propioni Formula (IV) may be at least about 50%, at least about 60%, trile, propyl acetates, sulfolane, tetrahydrofuran (THF), at least about 70%, at least about 80%, at least about 90%, or 2-methyl tetrahydrofuran, tetrahydropyran, trichlo at least about 95%. romethane, and combinations thereof. Non-limiting (c) Step B Reaction Mixture examples of suitable protic polar solvents include diols such Step B of the process comprises contacting the compound as propylene glycol, ethylene glycol, propanediol, and so comprising Formula (IV) with a reducing agent to form the forth; amides such as acetamide, benzamide, and the like; and compound comprising Formula (V). combinations of any of the above. Non-limiting examples of A variety of reducing agents may be used in this step of the Suitable nonpolar solvents include benzene, butyl acetate, 25 process. The reducing agent may be chiral or achiral. Non tert-butyl methyl ether, chlorobenzene, chloroform, chlo limiting examples of Suitable reducing agents for use in romethane, cyclohexane, dichloromethane, dichloroethane, chemical reduction include hydrides (e.g., Sodium borohy di-tert-butyl ether, dimethyl ether, diethylene glycol, diethyl dride, Sodium cyanoborohydride, lithium aluminum hydride, ether, diglyme, diisopropyl ether, ethyl tert-butyl ether, eth disobutylaluminum hydride, hydrogen iodide, hydrogen Sul ylene oxide, fluorobenzene, heptane, hexane, methyl tert fide, and the like), phosphites, hypophosphites, Sulfites, and butyl ether, toluene, and combinations thereof. In exemplary 30 combinations of a metal (e.g., tin, Zinc, or iron) or a metal embodiments, the solvent may be acetonitrile. compound (e.g., chromium chloride, chromium acetate, and In general, the volume to mass ratio of the solvent to the the like) with an organic or inorganic acid (e.g., formic acid, compound comprising Formula (II) ranges from about 0.5:1 acetic acid, propionic acid, trifluoroacetic acid, p-toluene to about 100:1. In various embodiments, the volume to mass Sulfonic acid, hydrochloric acid, and the like). In exemplary ratio of the solvent to the compound comprising Formula (II) 35 embodiments, the reducing agent may be sodium borohy may range from 0.5:1 to about 5:1, from about 5:1 to about dride or sodium cyanoborohydride. 25:1, or from about 25:1 to about 100:1. In exemplary The amount of reducing agent used in this step of the embodiments, the volume to mass ratio of the solvent to the process can and will vary. In general, the mole to mole ratio of compound comprising Formula (II) may range from about the compound comprising Formula (II) to the reducing agent 5:1 to about 20:1. 40 may range from about 1:0.5 to about 1:3. In various embodi (b) Step A Reaction Conditions ments, the mole to mole ratio of the compound comprising In general, the reaction is conducted at a temperature that Formula (II) to the reducing agent may range about 1:0.5 to ranges from about 0°C. to about 120°C. In various embodi about 1:1, from about 1:1 to about 1:1.5, from about 1:1.5 to ments, the reaction may be conducted at a temperature from about 1:2, from about 1:2 to about 1:2.5, or from about 1:2.5 about 0°C. to about 20°C., from about 20°C. to about 40°C., 45 to about 1:3. In exemplary embodiments, the mole to mole from about 40°C. to about 60°C., from about 60° C. to about ratio of the compound comprising Formula (II) to the reduc 80°C., from about 80°C. to about 100° C., or from about 100° ing agent may be from about 1:1 to about 1:2. C. to about 120° C. The reaction may be conducted at a first The reduction reaction generally is conducted in the pres temperature and then a second temperature. In exemplary ence of a solvent. Suitable solvents and ratios of solvent to the embodiments, the temperature of the reaction may range from 50 starting substrate are listed above in section (I)(a)(ii). The about 20° C. to about 60° C. The reaction generally is per solvent may be the same as the solvent used in step A of the formed under ambient pressure. process. For example, the solvent may be carried over from Typically, the reaction is allowed to proceed for a sufficient step A and/or additional solvent may be added to the reaction period of time until the reaction is complete, as determined by mixture prior to step B of the process. Alternatively, the chromatography (e.g., HPLC) or another suitable method. In 55 solvent used during step B of the process may be different this context, a "completed reaction' generally means that the from that used in step A of the process. In one embodiment, reaction mixture contains a significantly diminished amount the solvent used during step B may be acetonitrile. In another of the compound comprising Formula (II), and a significantly embodiment, the solvent used during step B may be methanol increased amount of the compound comprising Formula (IV) compared to the amounts of each present at the beginning of or a mixture of methanol and water. the reaction. Typically, the amount of the compound compris 60 (d) Step B Reaction Conditions ing Formula (II) remaining in the reaction mixture after the The temperature at which the reduction reaction is per reaction is complete may be less than about 3%, or less than formed may vary. In general, the temperature of the reaction about 1%. In general, the reaction may proceed for about 2 ranges from about 0°C. to about 120°C. In various embodi hours to about 24 hours. In certain embodiments, the reaction ments, the reaction may be conducted at a temperature from may be allowed to proceed for about a period of time ranging 65 about 0°C. to about 20°C., from about 20°C. to about 40°C., from about 2 hours to about 4 hours, from about 4 hours to from about 40°C. to about 60°C., from about 60° C. to about about 8 hours, from about 8 hours to about 12 hours, from 80°C., from about 80°C. to about 100°C., or from about 100° US 9, 181,237 B2 21 22 C. to about 120°C. In exemplary embodiments, the tempera ments, the yield of the compound comprising Formula (V) ture of the reaction may range from about 20°C. to about 60° may be at least about 60%, at least about 70%, at least about C. For example, the reaction may be conducted at room 80%, at least about 90%, or at least about 95%. temperature. The reaction generally is performed under ambi Each chiral carbon in the compounds described above may ent pressure. have an R configuration oran S configuration. That is, C-14 in Typically, the reaction is allowed to proceed for a sufficient the compounds comprising Formulas (II), (III), and (IV) may period of time until the reaction is complete, as detailed have an R oran S configuration. The configuration of C-8 and above. In a completed reaction, the amount of the compound C-14 in the compound comprising Formula (V) may be RR, comprising Formula (IV) remaining in the reaction mixture RS, SR, or SS. In particular embodiments, positions C-8 and may be less than about 3%, or less than about 1%. In general, 10 C-14 of the compound comprising Formula (V) have a syn the reaction may proceed for about 0.5 hour to about 72 hours. Stereochemistry. In some embodiments, the reaction may proceed for about 0.5 (e) Optional Additional Steps hour to about 4 hours, from about 4 hours to about 12 hours, Upon formation of the compound comprising Formula (V). from about 12 hours to about 24 hours, from about 24 hours to the compound comprising Formula (V) may undergo addi about 48 hours, or from about 48 hours to about 72 hours. 15 tional reactions. For example, the R (or R) group may be The compound comprising Formula (V) may be isolated converted to an ether, an amine, or an amide. from the reaction mixture using techniques known to those of (i) Synthesis of Ethers or Amines skill in the art. Non-limiting examples of Suitable techniques In embodiments in which R” of the compound comprising include precipitation, extraction, evaporation, distillation, Formula (V) is halogen, the compound comprising Formula chromatography, and crystallization. (V) may be contacted with ROH or R'NH2 to form a Typically, the yield of the compound comprising Formula compound comprising Formula (VIa) or Formula (VIb), (V) will be at least about 40% by weight. In certain embodi respectively, as shown in Reaction Scheme 2 below:

Reaction Scheme 2:

