EBLING L\BRARY
`UNIVERSITY OF WISCONSIN
`
`MAR 1 3 2007
`750 Highland Avenue
`Madison. WI 53705
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2042 - 1/10
`
`

`

`journal of
`Medicinal
`Chemistry
`
`Journal of Medicinal Chemisrrr (ISSN 0022-2623)
`is published biweekly by the American Chemica l
`Society at 11 55 16th St. , N. W .. Washington. DC
`20036. Periodica ls postage paid at Washington.
`DC, and add iti onal mailing offi ces. POSTMAS(cid:173)
`TER: Send address changes to Journal r!{ Medici(cid:173)
`nal Chemisrrr, Member & Subsc riber Se rvices.
`P.O. Box 3337. Co lumbus, OH 432 10.
`
`American Chemical S ociety
`1155 16th S t. , N .W.
`Washington, DC 20036
`(20 2) 872-4600
`TDD (2 02) 872-6076
`Fax (202) 776-8264
`
`Journal Publications
`Am e ri can C he mi ca l Society
`2540 Ole nt angy Ri ver Road
`P.O . Box 3330 , Co lumbus, OH 432 10
`(6 14) 447-3665
`Fax (61 4) 447-3745
`E- m a il acsproof@acs.org
`
`Memb er & S ubscriber Services
`P.O. Box 3337
`Columbus, OH 432 10
`
`Me111bers conwct:
`(6 14) 447-3776; (800) 333-95 11
`Fa x (6 14) 447-367 1
`E-ma il service@acs.org
`
`Agencies & institutions contact:
`(6 14) 447-3674; (888) 338-00 12
`Fax (6 14) 447-367 1
`E- m ai l liblink @acs.org
`
`Adve rtising Office
`Centcom , Ltd.
`676 East Swedes ford Road
`Suite 202 , Way ne , PA 19087- 16 12
`(6 1 0) 964-8061
`
`Publications Di vision
`Robe rt D. Bovensc hulte , Presider1t
`Bri a n D. C raw ford, Senior \lice President
`
`Journal Production & Manufacturing
`Operations
`Ann e C . O ' Me li a, \lice President;
`T e resa K. Lewandowsk i, Director;
`Diane E . Needham , Jouma/s Editing
`Ma nager; Lore lla M. Ya m, Associate Editor;
`Lisa H. C lay, Assistant Edito r;
`Karen S. Ge ist, Production Assiswnt
`
`S ales & Ma rke ting/Publications
`Dea n J. Smith , \lice President
`Ma tthew J . Price, Director,
`Product Ma rketing
`Jonathan J. Morga n, Manager.
`Product Ma rketing
`
`Copyright Permission: See copyri ght statu s form for certain ri ghts (http://pubs.acs.o rg). Repro(cid:173)
`graphic copy ing beyo nd that permitted by Sec ti on 107 or 108 of the U.S. Copyri ght Act is all owed,
`provided that the current per arti cle fee is paid to the Copyri ght C lea rance Cen ter. Te l: (978) 750-
`8400. Republica ti on or reproducti on for sale of articl es or abstracts in thi s journal is permitted only
`by written permi ssion from the Copy ri ght Office, ACS , Washington. DC. Te l: (202) 872-4368. Fax:
`(202) 776-8 11 2. E-mail: copyri ght @acs.org.
`
`EDITOHIAL I NFOH~ I ATION
`
`G uidelines for A uthors and Copyright Status Form: See the first printed issue o r eac h volu me or
`visit the Publications Division Web site (http://pubs.acs.org). Please conform to these instructions
`when submittin g manuscript s.
`
`Ma nuscript S ubmission: Submit via the secure ACS Web site (http://pubs.acs.org/jmc).
`
`Acce pted Papers and Proofs: Direc t correspondence to Journ al Publicati ons. Co lumbus, OH. Tel:
`(6 14) 447-3665. Fax: (6 14) 447-3745. E-mail: acsproof@acs.org.
`
`Journal Policies: The American Chemica l Society and its Ed it ors assume no responsibilit y for the
`statement s and op ini ons advanced by contributors. Reg istered names and trademarks, etc., used in this
`publication, e ven without specific indi cation thereo f, are not to be considered unprotected by law.
`
`Document Numb er: At the end of eac h doc ument is a 9- or 14-c harac ter code that serves as a link
`between the printed and elec tronic produc ts and fa cilitates retri eval of the doc ument in e lec tronic
`for m.
`
`Digital Object Identifier (DOl): T he DOl identi ficat ion system for digital media has been des igned
`to pro vide persistent and re liable identifi ca ti on of di gital objec ts. Information on the DOl and its go v(cid:173)
`erning body, the Intern ational DOl Foundati on, can be found at hllp://www.doi.org . In the Web edi(cid:173)
`ti ons o f ACS journals. the DOl appears at th e top of th e HTML version o f an arti cle and at th e bot(cid:173)
`tom of the first page in its PDF version; in th e printed edition s, the DOl appears in the same locati on
`as in the PD F version.
`
`2007 SunSCRIPTION AN D 0HDEHI NG INFO R~ IATION
`
`Printed E dition
`
`!VI embers
`Student l'vlembers
`Institutional
`
`North
`A meri ca
`
`$ 253
`$ 190
`$2073
`
`Outside
`North America'''
`
`$ 47 1
`$ 408
`$229 1
`
`Web Editiont
`$ 85
`Me mbers
`$ 85
`''' Air service in cluded. t For In stituti onal rates, ca ll M&SS Agency & In stituti onal Customer Servi ce.
`
`Web Edition: Thi s journal is ava il able to subsc ribers via the Internet. Members may contact Cus(cid:173)
`tomer Service. Tel: (6 14) 447-3776 or (800) 333-95 11. E-mail : service@acs.org. Institutional sub(cid:173)
`scribers may cont ac t Age ncy & Institutional Customer Service. Te l: (6 14) 447-3674 or (888) 338-
`00 12. Fax: (6 14) 447-367 1. E- mail : liblin k@acs.org. For add iti onal detail s. visit th e Publications
`Di vision Web site (hllp://pubs.acs.org).
`
`New a~Jd . Renewal Subscriptions: Se nd with payment to American Chemica l Society, P. O. Box
`182426: Columbus. OH 432 18-2426.
`
`S ubscriptic:in Dona tions: Members may donate/share their personal subsc ription s to/wi th librari es but
`onl y after 5 years from the dale of publicati on.
`
`Change. of-Address: Notify l'vlember & Subsc ri ber Services, ACS. Columbus, OH. Te l: (6 14) 447-
`3776 or (800) 333-95 11. Fax: (6 14) 447-367 1. E- mail: address@acs.org. Incl ude both old and new
`add resses and a mailing label rrom a rece nt issue.
`
`M icrofilm, M icrofiche, Back Issue, and Printed Edition Single Issue Orders: Send requests to
`Publications Support Services, ACS, Washin gton. DC. Tel: (202) 872-4376. Fax: (202) 872-6325.
`E-mail: pss@acs.org. Printed editi on not ava il able prior to 2003.
`
`Bulk Reprint Orders: For quotes and information. contact Cad mus Reprints. Te l: (888) 257-21 34 or
`(4 10) 8 19-3995. Fax: (4 10) 820-9765.
`
`C la ims for Issues Not Recei ved : C laims will be honored onl y if submilled within 90 days of the
`issue date fo r subscri bers in North Ameri ca or within 180 days of th e iss ue date for a ll other sub(cid:173)
`scribers. Members may cont act Customer Service. Te l: (6 14) 447-3776 or (800) 333-95 11. E-mail:
`service@acs.org. In stitutiona l subscribers may contact Age ncy & Institutional Customer Servi ce. Te l:
`(6 14) 447-3674 or (888) 338-00 12. Fax: (6 14) 447-367 1. E- mail: li blin k@acs.org.