US 9, 181,237 B2 23 24 wherein: ruthenium black, palladium black, etc. In still other embodi R is hydrogen, hydrocarbyl, or substituted hydrocarbyl, ments, the transition metal element may be immobilized on a R. R. R. and R independently are hydrogen, OR', solid surface or support. Suitable examples include, but are NR'N', nitro, cyano, thiol, hydrocarbyl, substituted not limited to, ruthenium on carbon, rhodium on carbon, hydrocarbyl, or together RandR along with the ring palladium on carbon, ruthenium on alumina, rhodium on carbons to which they are attached form a ring com alumina, platinum on alumina, palladium on alumina, prising { }O(CH), O—}: rhodium on silica, palladium on silica, palladium on charcoal, R. R. and R independently are hydrogen, OR', palladium on pumice, and so forth. In exemplary embodi NR'N', nitro, cyano, thiol, hydrocarbyl, or substi ments, the transition metal catalyst may be palladium Sup tuted hydrocarbyl; 10 ported on carbon. R. R', R', and R' independently are hydrogen, In other embodiments, the transition metal catalyst may be hydrocarbyl, or substituted hydrocarbyl, a transition metal salt. Non-limiting examples of suitable salts R" is hydrocarbyl or substituted hydrocarbyl: include acetates, acetyacetonates, alkoxides, butyrates, car n is an integer from 1 to 3; and bonyls, dioxides, halides, hexonates, hydrides, mesylates, the dashed lines represent optional double bonds. 15 octanates, nitrates, nitrosyl halides, nitrosyl nitrates, Sulfates, A similar reaction may be used to generate compounds in Sulfides, Sulfonates, phosphates, trifluoromethanesulfonates, which R is the ether or amine. In such embodiments, R in trimethylacetates, tosylates, and combinations thereof. The the compound comprising Formula (V) is halogen and R is transition metal salt may be soluble (i.e., homogeneous). hydrogen, OR, NR'N', nitro, cyano, thiol, hydrocarbyl, Alternatively, the transition metal salt may be immobilized on or substituted hydrocarbyl. a solid Support (i.e., heterogeneous). The transition metal salt Reaction mixture. The reaction commences with the for may be immobilized on the solid Support via noncovalent or mation of a reaction mixture comprising the compound com covalent bonds. In some embodiments, the solid Support may prising (V) in which R" is halogen (e.g., chloro, bromo, or be an inorganic material. Suitable inorganic materials include iodo) and an alcohol (i.e., R'OH) oranamine (i.e., R'NH2). silicas, alumina, titania, carbondium, Zirconia, activated char In some embodiments, R' may be alkyl, cycloalkyl, alkenyl, 25 coal, Zeolites, clays, polymers, ceramics, and activated car alkynyl, aryl, heteroaryl, substituted alkyl, substituted bon. Suitable silicas include silicon dioxide, amorphous cycloalkyl, substituted alkenyl, substituted alkynyl, substi silica, and microporous or mesoporous silicas. In other tuted aryl, or substituted heteroaryl. In other embodiments, embodiments, the Solid Support may be a polymer. The poly R" may be C-C alkyl, which may be substituted, linear or mer may be a natural polymer, a synthetic polymer, a semi cyclic. In still other embodiments, R' may be aryl or aryl 30 synthetic polymer, or a copolymer. Non-limiting examples of Substituted with halo, nitro, hydroxyl, keto or oxo, C-C, polymers include agarose, cellulose, nitrocellulose, methyl alkyl, C-C alkoxy, or C-C alkenyl. Suitable aryl groups cellulose, polyacrylic, polyacrylamide, polyacrylonitrile, include phenyl, benzyl, pyridyl, pyrimidyl, pyrrolyl, and imi polyamide, polyether, polyester, polyethylene, polystyrene, dazolyl. polysulfone, polyvinyl chloride, polyvinylidene, methacry The amount of ROH or R'NH, added to the reaction 35 late copolymer, and polystyrene-vinyl chloride copolymer. mixture can and will vary. In general, the mole to mole ratio In further embodiments, the transition metal catalyst may of the compound comprising Formula (V) to R'OH or be a transition metal complex. In general, a transition metal R''NH may range from about 1:0.5 to about 1:20. In various complex comprises the transition metal and 4, 5, or 6 coordi embodiments, the mole to mole ratio of the compound com nate species with oxidation states ranging from 0 to 8. The prising Formula (II) to ROH or R'NHagent may range 40 complexes may be ionic, or the complexes may comprise about 1:0.5 to about 1:1, from about 1:1 to about 1:2, from covalently bound ligands and counter ions. Alternatively, the about 1:2 to about 1:5, from about 1:5 to about 1:10, or from complexes may comprise a mixture of ionic and covalent about 1:10 to about 1:20. In exemplary embodiments, the bonds between the metal, ligand(s), and/or counter ion(s). mole to mole ratio of the compound comprising Formula (II) The ligand may be monodentate or polydentate. Non-limiting to ROH or R'NH may be from about 1:1 to about 1:5. 45 examples of Suitable ligands include arene ligands, olefin The reaction mixture further comprises a transition metal ligands, alkyne ligands, heterocycloalkyl ligands, heteroaryl catalyst. As used herein, the term “transition metal catalyst” ligands, alkyl ligands, cyclopentadienyl ligands, hydride refers to a transition metal element, transition metal salt, or a ligands, amine ligands, carbonyl ligands, nitrogen donor transition metal complex. In general, the transition metal may ligands, phosphorous donor ligands, oxygen donor ligands, be any transition metal. In some embodiments, the transition 50 and so forth. The ligand may also be a solvent such as, e.g., metal may be iridium, iron, nickel, osmium, palladium, plati DMSO, methanol, methylene chloride, tetrahydrofuran, num, ruthenium and rhodium. In one exemplary embodiment, acetone, ethanol, pyridine, or a tetraalkylammonia com the transition metal may be ruthenium, iridium, or rhodium. A pound. Suitable counter ions include, but are not limited to, skilled artisan appreciates that the oxidation state of transition halides, BF, PF, CIO, CHO, CFSO, CHCO, ArCO, metal may vary, and may be, for example, (0), (I), (II), (III), 55 CHSO. p-tolylSO, HSO, HPO, and hydrocarbylanions. (IV), (V), (VI) or (VII). For example, non-limiting examples Numerous transition metal complexes are detailed in “Trans of suitable transition metals include ruthenium(0), ruthenium position of Allylic Alcohols into Carbonyl Compounds Medi (II), ruthenium(III), ruthenium(IV), rhodium(0), rhodium(I), ated by Transition Metal Complexes” by Uma et al., Chem. rhodium(III), iridium(0), iridium(III), iridium(IV), palla Rev. 103: 27-51 (2003). dium(0), palladium(II), palladium(IV), platinum(0), platinu 60 In exemplary embodiments, the transition metal catalyst m(II), platinum(IV), and nickel(O). may comprise palladium. Non-limiting examples of palla In Some embodiments, the transition metal catalyst may be dium catalysts include Pd(acac), Pd(allyl)Cl. the transition metal element itself. For example, the transition Pd(MeCN),C1, Pd(dba), Pd(TFA), Pd(dba), CHC1, metal element may be a powder or a sponge, Such as, e.g., Pd(PPh), Pd(OAc), Pd(PCy)C1, Pd(PPh)C1, Pd(P(o- ruthenium powder, rhodium powder, ruthenium sponge, 65 tol)C1, Pd(amphos)Cl. Pd(dppf)Cl. Pd(dtpf)Cl. rhodium sponge, palladium sponge, and so forth. Alterna Pd(MeCN)(BF), PdBr, PdCl (SPhos) Pd(II) phenethy tively, the transition metal element may be rhodium black, lamine chloride, (XPhos) Pd(II) phenethylamine chloride, US 9, 181,237 B2 25 26 (RuPhos) Pd(II) phenethylamine chloride, (t-BuxPhos) Typically, the reaction is allowed to proceed for a sufficient Pd(II) phenethylamine chloride, and (BrettPhos) Pd(II) phen period of time until the reaction is complete, as detailed ethylamine chloride. above. In a completed reaction, the amount of the compound The amount of transition metal catalyst added to the reac comprising Formula (V) remaining in the reaction mixture tion mixture can and will vary. In general, the amount of 5 may be less than about 3%, or less than about 1%. In general, transition metal catalyst added to the reaction mixture may the reaction may proceed for about 1 hour to about 72 hours. range from about 0.005% to about 10% by weight. In various In some embodiments, the reaction may proceed for about 1 embodiments, the amount of transition metal catalyst added hour to about 4 hours, from about 4 hours to about 12 hours, to the reaction mixture may range from about 0.005% to from about 12 hours to about 24 hours, from about 24 hours to about 0.05%, from about 0.05% to about 0.5%, from about 10 0.5% to about 2%, or from about 2% to about 10% by weight. about 48 hours, or from about 48 hours to about 72 hours. In certain embodiments, the amount of transition metal cata The compound comprising Formula (VIa) or (VIb) may be lyst added to the reaction mixture may range from about isolated from the reaction mixture using techniques known to 0.01% to about 1% by weight. those of skill in the art. Non-limiting examples of suitable The reaction mixture further comprises a proton acceptor. 15 techniques include precipitation, extraction, evaporation, dis Suitable proton acceptors include borate salts (such as, for tillation, chromatography, and crystallization. example, NaBOs), di- and tri-basic phosphate salts (such as, Typically, the yield of the compound comprising Formula for example, NaHPO and NaPO, and the like), bicarbon (VIa) or (VIb) will be at least about 40% by weight. In certain ate salts (such as, for example, NaHCO, KHCO, LiHCO embodiments, the yield of the compound comprising For and so forth), carbonate salts (such as, for example, Na2CO, mula (VIa) or (VIb) may be at least about 60%, at least about KCO, LiCO, and the like), butoxides (such as, e.g., 70%, at least about 80%, at least about 90%, or at least about Sodium tert-butoxide, potassium tert-butoxide), organic 95%. bases (such as, for example, pyridine, triethylamine, diiso (ii) Synthesis of Amides propylethylamine, N-methylmorpholine, N,N-dimethylami In embodiments in which R” of the compound comprising nopyridine), and mixtures thereof. In exemplary embodi 25 Formula (V) is NH, the compound comprising Formula (V) ments, the proton acceptor may be sodium tert-butoxide, may be contacted R'C(O)X" to form a compound comprising KCOs, or triethylamine. Formula (VIc) according to Reaction Scheme 3: The amount of proton acceptor added to the reaction mix ture may vary. In general, the mole to mole ratio of the compound comprising Formula (V) to the proton acceptor 30 Reaction Scheme 3: may range from about 1:0.5 to about 1:10. In various embodi ments, the mole to mole ratio of the compound comprising RI R12 R11 Formula (V) to the proton acceptor may range from about 1:0.5 to about 1:2, from about 1:2 to about 1:5, or from about 1:5 to about 1:10. In exemplary embodiments, the mole to 35 mole ratio of the compound comprising Formula (V) to the proton acceptor may range from about 1:1 to about 1:4. -e- The reaction mixture also comprises a solvent. Suitable Solvents include aprotic polar solvents, non-polar solvents, or combinations thereof. Examples of aprotic polar solvents and 40 non-polar solvents are presented above in section (II)(a)(ii). In exemplary embodiments, the solvent may be toluene, tet rahydrofuran (THF), N,N-dimethylformamide (DMF), or, N,N-dimethylacetamide (DMAC), In general, the Volume to mass ratio of the solvent to the 45 compound comprising Formula (V) may range from about 0.5:1 to about 100:1. In various embodiments, the volume to mass ratio of the solvent to the compound comprising For mula (V) may range from 0.5:1 to about 5:1, from about 5:1 to about 25:1, or from about 25:1 to about 100:1. In exem 50 plary embodiments, the volume to mass ratio of the solvent to the compound comprising Formula (V) may range from about 5:1 to about 20:1. Reaction conditions. The temperature at which the reaction is conducted may vary depending upon the identity of the 55 Solvent and the nature of the Substituents on the compound comprising Formula (V). In general, the temperature of the reaction may range from about 0° C. to about 200° C. In wherein: various embodiments, the reaction may be conducted at a R is hydrogen, hydrocarbyl, or substituted hydrocarbyl, temperature from about 0°C. to about 20°C., from about 20° 60 R. R. R. and Rindependently are hydrogen, halogen, C. to about 40°C., from about 40° C. to about 60° C., from OR', amine, nitro, cyano, thiol, hydrocarbyl, substi about 60° C. to about 80°C., from about 80°C. to about 100° tuted hydrocarbyl, or together RandR along with C., from about 100° C. to about 120° C., from about 120° C. the ring carbons to which they are attached form a ring to about 150° C., or from about 150° C. to about 200° C. In comprising { }O(CH2)O{-}; specific embodiments, the reaction may be conducted at a 65 R. R. and R independently are hydrogen, halogen, temperature ranging from room temperature to reflux. The OR', amine, nitro, cyano, thiol, hydrocarbyl, or sub reaction generally is performed under ambient pressure. stituted hydrocarbyl: US 9, 181,237 B2 27 28 R. R', R'', R', and R' independently are hydrogen, The amount of proton acceptor added to the reaction mix hydrocarbyl, or substituted hydrocarbyl, ture may vary. In general, the mole to mole ratio of the R" is hydrocarbyl or substituted hydrocarbyl: compound comprising Formula (V) to the proton acceptor X" is halogen; ranges from about 1:0.5 to about 1:10. In various embodi n is an integer from 1 to 3; and ments, the mole to mole ratio of the compound comprising the dashed lines represent optional double bonds. Formula (V) to the proton acceptor may range from about In other embodiments, R” in the compound comprising 1:0.5 to about 1:2, from about 1:2 to about 1:5, or from about 1:5 to about 1:10. In exemplary embodiments, the mole to Formula (V) may be NO, which can be reduced to NH by mole ratio of the compound comprising Formula (V) to the contact with a hydrogen source (e.g., gaseous hydrogen and a 10 proton acceptor may range from about 1:1 to about 1:4. Suitable catalyst, e.g., palladium on carbon). The reaction mixture also comprises a solvent. Suitable A similar reaction may be used to generate compound in Solvents include aprotic polar solvents, non-polar solvents, or which R is the amide. In such embodiments, R in the com combinations thereof. Examples of aprotic polar solvents and pound comprising Formula (V) is NH (or NO) and R7 is 15 non-polar solvents are presented above in section (II)(a)(ii). hydrogen, halogen, OR', amine, cyano, thiol, hydrocarbyl, In exemplary embodiments, the solvent may be tetrahydro or substituted hydrocarbyl. furan, acetonitrile, dichloromethane, or chloroform. Reaction mixture. The reaction commences with the for In general, the Volume to mass ratio of the Solvent to the mation of a reaction mixture comprising the compound com compound comprising Formula (V) may range from about prising (V) in which R" is NH and an acyl halide (e.g., 0.5:1 to about 100:1. In various embodiments, the volume to RC(O)x"). In some embodiments, R' may be alkyl, mass ratio of the solvent to the compound comprising For cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, Substituted mula (V) may range from 0.5:1 to about 5:1, from about 5:1 alkyl, substituted cycloalkyl, substituted alkenyl, substituted to about 25:1, or from about 25:1 to about 100:1. In exem alkynyl, substituted aryl, or substituted heteroaryl. In certain 25 plary embodiments, the volume to mass ratio of the solvent to embodiments, R' may be C-C alkyl, which may be substi the compound comprising Formula (V) may range from tuted, linear orcyclic. In other embodiments, R' may be aryl about 5:1 to about 20:1. or aryl substituted with halo, nitro, hydroxyl, keto or oxo, Reaction conditions. The temperature at which the reaction C-C alkyl, C-C alkyOXy, or C-C alkenyl. Suitable aryl is conducted can and will may. In general, the temperature of groups include phenyl, benzyl, pyridyl, pyrimidyl, pyrrolyl, 30 the reaction may range from about -50° C. to about 50° C. In and imidazolyl. various embodiments, the temperature of the reaction may A variety of acylhalides are suitable for use in this reaction. range from about -500° C. to about -20°C., from about -20° Suitable acylhalides include, without limit, alkyl acyl halides C. to about 0°C., from about 0°C. to about 20° C., or from (such as, e.g., formyl halide, acetyl halide, propionylhalide, 35 about 20° C. to about 50° C. In specific embodiments, the butyryl halide, hexanoyl halide, cyclopentane carbonyl reaction may be conducted at room temperature. The reaction halide, and the like) and aryl acyl halides (such as, e.g., generally is performed under ambient pressure. benzoyl halide, phenyl acetyl halide, phenyl haloformate, Typically, the reaction is allowed to proceed for a sufficient toluoyl halide, toluenesulfonylhalide, 2-furoyl halide, nico period of time until the reaction is complete, as detailed 40 above. In a completed reaction, the amount of the compound tinoyl halide, piperonyloyl halide, and so forth). comprising Formula (V) remaining in the reaction mixture The amount of acyl halide utilized in the reaction can and may be less than about 3%, or less than about 1%. In general, will vary. In general, the mole to mole ratio of the compound the reaction may proceed for about 1 hour to about 72 hours. comprising Formula (V) to the acyl halide may range from In some embodiments, the reaction may proceed for about 1 about 1:0.8 to about 1:2. In various embodiments, the mole to 45 hour to about 4 hours, from about 4 hours to about 12 hours, mole ratio of the compound comprising Formula (V) to the from about 12 hours to about 24 hours, from about 24 hours to acyl halide may range from about 1:0.8 to about 1:1.0, from about 48 hours, or from about 48 hours to about 72 hours. about 1:1.0 to about 1:1.2, from about 1:1.2 to about 1:14, The compound comprising Formula (VIc) may be isolated from about 1:1.4 to about 1:1.8, or from about 1:1.8 to about from the reaction mixture using techniques known to those of 50 skill in the art. Non-limiting examples of Suitable techniques 1:2. In exemplary embodiments, the mole to moleratio of the include precipitation, extraction, evaporation, distillation, compound comprising Formula (V) to the acyl halide may chromatography, and crystallization. range from about 1:1.0 to about 1:1.2. Typically, the yield of the compound comprising Formula The reaction mixture further comprises a proton acceptor. (VIc) will be at least about 40% by weight. In certain embodi Suitable proton acceptors include borate salts (such as, for 55 ments, the yield of the compound comprising Formula (VIc) example, NaBO), di- and tri-basic phosphate salts (such as, may be at least about 60%, at least about 70%, at least about for example, NaHPO and NaPO, and the like), bicarbon 80%, at least about 90%, or at least about 95%. ate salts (such as, for example, NaHCO, KHCO, LiHCO (III) Process for Preparing a Compound Comprising Formula and so forth), carbonate salts (such as, for example, Na2CO, (II) 60 A further aspect of the present disclosure encompasses a KCO, LiCO, and the like), butoxides (such as, e.g., process for preparing a compound comprising Formula (II). Sodium tert-butoxide, potassium tert-butoxide), organic The process comprises contacting a compound comprising bases (such as, for example, pyridine, triethylamine, diiso Formula (I) with a carbonyl donor for preparing a compound propylethylamine, N-methylmorpholine, N,N-dimethylami of Formula (V) may comprise contacting a compound of nopyridine), and mixtures thereof. In exemplary embodi 65 Formula (I) with a carbonyl donor to form the compound ments, the proton acceptor may be triethylamine, comprising Formula (II), according to Reaction Scheme 4 diisopropylethylamine, or N-methylmorpholine. below: US 9, 181,237 B2 29 30 ments, each of R. R', R', and R' may be hydrogen. In Reaction Scheme 4: various embodiments, the ring containing the dashed lines may have one, two, or three double bonds. The configuration R1 R12 R11 of C-14 may be R or S. (a) Reaction Mixture The process commences with formation of a reaction mix ture. The reaction mixture comprises the compound compris Carbonyl donor ing Formula (I) as detailed above. (i) Carbonyl Donor 10 The reaction mixture further comprises a carbonyl donor. A variety of carbonyl donors are suitable for use in this process. In some embodiments, the carbonyl donor may be an acyl halide, i.e., RC(O)x, wherein R' is hydrocarbylor substituted hydrocarbyl and X is halogen (and wherein R' is transferred 15 along with the carbonyl to the compound comprising Formula

(II)). In other embodiments, the carbonyl donor may be a formate, i.e., R"OC(O)H, wherein R" is hydrocarbyl or sub stituted hydrocarbyl (and wherein H is transferred along with the carbonyl to the compound comprising Formula (II)). In further embodiments, the carbonyl donor may be an alde hyde, i.e., R'CHO, wherein R is hydrocarbyl or substituted hydrocarbyl (and wherein R' is transferred along with the carbonyl to the compound comprising Formula (II)). In some embodiments the carbonyl donor may be an acyl 25 halide. Non-limiting examples of suitable acyl halides include alkyl acyl halides (such as, e.g., formyl halide, acetyl halide, propionyl halide, butyryl halide, hexanoyl halide, cyclopentane carbonyl halide, and the like) and aryl acyl halides (such as, e.g., benzoyl halide, Substituted benzoyl 30 halide, phenyl acetyl halide, phenyl haloformate, toluoyl halide, toluenesulfonyl halide, 2-furoyl halide, nicotinoyl wherein: halide, piperonyloyl halide, and so forth). The amount of acyl R is hydrogen, hydrocarbyl, or substituted hydrocarbyl, halide added to the reaction mixture may vary. In general, the R. R. R. and Rindependently are hydrogen, halogen, mole to mole ratio of the compound comprising Formula (I) OR', NR'N', nitro, cyano, thiol, hydrocarbyl, sub 35 to the acyl halide may range from about 1:0.5 to about 1:4. In stituted hydrocarbyl, or together RandR along with various embodiments, the mole to mole ratio of the com the ring carbons to which they are attached form a ring pound comprising Formula (I) to the acyl halide may range comprising { }O(CH2)O{-}; from about 1:0.1 to about 1:1, from about 1:1 to about 1:2, R. R. R', and Rindependently are hydrogen, halogen, from about 1:2 to about 1:3, or from about 1:3 to about 1:4. In OR', NR'N', nitro, cyano, thiol, hydrocarbyl, sub 40 exemplary embodiments, the mole to mole ratio of the com stituted hydrocarbyl, or together RandR along with pound comprising Formula (I) to the acyl halide may range the ring carbons to which they are attached form a ring from about 1:1 to about 1:2. comprising { }O(CH2)O{-}; In other embodiments, the carbonyl donor may be a for R. R', R'', R', R', R', and R' independently are mate. Non-limiting examples of Suitable aldehydes include hydrogen, hydrocarbyl, or substituted hydrocarbyl: 45 methyl formate, ethyl formate, propyl formate, butyl formate, n is an integer from 1 to 3; and pentyl formate, hexyl formate, phenyl formate, benzyl for the dashed lines represent optional double bonds. mate, and so forth. The amount of formate contacted with the In some embodiments, R may be hydrogen, alkyl, hetero compound comprising Formula (I) can and will vary. In gen cylic, aryl, heteroaryl, substituted alkyl, substituted hetero eral, the mole to mole ratio of the compound comprising cyclic, substituted aryl, or substituted heteroaryl. In various 50 Formula (I) to the formate may range from about 1:5 to about iterations, R may be lower alkyl, which is defined herein as 1:50. In certain embodiments, the mole to mole ratio of the C-C, and may be linear or cyclic. In otheriterations, R may compound comprising Formula (I) to the formate may range be morpholinyl, piperizinyl, phenyl, benzyl, pyridyl, from about 1:5 to about 1:10, from about 1:10 to about 1:30, pyridazinyl, pyranyl, oxazinyl, piperonyl, etc. Any of the or from about 1:30 to about 1:50. In exemplary embodiments, foregoing may be substituted with at least one alkyl, alkenyl, 55 the mole to mole ratio of the compound comprising Formula alkynyl, aryl, halogen, Oxo, keto, hydroxy, acyl, acyloxy, (I) to the formate may range from about 1:10 to about 1:30. alkoxy, alkenoxy, alkynoxy, aryloxy, nitro, amino, amine, In additional embodiments, the carbonyl donor may be an amide, thiol, cyano, ketal, acetal, ester, or ether. aldehyde. Non-limiting examples of suitable aldehydes In various embodiments R* and R independently may be include formaldehyde, acetaldehyde, propionaldehyde, hydrogen, halogen, hydroxy, alkyoxy, alkyl or together R 60 butyraldehyde, cyclopropane carboxaldehyde, cyclobutane and R may form { }O CH, O{ }. In other embodi carboxaldehyde, benzaldehyde, glyoxal, glyoxylic acid, ments, RandR independently may be hydrogen, halogen, 2-furaldehyde, nicotinaldehyde, and so forth. The amount of hydroxy, alkoxy, or alkyl. In further embodiments, RandR aldehyde added to the reaction mixture may vary. In general, independently may be hydrogen, halogen, hydroxy, alkoxy, the mole to mole ratio of the compound comprising Formula alkyl, aryloxy, Substituted aryloxy, nitro, amino, amine, or 65 (I) to the aldehyde may range from about 1:0.2 to about 1:4. amide. In some embodiments, R' may be hydrogen, alkyl, In various embodiments, the mole to mole ratio of the com aryl, substituted alkyl, or substituted aryl. In other embodi pound comprising Formula (I) to the aldehyde may range US 9, 181,237 B2 31 32 from about 1:0.1 to about 1:1, from about 1:1 to about 1:2, The amount of proton acceptor or proton donor added to from about 1:2 to about 1:3, or from about 1:3 to about 1:4. In the reaction mixture comprising an aldehyde may vary. In exemplary embodiments, the mole to mole ratio of the com general, the mole to mole ratio of the of the compound com pound comprising Formula (I) to the aldehyde may range prising Formula (I) to the proton acceptor ranges from about from about 1:0.5 to about 1:2. 1:0.05 to about 1:10. In various embodiments, mole to mole (ii) Optional Proton Acceptor or Proton Donor ratio of the compound comprising Formula (I) to the proton Depending upon the carbonyl donor used, the reaction acceptor or proton donor may range from about 1:0.05 to mixture may further comprise a proton acceptor or proton about 1:1, from about 1:1 to about 1:5, or from about 1:5 to donor. In embodiments in which the carbonyl donor is an acyl 10 about 1:10. In exemplary embodiments, the mole to mole halide, the reaction mixture may further comprise a proton ratio of the compound comprising Formula (I) to the proton acceptor. The proton acceptor typically has a pKa between acceptor or proton donor may range from about 1:0.1 to about about 7 and about 13. Suitable proton acceptors having this characteristic include borate salts (such as, for example, 1:5. NaBO), di- and tri-basic phosphate salts (such as, for 15 (iii) Solvent example, NaHPO and NaPO, and the like), bicarbonate In some embodiments, the reaction mixture may further salts (such as, for example, NaHCO, KHCO, LiCO, and comprise a solvent. Suitable solvents and ratios of solvent to the like), carbonate salts (such as, for example, NaCO, the starting substrate are listed above in section (II)(a)(ii). In KCOs, LiCO, and the like), organic bases (such as, for exemplary embodiments, the solvent may be tetrahydrofuran, example, pyridine, triethylamine, diisopropylethylamine, and the Volume to mass ratio of the solvent to the compound N-methylmorpholine, N,N-dimethylaminopyridine), and comprising Formula (I) may range from about 2:1 to about mixtures of any of the above. In exemplary embodiments, the 20:1. proton acceptor may be triethylamine. (b) Reaction Conditions The amount of proton acceptor added to the reaction mix 25 ture comprising an acyl halide may vary. In general, the mole In general, the reaction is conducted at a temperature that to mole ratio of the compound comprising Formula (I) to the ranges from about 10°C. to about 80°C. In various embodi proton acceptor ranges from about 1:0.5 to about 1:10. In ments, the reaction may be conducted at a temperature from various embodiments, mole to mole ratio of the compound about 10° C. to about 20°C., from about 20° C. to about 30° comprising Formula (I) to the proton acceptor may range 30 C., from about 30°C. to about 50° C., or from about 50° C. to from about 1:0.5 to about 1:2, from about 1:2 to about 1:5, or about 80°C. In exemplary embodiments, the temperature of from about 1:5 to about 1:10. In exemplary embodiments, the the reaction may range from about 20°C. to about 30°C. The mole to mole ratio of the compound comprising Formula (I) reaction generally is performed under ambient pressure. to the proton acceptor may range from about 1:1 to about 1:4. 35 Typically, the reaction is allowed to proceed for a sufficient In embodiments in which the carbonyl donor is an alde period of time until the reaction is complete, as detailed hyde, the reaction mixture may further comprise a proton above. In a completed reaction, the amount of the compound acceptor or a proton donor. In general, the proton donor or comprising Formula (I) remaining in the reaction mixture proton acceptor has a pKa of less than about 9. Suitable may be less than about 3%, or less than about 1%. In general, proton donors include, but are not limited to, HOAc, HCO.H. 40 the reaction may proceed for about 2 hours to about 24 hours. n-PrCOH, PhCOH, MeSOH, poly HPO, HPO, In some embodiments, the reaction may proceed for about 2 HSO, HCl, HBr, H, CFSOH, p-methyltoluenesulfonic hours to about 6 hours, from about 6 hours to about 12 hours, acid, and combinations thereof. Suitable proton acceptors or from about 12 hours to about 24 hours. include borate salts (such as, for example, NaBO), di- and 45 The compound comprising Formula (II) may be isolated tri-basic phosphate salts (such as, for example, Na HPO and from the reaction mixture using techniques known to those of NaPO, and the like), bicarbonate salts (such as, for skill in the art. Non-limiting examples of Suitable techniques example, NaHCO, KHCO, LiHCO, and so forth), carbon include precipitation, extraction, evaporation, distillation, ate salts (such as, for example, NaCO, KCO, LiCO, and chromatography, and crystallization. the like), butoxides (such as, e.g., sodium tert-butoxide, potassium tert-butoxide), organic bases (such as, for 50 In general, the yield of the compound comprising Formula example, pyridine, triethylamine, diisopropylethylamine, (II) will be at least about 40% by weight. In certain embodi N-methylmorpholine, N,N-dimethylaminopyridine), and ments, the yield of the compound comprising Formula (II) mixtures thereof. Other suitable proton acceptors/proton may be at least about 60%, at least about 70%, at least about donors include N,N-bis-(2-hydroxyethyl)-glycine (BICINE), 55 80%, at least about 90%, or at least about 95%. N-tris(hydroxymethyl)methylglycine (TRICINE), tris(hy C-14 in the compounds comprising Formulas (I) and (II) droxymethyl)aminomethane (TRIS), 3-(cyclohexylamino)- may have an R or an S configuration. 1-propanesulfonic acid (CAPS), 3-(cyclohexylamino)-2-hy (IV) Methods of Using Compounds Comprising Formula (V) droxy-1-propanesulfonic acid (CAPSO), N-(2- Yet another aspect of the present disclosure provides meth hydrooxyethyl)piperazine-N'-(3-propanesulfonic acid) 60 (EPPS), N2-hydroxyethylpiperazine-N'-2-ethanesulfonic ods of using the compounds comprising Formula (V). acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES). (a) Inhibiting Cancer Cell Growth 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine In one embodiment, a compound comprising Formula (V) N,N'-bis(2-ethanesulfonic acid) (PIPES), 3-tris(hy or a pharmaceutically acceptable salt thereof may be used to droxymethyl)amino-1-propanesulfonic acid (TAPS), and 65 inhibit cancer cell growth, wherein the method comprises N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid contacting a cancer cell with an effective about of a com (TES). pound comprising Formula (V): US 9, 181,237 B2 34 precise amount to be used can be determined by the skilled