`
`Su pporting Information (SI): S l from 1995 to th e present is avail able free of charge from th e jour(cid:173)
`nal ' s home page (http://pubs.acs.org/j mc). For in fo rm ation on e lec troni c access. se nd e-m ail to
`journalhel p @acs.org. S l prior to 1995 is ava il ab le, for a fee, from Publi cation s Support Services. Tel:
`(202 ) 872-4376. Fax: (202) 872-63 25. E- mail : pss@ acs.org.
`
`Ca nad ian GST Reg. No. 12757 1347
`Prin ted in th e us};:
`
`© Copyri ght 2007 Ameri can Chem ica l Soc iety
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2042 - 2/10
`
`

`

`820
`
`J. Med. Chem. 2007, 50, 820-827
`
`Synthesis and Comparative Toxicology of a Series of Polyhedral Borane Anion-Substituted
`Tetraphenyl Porphyrins
`
`Myoung-Seo Koo,t TQmoko Ozawa,+ Raquel A. Santos,+ Kathleen R. Lamborn,+ Andrew W. Bollen,§ Dennis F. Deen,+.ll and
`Stephen B. Kahl *·'
`
`Departmeuts of Pharmaceutical Chemislly, Neurological Surgery, Pathology, aud Radiatiou Oucology, Uuiuersity of Ca/(fomia,
`Sau Fraucisco, Sau Fraucisco, Califomia 94143
`
`Received July 27, 2006
`
`Three structurally similar tetraphenylporphyrins bearing polyhedral borane anions have been synthesized
`and their toxicological profiles obtained in rats. These conjugates were found to have quite different acute
`toxicities as manifested at the maximum tolerated dose (MTD). When given at the MTD and observed over
`28 days, the most acutely toxic porphyrin was found to be devoid of toxicity, as measured by blood chemistry
`panels. The remaining two less acutely toxic compounds both elicited significant changes, characterized by
`moderate to severe thrombocytopenia, failure to gain weight normally and changes in liver enzymes indicative
`of mild hepatotoxicity. All toxic effects were transient, with platelets rebounding to above normal levels at
`day 28 . We conclude that thrombocytopenia is the dose lirniting toxicity for boronated porphyrins in mammals
`and suggest that these effects may be due to the porphyrin, not the borane or carborane.
`
`Introduction
`Over the past two decades, porphyrins, metalloporphyrins,
`and their derivatives have found increasing applications in
`medicine.1•2 In binary therapies of cancer, such as photodynamic
`therapy (PDP) and boron neutron capture therapy (BNCT),
`these compounds are used for their abilities to act as sensitizers
`or intermediaries for local damage initiated by the application
`of some external form of radiation . In PDT, the activating agent
`is a photon in the 630-750 nm region, but the actual cytotoxic
`species is generally accepted as being a reactive oxidizing
`species, primarily singlet oxygen . In BNCT, thermal neutron
`fission with the 10B nucleus leads to prompt emission of 4He
`(alpha particles) and 7Li nuclei , together with a gamma ray,
`whose combined energies exceed 2.3 MeV. In both PDT and
`BNCT, the lethal radiation damage is confined to target cells
`by two processes. Because the mean free paths of singlet oxygen
`and the fission products are very short ( < 0.05 ftm and <I 0
`ftm , respectively), the resultant apoptosis and necrosis occur
`only in cells that have taken up the sensitizer and not in
`surrounding normal cells. And because the activating beam of
`photons or neutrons can be sufficiently collimated to irradiate
`only the tumor plus a surrounding margin , sensitizer uptake in
`other remote tissues and organs does not lead to damage. The
`latter is only true, however, if the porphyrin itself does not cause
`toxicity to these other tissues and organs. Systemic administra(cid:173)
`tion of porphyrins and related compounds leads inevitably to
`exposure of a histologically diverse population of tissues and
`organs completely unrelated to the actual target tissues. This
`typically, but not necessarily, leads to observable and quantifi(cid:173)
`able alterations in plasma and/or cellular chemotoxic indicators.
`An example of a direct, medically beneficial chemotoxic effect
`of porphyrins in human pathologies is the application of tin
`
`" To whom correspondence should be addressed: Tel.: 415-476-4684.