(V) practitioner in view of desired dosages and side effects of the compound. In some embodiments, the cancer cell may be in vitro. The cancer cell may be a primary cancer cell or a cultured cancer cell line cell. The cancer cell line may be a human cancer cell line or a mammalian cancer cell line. Human or other mam malian cancer cell lines are commercially available and/or are well known to those skilled in the art. The in vitro cancer cell 10 may be contacted with the compound comprising Formula (V) continuously, for a short period of time, intermittently, or any of a variety of regimes. In other embodiments, the cancer cell may be in Vivo, i.e., the cancer cell may be disposed in a Subject. In some embodi 15 ments, the Subject may be a human. In other embodiments, the Subject may be a non-human animal. Non-limiting wherein: examples of non-human animals include companion animals R is hydrogen, hydrocarbyl, or substituted hydrocarbyl, (e.g., cats, dogs, horses, rabbits, gerbils), agricultural animals R. R. R., and Rindependently are hydrogen, halogen, (e.g., cows, pigs, sheep, goats, fowl), research animals (e.g., OR', NR'N', nitro, cyano, thiol, hydrocarbyl, sub rats, mice, rabbits, primates), and Zoo animals (e.g., lions, stituted hydrocarbyl, or together RandR along with tiger, elephants, and the like). the ring carbons to which they are attached form a ring The cancer in the Subject may be primary or metastatic; the comprising { }O(CH2)O{-}; tumor may be malignant or benign. The cancer may be early R. R. R', and Rindependently are hydrogen, halogen, stage or late stage. Non-limiting examples of cancers that OR', NRN nitro, cyano, thiol, hydrocarbyl, substi 25 may be treated include acute lymphoblastic leukemia, acute tuted hydrocarbyl, or together RandR along with myeloid leukemia, adrenocortical carcinoma, AIDS-related the ring carbons to which they are attached form a ring cancers, AIDS-relatedlymphoma, anal cancer, appendix can comprising { }O(CH), O—}; cer, astrocytomas (childhood cerebellar or cerebral), basal R. R', R'', R', R', and R' independently are hydro cell carcinoma, bile duct cancer, bladder cancer, bone cancer, gen, hydrocarbyl, or substituted hydrocarbyl; 30 brainstem glioma, brain tumors (cerebellar astrocytoma, n is an integer from 1 to 3; and cerebral astrocytoma/malignant glioma, ependymoma, the dashed lines represent optional double bonds. medulloblastoma, supratentorial primitive neuroectodermal In some embodiments, R may be hydrogen, alkyl, hetero tumors, visual pathway and hypothalamic gliomas), breast cylic, aryl, heteroaryl, substituted alkyl, substituted hetero cancer, bronchial adenomas/carcinoids, Burkitt lymphoma, cyclic, substituted aryl, or substituted heteroaryl. In various 35 carcinoid tumors (childhood, gastrointestinal), carcinoma of iterations, R may be lower alkyl, which is defined herein as unknown primary, central nervous system lymphoma (pri C-C, and may be linear or cyclic. In otheriterations, R may mary), cerebellar astrocytoma, cerebral astrocytoma/malig be morpholinyl, piperizinyl, phenyl, benzyl, pyridyl, nant glioma, cervical cancer, childhood cancers, chronic lym pyridazinyl, pyranyl, oxazinyl, piperonyl, etc. Any of the phocytic leukemia, chronic myelogenous leukemia, chronic 40 myeloproliferative disorders, colon cancer, cutaneous T-cell foregoing may be substituted with at least one alkyl, alkenyl, lymphoma, desmoplastic Small round cell tumor, endometrial alkynyl, aryl, halogen, Oxo, keto, hydroxy, acyl, acyloxy, cancer, ependymoma, esophageal cancer, Ewings sarcoma alkoxy, alkenoxy, alkynoxy, aryloxy, nitro, amino, amine, in the Ewing family of tumors, extracranial germ cell tumor amide, thiol, cyano, ketal, acetal, ester, or ether. (childhood), extragonadal germ cell tumor, extrahepatic bile In various embodiments R and R independently may be 45 duct cancer, eye cancers (intraocular melanoma, retinoblas hydrogen, halogen, hydroxy, alkyoxy, alkyl or together R toma), gallbladder cancer, gastric (stomach) cancer, gas and R may form { }O CH O{ }. In other embodi trointestinal carcinoid tumor, gastrointestinal stromal tumor, ments, RandR independently may be hydrogen, halogen, germ cell tumors (childhood extracranial, extragonadal, ova hydroxy, alkoxy, or alkyl. In further embodiments, RandR rian), gestational trophoblastic tumor, gliomas (adult, child independently may be hydrogen, halogen, hydroxy, alkoxy, 50 hood brain stem, childhood cerebral astrocytoma, childhood alkyl, aryloxy, Substituted aryloxy, nitro, amino, amine, or visual pathway and hypothalamic), gastric carcinoid, hairy amide. In other embodiments, each of R. R', R'', and R' cell leukemia, head and neck cancer, hepatocellular (liver) may be hydrogen. In various embodiments, the ring contain cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypo ing the dashed lines may have one, two, or three double thalamic and visual pathway glioma (childhood), intraocular bonds. The configuration of each of C-14 and C-8 may be R 55 melanoma, islet cell carcinoma, Kaposi sarcoma, kidney can or S. In exemplary embodiments, C-14 and C-8 have a syn cer (renal cell cancer), laryngeal cancer, leukemias (acute Stereochemistry. lymphoblastic, acute myeloid, chronic lymphocytic, chronic In specific embodiments, the compound comprising For myelogenous, hairy cell), lip and oral cavity cancer, liver mula (V) may be a compound comprising Formula (V-1). In cancer (primary), lung cancers (non-Small cell, Small cell), other embodiments, the compound comprising Formula (V) 60 lymphomas (AIDS-related, Burkitt, cutaneous T-cell, may be a compound comprising Formula (V-1a) or Formula Hodgkin, non-Hodgkin, primary central nervous system), (V-1 b). macroglobulinemia (Waldenström), malignant fibrous histio The method comprises contacting a cancer cell with an cytoma of bonefosteosarcoma, medulloblastoma (child effective amount of the compound comprising Formula (V). hood), melanoma, intraocular melanoma, Merkel cell carci An “effective” amount refers to the dose of the compound that 65 noma, mesotheliomas (adult malignant, childhood), affects (i.e., positively or negatively) a process of interest metastatic Squamous neck cancer with occult primary, mouth (e.g., cell proliferation or a process involved therein). The cancer, multiple endocrine neoplasia syndrome (childhood), US 9, 181,237 B2 35 36 multiple myeloma/plasma cell neoplasm, mycosis fungoides, has been diagnosed as having a pain condition. As used myelodysplastic syndromes, myelodysplastic/myeloprolif herein, the term “pain” refers to the unpleasant sensory and erative diseases, myelogenous leukemia (chronic), myeloid emotional experience associated with actual or perceived tis leukemias (adult acute, childhood acute), multiple myeloma, Sue damage by a noxious stimulus. The pain may be acute or myeloproliferative disorders (chronic), nasal cavity and para chronic pain. For example, the pain may be traumatic or nasal sinus cancer, nasopharyngeal carcinoma, neuroblas inflammatory pain, which results from injury to non-neural toma, non-Hodgkin lymphoma, non-Small cell lung cancer, tissue. Non-limiting examples of traumatic or inflammatory oral cancer, oropharyngeal cancer, osteosarcoma/malignant pain include arachnoiditis, arthritis, back pain, burn pain, fibrous histiocytoma of bone, ovarian cancer, ovarian epithe central pain syndrome, cancer pain, headaches (including lial cancer (Surface epithelial-stromal tumor), ovarian germ 10 cell tumor, ovarian low malignant potential tumor, pancreatic migraines, cluster, and tension headaches); head and facial cancer, pancreatic cancer (islet cell), paranasal sinus and pain, muscle pain (including fibromyalgia), myofascial pain nasal cavity cancer, parathyroid cancer, penile cancer, pha syndromes; reflex sympathetic dystrophy syndrome, repeti ryngeal cancer, pheochromocytoma, pineal astrocytoma, tive stress injuries, Sciatica, shingles and other skin disorders, pineal germinoma, pineoblastoma and Supratentorial primi 15 sports injuries, spinal Stenosis, Surgical pain, temporoman tive neuroectodermal tumors (childhood), pituitary adenoma, dibular disorders, trauma, and/or vascular disease or injury. plasma cell neoplasia, pleuropulmonary blastoma, primary Alternatively, the pain may be neuropathic pain, which central nervous system lymphoma, prostate cancer, rectal results from injury to or inflammation of the central or periph cancer, renal cell carcinoma (kidney cancer), renal pelvis and eral nervous system. Neuropathic pain may occur in any part ureter transitional cell cancer, retinoblastoma, rhabdomyosa of the body and is frequently described as a hot, burning rcoma (childhood), salivary gland cancer, sarcoma (Ewing sensation, which can be devastating to the affected individual. family of tumors, Kaposi, soft tissue, uterine), Sezary syn Neuropathic pain may be acute or chronic; it may result from drome, skin cancers (nonmelanoma, melanoma), skin carci diseases that affect nerves (such as diabetes), from trauma, noma (Merkel cell), Small cell lung cancer, Small intestine Surgical procedures, arthritis, AIDS, burn injuries, cerebral or cancer, soft tissue sarcoma, squamous cell carcinoma, squa 25 lumbar spine disease, fibromyalgia, post-ischemic pain, mous neck cancer with occult primary (metastatic), stomach tumors, viral neuralgias, or, because chemotherapy drugs can cancer, Supratentorial primitive neuroectodermal tumor affect nerves, it may be a consequence of cancer treatment. (childhood), T-Cell lymphoma (cutaneous), testicular cancer, Among the many neuropathic pain conditions are diabetic throat cancer, thymoma (childhood), thymoma and thymic neuropathy (which results from nerve damage secondary to carcinoma, thyroid cancer, thyroid cancer (childhood), tran 30 sitional cell cancer of the renal pelvis and ureter, trophoblastic vascular problems that occur with diabetes); reflex sympa tumor (gestational), unknown primary site (adult, childhood), thetic dystrophy syndrome, which may follow injury; phan ureter and renal pelvis transitional cell cancer, urethral can tom limb and post-amputation pain, which may result from cer, uterine cancer (endometrial), uterine sarcoma, vaginal the Surgical removal of a limb; post-herpetic neuralgia, which cancer, visual pathway and hypothalamic glioma (child 35 may occur after an outbreak of shingles; and complex hood), Vulvar cancer, Waldenström macroglobulinemia, and regional pain syndrome or central pain syndrome, which may Wilms tumor (childhood). result from trauma to the brain or spinal cord. In embodiments in which the cancer cell is in vivo, the Characteristic symptoms of neuropathic pain include cancer cell generally is contacted with the compound by hyperesthesia (i.e., enhanced sensitivity to a natural stimu administering an effective amount of the compound compris 40 lus); allodynia (i.e., widespread tenderness or hypersensitiv ing Formula (V) to the Subject. The compound may be admin ity to tactile stimuli), hyperalgesia (i.e., abnormal sensitivity istered orally (as a solid or a liquid), parenterally (which to pain); spontaneous burning pain; and/orphantom pain (i.e., includes intramuscular, intravenous, intradermal, intraperito perception of pain that is non-existent). Hyperesthesia neal, and Subcutaneous), or topically (which includes trans involves an unusual increased or altered sensitivity to sensory mucosal and transdermal). An "effective' amount refers to 45 stimuli, including for example, acoustic, cerebral, gustatory, the dose of the compound that inhibits the growth of the muscular, olfactory, onelric, optic, or tactile. As an example, cancer cell. The amount to be used can be determined by the a painful sensation from a normally painless touch stimulus. skilled practitioner in view of desired dosages and side effects Allodynia involves an intensified, unpleasant, and painful of the compound. The compound comprising Formula (V) perception of stimuli triggered by heat or by contact, which is may be administered once or repeatedly to the Subject. 50 based on a lowering of the pain threshold for these stimuli, Repeated administrations may be at regular intervals of 2 including, for example, a non-noxious stimulus to normal hours, 6 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 30 skin. Hyperalgesia involves the excessive perception of a days, and so forth. variety of stimuli, again based on a lowering of the pain Following contact with the compound, the growth of the threshold and thus an abnormally increased pain sense, cancer cell generally is inhibited. In some embodiments, can 55 including for example, auditory or muscular stimuli. Phan cer cell growth may be inhibited about 0.5-fold, about 1-fold, tom pain involves a perception of pain in a limb that is non about 2-fold, about 5-fold, about 10-fold, or more than existent, such as perceived pain in a limb that has been ampu 10-fold. In other embodiments, cancer cell growth may be tated, i.e. phantom limb syndrome. inhibited to such a degree that the cell undergoes cell death The additional therapeutic agent may be an opiate analge (via apoptosis or necrosis). 60 sic (e.g., morphine, oxycodone, oxymorphone, hydrocodone, (b) Analgesia hydromorphone, codeine, etc.) or a nonopiate analgesic (e.g., In another embodiment, a compound comprising Formula tramadol, tapentadol, acetaminophen, a non-steroidal anti (V) or a pharmaceutically acceptable salt thereofmay be used inflammatory agent). A person skilled in the art is able to alone or in combination with at least one additional therapeu determine an effective amount of the compound to be admin tic agent for the treatment of a pain condition in a subject. The 65 istered to the Subject. In general, the Subject may be a human method comprises administering an effective amount of the or a non-human mammalian animal (example of which are compound(s) to a Subject. In general, the Subject to be treated presented above). US 9, 181,237 B2 37 38 Definitions ring. The heterocyclo group preferably has 1 or 2 oxygen The compounds described herein may have asymmetric or atoms and/or 1 to 4 nitrogenatoms in the ring, and is bonded chiral centers. Compounds of the present invention contain to the remainder of the molecule through a carbon or heteroa ing an asymmetrically substituted atom may be isolated in tom. Exemplary heterocyclo groups include heteroaromatics optically active or racemic form. All chiral, diastereomeric, 5 as described below. Exemplary substituents include one or racemic forms and all geometric isomeric forms of a structure more of the following groups: hydrocarbyl, substituted are intended, unless the specific stereochemistry or isomeric hydrocarbyl, hydroxy, protected hydroxy, acyl, acyloxy, form is specifically indicated. All processes used to prepare alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, amido, amino, compounds of the present invention and intermediates made 10 cyano, ketals, acetals, esters and ethers. therein are considered to be part of the present invention. The term "heteroaryl” as used herein alone or as part of The term “acyl, as used herein alone or as part of another group, denotes the moiety formed by removal of the hydroxy another group denotes optionally Substituted aromatic groups group from the group COOH of an organic carboxylic acid, having at least one heteroatom in at least one ring, and pref e.g., RC(O) , wherein R is R', RO , R. RN or R'S , 15 erably 5 or 6 atoms in each ring. The heteroaryl group pref R" is hydrocarbyl, heterosubstituted hydrocarbyl, or hetero erably has 1 or 2 oxygen atoms and/or 1 to 4 nitrogen atoms cyclo, and R is hydrogen, hydrocarbyl or substituted hydro in the ring, and is bonded to the remainder of the molecule carbyl. through a carbon. Exemplary heteroaryls include furyl, ben The term “acyloxy.” as used herein alone or as part of Zofuryl, oxazolyl, isoxazolyl, oxadiazolyl, benzoxazolyl, another group, denotes an acyl group as described above benzoxadiazolyl pyrrolyl pyrazolyl, imidazolyl, triazolyl, bonded through an oxygen linkage (O), e.g., RC(O)O— tetrazolyl pyridyl, pyrimidyl, pyrazinyl, pyridaZinyl, indolyl, wherein R is as defined in connection with the term “acyl.” isoindolyl, indolizinyl, benzimidazolyl, indazolyl, benzotria The term “alkyl as used herein describes groups which are Zolyl, tetraZolopyridazinyl, carbazolyl, purinyl, quinolinyl, preferably lower alkyl containing from one to eight carbon 25 isoquinolinyl, imidazopyridyl and the like. Exemplary Sub atoms in the principal chain and up to 20 carbonatoms. They stituents include one or more of the following groups: hydro may be straight or branched chain or cyclic and include carbyl, substituted hydrocarbyl, hydroxy, protected hydroxy, methyl, ethyl, propyl, isopropyl, butyl, hexyl and the like. acyl, acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, The term “alkenyl as used herein describes groups which 30 amido, amino, cyano, ketals, acetals, esters and ethers. are preferably lower alkenyl containing from two to eight The terms “hydrocarbon' and “hydrocarbyl as used carbon atoms in the principal chain and up to 20 carbon herein describe organic compounds or radicals consisting atoms. They may be straight or branched chain or cyclic and exclusively of the elements carbon and hydrogen. These moi include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, eties include alkyl, alkenyl, alkynyl, and aryl moieties. These hexenyl, and the like. 35 moieties also include alkyl, alkenyl, alkynyl, and aryl moi The term “alkynyl' as used herein describes groups which eties substituted with other aliphatic or cyclic hydrocarbon are preferably lower alkynyl containing from two to eight groups, such as alkaryl, alkenaryland alkynary1. Unless oth carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include erwise indicated, these moieties preferably comprise 1 to 20 ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like. 40 carbon atoms. The “substituted hydrocarbyl moieties described herein The term “aromatic” as used herein alone or as part of are hydrocarbyl moieties which are substituted with at least another group denotes optionally substituted homo- or het one atom other than carbon, including moieties in which a erocyclic aromatic groups. These aromatic groups are pref carbon chain atom is substituted with a hetero atom such as erably monocyclic, bicyclic, or tricyclic groups containing 45 nitrogen, oxygen, silicon, phosphorous, boron, Sulfur, or a from 6 to 14 atoms in the ring portion. The term “aromatic' halogen atom. These Substituents include halogen, heterocy encompasses the “aryl and "heteroaryl groups defined clo, alkoxy, alkenoxy, aryloxy, hydroxy, protected hydroxy, below. acyl, acyloxy, nitro, amino, amido, nitro, cyano, ketals, The term “aryl' or 'Ar' as used herein alone or as part of 50 acetals, esters and ethers. another group denote optionally substituted homocyclic aro When introducing elements of the present invention or the matic groups, preferably monocyclic or bicyclic groups con preferred embodiments(s) thereof, the articles “a”, “an', taining from 6 to 12 carbons in the ring portion, such as “the' and “said are intended to mean that there are one or phenyl, biphenyl, naphthyl, substituted phenyl, substituted more of the elements. The terms “comprising”, “including biphenyl or substituted naphthyl. Phenyl and substituted phe 55 and “having are intended to be inclusive and mean that there nyl are the more preferred aryl. may be additional elements other than the listed elements. The terms “halogen' or “halo' as used herein alone or as Having described the invention in detail, it will be apparent part of another group refer to chlorine, bromine, fluorine, and that modifications and variations are possible without depart iodine. ing from the scope of the invention defined in the appended The term "heteroatom' shall mean atoms other than carbon 60 claims. and hydrogen. The terms "heterocyclo” or "heterocyclic” as used herein EXAMPLES alone or as part of another group denote optionally Substi The following examples are simply intended to further tuted, fully saturated or unsaturated, monocyclic or bicyclic, 65 illustrate and explain the present invention. The invention, aromatic or non-aromatic groups having at least one heteroa therefore, should not be limited to any of the details in these tom in at least one ring, and preferably 5 or 6 atoms in each examples. US 9, 181,237 B2 39 40 Example 1 -continued HCO Preparation of Compound 7 Methylamide from Compound 7 5 N HO O

H3CO O 10 H3CO NH2 - Chemical Formula: C20H23NO4 -- Hs Exact Mass: 341.16 NEt, THF, rt Molecular Weight: 341.40 HO Cmpd 7 methylamide HCO 15 Compound 7 (20.01 g, 0.06 moles) was dissolved in anhy drous tetrahydrofuran (100 mL). To this solution was added HCO triethylamine (17.59 g, 0.17 moles, 24.22 mL) dropwise. Chemical Formula: CoH23NOs Using an addition funnel, acetylchloride (9.10g, 0.12 moles, Exact Mass: 345.16 8.24 mL) was added dropwise. Then, the reaction was stirred Molecular Weight:345.39 20 at room temperature for 4 hours. Distilled water (10 mL) was Cmpd 7 added and the entire mixture was evaporated to thick oil under reduced pressure. Added methanol (50 mL) and distilled H3CO water (10 mL). To this solution was added 50% aqueous 1. NaOH sodium hydroxide (1 mL) to pH 130. The mixture was stirred O N ... Y8. overnight at room temperature. The pH of the solution was H2O, CH3OH 25 adjusted to pH 5.0 using a dropwise addition of 36% hydro us 2. HCI chloric acid. The mixture was extracted using ethyl acetate O O (2x100 mL). The extracts were combined, washed with dis tilled water (3x100 mL), dried over anhydrous magnesium Sulfate, and then filtered. Upon standing, a precipitation began to form from the ethyl acetate solution. The precipitate HCO 30 was isolated by filtration and dried on the funnel producing the methylamide of compound 7 (17.60 g, 89% yield). Chemical Formula: C22H25NOs Exact Mass: 383.17 Example 2 Molecular Weight: 383.44 Preparation of Methyl-Berbine from Compound 7 35 Methylamide

H3CO H3CO POCl3 toluene N reflux N -e- HO O HO C

HCO H3CO Chemical Formula: C20H23NO4 Chemical Formula: C20H23CINO" Exact Mass: 341.16 Exact Mass: 360.14 Molecular Weight: 341.41 Molecular Weight: 360.86 | HCO H3CO C N NaBH4 +n CH3OH, H2O

OCH OCH OH OH Chemical Formula: C20H2CINO3 Chemical Formula: C20H23NO3 Exact Mass: 359.13 Exact Mass: 325.17 Molecular Weight: 359.85 Molecular Weight: 325.41 Berbine US 9, 181,237 B2 41 42 Compound 7 methylamide (3.50 g., 0.01 moles) was slur foam was dried overnight at room temperature yielding the ried in toluene (50 mL). The slurry was warmed to 70° C. N-formyl compound (5.30 g, 16.2 mmol. 100% yield) as a then cooled to 50° C. To the cooled solution was added mixture of rotamers. phosphorus oxychloride (1.96 g., 0.01 moles, 1.19 mL). The mixture was warmed to reflux and held at reflux 5 hours, then Example 4 cooled to room temperature. An orange precipitate formed. The toluene was decanted and methanol (100 mL) was added. Preparation of Berbine from Compound (R)-7 The mixture was evaporated to thick oil. To this thick oil was N-Formate added methanol (20 mL), distilled water (10 mL), then sodium borohydride (780 mg, 0.02 moles). The reaction was 10 stirred for 30 minutes at room temperature. The mixture was poured into chloroform (100 mL), then distilled water (50 H3CO mL) and 1% aqueous hydrochloric acid (1 mL) were added. N H The chloroform layer was removed and the remaining aque 15 ous layer was discarded. The chloroform layer was dried over POCl. CH3CN anhydrous magnesium sulfate (2.0 g), filtered, and evapo HO O -as rated to dryness to thick oil. The oil was dissolved in ethyl acetate (10 mL) and was allowed to stand at room tempera ture. Upon standing, a precipitate formed. The berbine 1 (2.40 HCO g, 72% yield) was isolated by filtration and washing the precipitate with ethyl acetate (1 mL). Chemical Formula: CoH2NO4 Exact Mass: 327.15 Example 3 Molecular Weight: 327.37 10825-101 25 Preparation of Compound (R)-7 N-Formate H3CO C N n H3CO 30 NaCNBH CHCN, rt Hs NH PrCHO OCH HO neat, reflux OH Chemical Formula: CoH20CINO3 Exact Mass: 345.11 H3CO Molecular Weight: 345.82 Chemical Formula: C18H1NO3 H3CO Exact Mass: 299.15 40 Molecular Weight: 299.36 (R)-7 N

H3CO OCH OH 50 Chemical Formula: CoH2NO3 N H Exact Mass: 311.15 HO O Molecular Weight: 311.37

HCO 55 Into a 3 neck round bottom flask was charged N-formyl Chemical Formula: CoH2NO4 (R)-7 (1.22 g, 3.73 mmol) and acetonitrile (15 mL). To this Exact Mass: 327.15 solution was added phosphorus oxychloride (2.0 eq., 7.45 Molecular Weight: 327.37 mmol. 1.14 g. 0.69 mL) dropwise. After the addition was (R)-7 CHO complete, the mixture was stirred at room temperature over 60 night. At that time, the solvent was removed under vacuum to forman orange oil. The oil was redissolved inacetonitrile (20 Compound (R)-7(4.85g. 16.20 mmol) was slurried in 30 mL) then sodium cyanoborohydride (2.0 eq., 7.45 mmol, 0.47 mL propyl formate. This mixture was warmed to reflux and g) was added. This mixture was stirred at room temperature maintained at reflux for 12 hours. The reaction was cooled to for 1 hour at which time the reaction was deemed complete. room temperature then evaporated under reduced pressure to 65 Distilled water (20 mL) was added and this mixture was an oil. To the crude oil, ethyl acetate (10 mL) was added and stirred for 1 hour. Extraction of this reaction mixture was the Solution was evaporated once again to form a foam. This accomplished using ethyl acetate (3x20 mL). The extracts US 9, 181,237 B2 43 44 were combined, dried over anhydrous sodium sulfate, fil ture for 16 hours, the pH was adjusted to 5.0 using 10% tered, and then evaporated. The product berbine (1.0 g, 3.21 HC1/HO, and then an additional 25 mL of distilled water was mmol. 85% yield) was isolated using column chromatogra added. The solution was extracted using EtOAc (3x25 mL), phy (SiO G60, 70 to 230 mesh) eluting with a gradient from the extracts were combined, dried over anhydrous NaSO, 0% EtOAc/hexanes to 50% EtOAc/hexanes, combining simi filtered, and then concentrated to an oil. The oil was placed in lar fractions by TLC, evaporating to a foam, and then drying vacuum at room temperature for 48 hours where the material the foam under vacuum at room temperature for 48 hours. solidified producing the N-propyl amide (14.23 g, 40.03 mmol, 97% yield). Example 5 10 Example 6 Preparation of Compound (R)-7 Propylamide Preparation of Ethyl-Berbine from Compound (R)-7 Propylamide

C 15 HCO 1. O HCO NH (2.0 eq) Chemical Formula: C3H5CIO N HO Exact Mass: 92.00 r POCl3 Molecular Weight: 92.52 HO O CHCN NEt, THF, rt 50 C. H3CO 25 Chemical Formula: C18H1NO3 HCO Exact Mass: 299.15 Chemical Formula: C2H5NO4 Molecular Weight: 299.36 Exact Mass: 355.18 (R)-7 Molecular Weight: 355.43 HCO 30 HCO Cl N O Nrn NaCNBH Nus. O CHCN, rt 35 -> 1. NaOH H3CO H2O, CH3OH OCH 2.10% HCI Chemical Formula: C24H29NOs He OH Exact Mass: 411.20 40 Chemical Formula: C2H24CINO3 Molecular Weight: 411.49 Exact Mass: 373.14 HCO Molecular Weight:373.87 H3CO

45 Sn N HO O

H3CO 50 Chemical Formula: C2H25NO4 OCH Exact Mass: 355.18 Molecular Weight: 355.43 OH Chemical Formula: C2H25NO3 55 Exact Mass: 339.18 Into a round bottom flask was charge (R)-7 (12.36 g., 41.29 Molecular Weight: 339.43 mmol) and tetrahydrofuran (100 mL). To this slurry was added triethylamine (2.10 eq., 86.71 mmol, 8.77g, 12.1 mL) and propionyl chloride (2.05 eq., 84.64 mmol. 7.83 g, 7.4 mL) Into a 3 neck round bottom flask was charged (R)-7 N-pro dropwise. The reaction was stirred at room temperature for 16 60 pylamide (1.54g, 4.33 mmol) and acetonitrile (15 mL). To hours, after which the reaction was deemed complete. The this solution was added phosphorus oxychloride (1.0 eq., 4.33 reaction mixture was filtered through a fritted funnel, and the mmol. 0.66 g., 0.40 mL) dropwise. After the addition was solid was washed with EtOAc (50 mL). The filtrate was complete, the mixture was warmed to 50° C. and stirred for 12 transferred to a round bottom flask and evaporated to an oil. hours, then cooled to room temperature and stirred for 4 To the oil was added methanol (25.0 mL) and distilled water 65 hours. At that time, sodium cyanoborohydride (1.0 eq., 4.33 (10.0 mL). To this solution was added solid sodium hydroxide mmol, 0.27g) was added. This mixture was stirred at room (2.0 eq., 82.58 mmol. 3.30 g). After stirring at room tempera temperature for 6 hours, at which time the reaction was US 9, 181,237 B2 45 46 deemed complete. Distilled water (30 mL) was added and the Into a round bottom flask was charge (R)-7 (1.92 g. 6.41 pH was adjusted to 1.0 using 10% HC1/HO. This reaction mmol) and tetrahydrofuran (15 mL). To this slurry was added mixture was stirred at room temperature for 24 hours. After triethylamine (2.10 eq., 13.46 mmol. 1.31 g, 1.88 mL) and adjusting the pH to 9.2 using 29% NH/HO, the reaction was thenbenzoyl chloride (2.05 eq., 13.15 mmol. 1.84g, 1.53 mL) extracted using ethyl acetate (3x25 mL). The extracts were dropwise. The reaction was stirred at room temperature for 16 combined, dried over anhydrous sodium sulfate, filtered, and then evaporated. The product berbine (1.3 g, 3.83 mmol, 88% hours, at which time the reaction was deemed complete. To yield) was isolated using column chromatography (SiO G60, the reaction was added distilled water (25.0 mL) and then 70 to 230 mesh) eluting with a gradient from 0% EtOAc/ extracted using EtOAc (3x25 mL), the extracts were com hexanes to 50% EtOAc/hexanes, combining similar fractions 10 bined, and evaporated to an oil. To the oil was added methanol by TLC, evaporating to a foam, and then drying the foam (10.0 mL) and distilled water (5.0 mL). To this solution was under vacuum at room temperature for 48 hours. added 50% NaOH/HO (5.0 mL). After stirring at room tem perature for 16 hours, the pH was adjusted to 5.0 using 10% Example 7 15 HCl/H2O, and then an additional 25 mL of distilled water was added. The solution was extracted using EtOAc (3x25 mL), the extracts were combined, dried over anhydrous NaSO, Preparation of Compound (R)-7 Benzamide filtered, and then concentrated to an oil. Isolation of the prod uct was accomplished through column chromatography (SiO, G60, 70 to 230 mesh) using gradient elution from 0% H3CO EtOAc/hexanes to 60% EtOAc/hexanes. Similar fractions monitored by TLC were combined, evaporated to a foam, and NH C placed under vacuum for 18 hours at room temperature yield 25 ing the product (1.88 g, 4.66 mmol. 73% yield) as an off white solid. HO O (2.0 eq) Chemical Formula: C7H5CIO Example 8 H3CO Exact Mass: 140.00 30 Chemical Formula: C18H1NO3 Molecular Weight: 140.57 Preparation of Phenyl-Berbine from Compound Exact Mass: 299.15 NEt, THF, rt (R)-7 Benzamide Molecular Weight: 299.36 (R)-7 6920-71 35 HCO H3CO

N POCl. CHCN O O 40 -- C’s O HO O 50 C. HCO HCO Chemical Formula: C3HoNOs 45 Chemical Formula: C5H5NO4 Exact Mass: 507.20 Exact Mass: 403.18 Molecular Weight: 507.58 Molecular Weight:403.47

1. NaOH 50 H2O, CH3OH 2.10% HC HCO HCO Cl 55 N NaCNBH CHCN, rt HO O O 60 O OCH H3CO Chemical Formula: C25H25NO4 OH Exact Mass: 403.18 Chemical Formula: C5H4CINO3 Molecular Weight: 403.47 65 Exact Mass: 421.14 Molecular Weight: 421.92 US 9, 181,237 B2 47 48 -continued 1.0 using concentrated HC1. This mixture was stirred at room HCO temperature for 24 hour, and then evaporated to ~/2 volume. After adjusting the pH to 9.2 using 29% NH/HO, the reac tion was extracted of using chloroform (3x25 mL). The extracts were combined, dried over anhydrous sodium Sul fate, filtered, and then evaporated. The product berbine (0.86 g, 2.22 mmol. 49% yield) and (R)-7 N-benzamide (0.84 g) was isolated using column chromatography (SiO, G60, 70 to OCH 10 230 mesh) eluting with a gradient from 0% EtOAc/hexanes to OH 25% EtOAc/hexanes, combining similar fractions by TLC, Chemical Formula: C5H5NO3 evaporation to a foam, and then drying the foam under Exact Mass: 387.18 vacuum at room temperature for 48 hours. Based on recovery Molecular Weight: 387.47 15 of the (R)-7 N-benzamide, the yield of the reaction was 89%. The present invention is not limited to the above embodi ments and may be variously modified. The above description of exemplary embodiments is intended only to acquaint oth ers skilled in the art with the invention, its principles and its practical application so that others skilled in the art may adapt Into a 3 neck round bottom flask was charged (R)-7 N-ben and apply the invention in its numerous forms, as may be best Zamide (1.84 g. 4.56 mmol) and acetonitrile (25 mL). The Suited to the requirements of a particular use. reaction was distilled until a vapor temperature of 82°C. was reached, then the solution was cooled to room temperature. Example 9 To this solution was added phosphorus oxychloride (1.0 eq., 25 4.56 mmol, 0.70 g., 0.43 mL) dropwise. After the addition was Preparation of Berbine Derivative from Compound complete, the mixture was warmed to 50° C. and stirred for 20 Br (R)-7 hours, then cooled to room temperature. At that time, sodium Different regioisomers of berbine can be prepared by cyanoborohydride (1.0 eq., 4.56 mmol, 0.30 g) was added. 30 including a halogen group in the starting compound, such that This mixture was stirred at room temperature for 18 hours. cyclization is directed to the least hindered position. The Distilled water (15 mL) was added and the pH was adjusted to reaction scheme below demonstrates this approach.

HCO

1. RCHO HO 2. NaBH4

H3CO Br

10%. Po?C HCO2H, NEt IPA, reflux

US 9, 181,237 B2 49 50 Reaction with RCHO (step 1) can be performed as detailed -continued above in Examples 1, 3, 5, and 7, and the cyclization and H3CO reduction step (2) can be performed as described above in Examples 2, 4, 6, and 8. The halogen group can be removed N by contact with 10% PD/C, formic acid, and triethylamine in 5 isopropyl alcohol at reflux. Example 10

10 Preparation of Substituted Phenyl-Berbine

Substituted aromatic derivatives of berbine can be pre pared according to the following scheme. X can be halogen, 15 NO, CH, or OCH.

HCO 1. 2O NH OHC

HO 2. NaBH4 25 Example 11 Preparation of Ether or Amine Derivatives of H3CO Phenyl-Berbine (R)-7 Ether and amine derivatives of phenyl berbine can be pre pared according to the following scheme.

HCO H3CO

O NH 1. PhCHO N 2. NaBH4

HO C

C OH Pd Cross Coupling Pd Cross Coupling ArCDH or ROH ArNH2 or RNH2

HCO H3CO

N N

Ar(R) O - Ar(R) N1H OH OH US 9, 181,237 B2 51 52 Step 1 can be performed as detailed above in Examples 1, Example 12 3, 5, and 7, and step 2 can be performed as described above in Examples 2, 4, 6, and 8. The product can be contacted with Preparation of Amide Derivatives of Phenyl-Berbine (1-5 eq) of alcohol or amine, (0.01-1%) transition metal catalyst, and (1-4 eq.) Sodium tert-butoxide, potassium car bonate, or triethylamine. The reaction can be conducted in the presence oftoluene, THF, DMF, or DMAC, and at a tempera Amide derivatives of phenyl berbine can be prepared ture ranging from room temperature to reflux. according to the following scheme. H3CO H3CO

NH 1. PhCHO N 2. NaBH4

HO NO NO OH Pol/C, ". HCO H3CO

N N

a O ls R NH2 H OH OH

Step 1 can be performed as detailed above in Examples 1, 3, 5, and 7, and step 2 can be performed as described above in Examples 2, 4, 6, and 8. Reduction of NO to NH can be carried out in the presence of hydrogen and a transition metal catalyst. The amide can be formed by contact with (1.0-1.2 eq.) acyl halide and (1-4 eq.) triethylamine, diisopropylethy lamine, or N-methyl morpholine. Reaction with the acyl halide can be conducted in the presence of THF, acetonitrile, dichloromethane, or chloroform and the reaction can be con ducted at room temperature. Example 13 B16 Cancer Cell Screening Assay The murine melanoma cell line, B16, was used to screen the activity of compounds comprising Formula (V). B16 cells were grown understandard conditions and exposed to several concentrations of each of the tested compounds. Table 1 shows the effects of the tested compounds on the inhibition of B16 proliferation and the inhibition of lactate dehydrogenase (LDH) release. TABLE 1

B16 Cancer Cell Screening Data.

Compound B16 Profile B16 LDH ID Structure IC50 IC50

HCO >600 M 175 M US 9, 181,237 B2 53 54 TABLE 1-continued

B16 Cancer Cell Screening Data.

Compound B16 Profile B16 LDH ID Structure IC50 IC50

M-R-0002 HCO 200 M 200 M

M-R-0003 H3CO >600 M 150 M

M-R-0004 HCO >600 M >600 M

OCH OH

M-R-0005 H3CO 35 M 30 M

N w

OCH OH

M-R-0006 H3CO 35 M 35 M

OCH OH US 9, 181,237 B2 55 56 TABLE 1-continued

B16 Cancer Cell Screening Data.

Compound B16 Profile B16 LDH ID Structure IC50 IC50

M-R-0007 HCO >400 M Inactive

N &Y-1

OCH OH

M-R-0008 HCO 38 M >320 M

OCH M-R-0009 HCO CO Inactive Inactive

OCH OH

M-R-0010 H3CO 300 M Inactive

M-R-OO11 O 120 M 70 M

{O N

OCH OCH US 9, 181,237 B2 57 58 TABLE 1-continued

B16 Cancer Cell Screening Data.

Compound B16 Profile B16 LDH ID Structure IC50 IC50

M-R-OO12 >600 M >700 M O

{O N

OCH

M-R-OO13 75 M 700 M

OCH

M-R-0014 H3CO 10 M 70 M

M-R-0015 HCO 25 M 130 M

N w

M-R-OO16 75 M 700 M

US 9, 181,237 B2 59 60 TABLE 1-continued

B16 Cancer Cell Screening Data.

Compound B16 Profile B16 LDH ID Structure IC50 IC50

M-R-0017 55 M 60 M H3CO

M-R-0018 O 35 M 30 M

OCH OH

M-R-0019 O 45 M 50 M

O N w

OCH OH

M-R-0020 H3CO Br 18 M 30 M

Br OCH

M-R-0021 65 IM -350 M HCO US 9, 181,237 B2 61 62 TABLE 1-continued

B16 Cancer Cell Screening Data.

Compound B16 Profile B16 LDH ID Structure IC50 IC50

M-R-0022 H3CO HN 25 M 30 M

M-R-0023 HCO 27 M 40 M

M-R-0024 H3CO 60 M 70 M

M-R-0025 O >160 M -350 M

{O N

OH OCH

M-R-0026 O 55 M 60 M

{O O N

OH OCH US 9, 181,237 B2 63 64 What is claimed is: wherein: 1. A compound of Formula (V-1): R’ is hydroxy or alkyoxy and R is hydrogen, or together R° and R form { }O CH-O{ }; R1 R12 R11 RandR independently are hydrogen, halogen, hydroxy, alkoxy, or alkyl; R and R independently hydrogen, halogen, hydroxy, alkyOXy, alkylaryloxy, Substituted aryloxy, nitro, amino, amine, or amide; 10 R" is hydrogen, halogen, hydroxy, alkoxy, alkyl, nitro, amino, or amine; and m is 0. 4. The compound of claim 1, wherein the compound has 15 Formula (V-1b): wherein: (V-1b) R. R. R. and R independently are hydrogen, halogen, OR',NR'N', nitro, cyano, thiol, hydrocarbyl, substi tuted hydrocarbyl, or together RandR along with the ring carbons to which they are attached form a ring comprising { }O(CH2)O{-}: R. R. R', and R independently are hydrogen, halogen, OR', NR'N', nitro, cyano, thiol, hydrocarbyl, substi tuted hydrocarbyl, or together RandR along with the 25 ring carbons to which they are attached form a ring comprising { }O(CH2), O—}; provided that at least two of R. R. and Rare other than methoxy; R. R', R'', and R' independently are hydrogen, hydro 30 carbyl, or substituted hydrocarbyl; wherein: R. R. R. R', and R independently are hydrogen, R is hydroxy or alkyoxy and R is hydrogen, or together halogen, OR, NR'N', nitro, cyano, thiol, hydrocar R° and R form { }O CH-O{ }; byl, or substituted hydrocarbyl: RandR independently are hydrogen, halogen, hydroxy, R" and R' independently are hydrogen, hydrocarbyl, or 35 alkoxy, or alkyl; substituted hydrocarbyl, R and R independently hydrogen, halogen, hydroxy, m is an integer of 0. alkyOXy, alkylaryloxy, Substituted aryloxy, nitro, amino, n is an integer from 1 to 3; and amine, or amide; the dashed lines represent optional double bonds. 40 R" is hydrogen, halogen, hydroxy, alkoxy, alkyl, nitro, 2. The compound of claim 1, wherein each of R. R. R. R', R', and R' is hydrogen; R and R independently are amino, or amine; and hydrogen, halogen, hydroxy, alkyoxy, alkyl or together R m is 0. and R form { }O—CH2—O{ }; R and R indepen 5. The compound of claim 1, wherein the compound is dently are hydrogen, halogen, hydroxy, alkoxy, or alkyl; R' chosen from the following compounds: and R' independently are hydrogen, halogen, hydroxy, 45 alkoxy, alkyl, aryloxy, Substituted aryloxy, nitro, amino, amine, or amide; and R. R. R. R', and R indepen 21 dently are hydrogen, halogen, hydroxy, alkoxy, alkyl, nitro, amino, or amine. 50 R3 N SX 3. The compound of claim 1, wherein the compound has Y Formula (V-1a):

(V-1a) 55 X OR 15 R2 21 60 R3 N SX Y OR 15

65 US 9, 181,237 B2 65 66 -continued -continued R2 R2 21 O 21 R3 O N S X 5 R3 N N X Y Y

O NO 10 O O O21N x OR 15 R2 21 R2 21 15 R3 O N Sx R3 N S. X Y Y OR 15 O 21 O 2O N S XZ NO OR 15 R2 R2

R3 O N 21N X 25 3 O N 21N X Y R Y OR 15

O NH2 30 O N OS X H Z OR 15 R2 R2 21

N 21 35 R3 O N S X R3 SX Y Y OR 15 40 O lO NH2 15 2 OR R 2 21 R 21

N N s 45 N R3 Y R3 SX Y OR 15 O

OR 15 N R 14 H OR 15

55 R2 R 21 21 N N R3 S x R2 SX Y 60 O 21 O N N O NxZ 65 OR 15 A. 21 OR 15 Z US 9, 181,237 B2 67 68 -continued R2

R3 O N rN x 5 OR 15

CON N 10 i | A-4 Z wherein: 15 R is hydroxy or alkyoxy and R is hydrogen, or together R° and R form { }O CH-O{-} R'' is alkyl or substituted alkyl. R" is hydrogen or C-C alkyl: X is halogen; and Y and Z independently are hydrogen, halogen, hydroxy, alkoxy, alkyl, nitro, amino, or amine. k k k k k