`Fax: 415-476-0688. E-mail: sbkahl @picasso.ucsf.edu.
`t Deparlment of Pharmaceutical Chemistry.
`* Department of Neurological Surgery.
`§ Departmenl of Palhology.
`11 Department of Radiation Oncology.
`" Abbreviations: BNCT, boron neulron capture lherapy; BSH , boro(cid:173)
`captate sodium ; PDT, phoiodynami c therapy; TPPS 4, tetraphenylporphyrin
`tetrasulfonale.
`
`porphyrins in the control of hyperbilirubinemia in neonates
`through inhibition of heme oxygenase.2
`Surprisingly, despite this· widespread interest in the medicinal
`chemical aspects of porphyrins and their derivatives, there is a
`paucity of published information on the toxicology of systemi(cid:173)
`cally administered porphyrins in mammals. In PDT, the medical
`field in which porphyrins have been most widely applied, there
`is an almost complete lack of any published information re(cid:173)
`garding their chemotoxicities. In a study of motexafin lutetium(cid:173)
`mediated intraperitoneal PDT in a canine model , Hahn and co(cid:173)
`workers .noted mild, transient liver function abnormalities 3
`Miura et al. observed a significant decrease in platelet counts
`of mice given tetraphenylporphyrin tetrasulfonate (TPPS4) 4 This
`compound has also been reported to be neurotoxic.5 Reports in
`the BNCT field suggest that many boronated porphyrins al so
`exhibit thrombocytopenia, as well as mild
`to moderate
`hepatotoxicity.6- 10 These compounds have included both c/osa(cid:173)
`and nido-carboranes , but none bearing the extremely stable and
`nontoxic derivatives of the c/oso-borane anions [B 12H 12]2- and
`[B10H1of- .
`We repmt here the syntheses and comparative chemotoxicities
`in normal rats of a series of three c/oso-borane anion derivatives
`of tetraphenylporphyrin . These compounds differ from each
`other only in the nature of the covalent chemical linkage between
`the porphyrin and the dodecaborate (B 12) anion. We observed
`that the three compounds had substantially different maximum
`tolerated doses (MTDs) in rats, which manifested itself in
`moderate to severe hemorrhagic liver necrosis in two cases and
`moderate to severe hemorrhage or congestion in the heart, lungs,
`or kidneys (but not livers) of the third compound. The three
`derivatives also had noticeably different patterns of toxic effects
`in normal rats when given at the MTD. Both the least acutely
`toxic and the middle compound produced profound thrombo(cid:173)
`cytopenia at the MTD within the first 24 h after administration,
`while the most acutely toxic compound produced no such effect.
`This effect was paralleled by suppression of normal weight gain
`in these animals. Platelet counts returned to normal in all
`affected animals by the end of the 28-day experiment. Our
`results , in combination with previous observations, suggest that
`thrombocytopenia may be the most significant and com mon
`
`I 0.1 021/jm060895b CCC: $37 .00 © 2007 American Che mical Society
`Published on We b 01 /2512007
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2042 - 3/10
`
`

`

`Borane Anion-Substituted Tetraph enyl P01phyri11s
`
`l ou mal of Medicinal Chemist!)', 2007, \lol. 50, No. 4 821
`
`tox ic ity assoc ia ted w ith high-dose porphyrin admini strati o n in
`mamma ls and that a lternati ve methods of administTation of these
`compounds may be necessary to avoid tox ic e ffec ts.
`
`E xperimental Methods
`Cs2B 12H 12 and Na2B 12H11SH were purchased from Katchem, Ltd .
`(Prague, Czech Republic), and used without further puri fication.
`Na[B 12H 11 NH3) was prepared by the method of Hertler and
`Raasch. 11 [Ph3C H3h[B 12H 11 0H] was prepared by the method of
`Hawthorne and co-workers. 12 meso-Tetra(4-ca rboxyphenyl) por(cid:173)
`phine was purchased from Frontier Sc ientific and used without
`further purification. All other reagents and solvents were purchased
`fro m Aldrich C he micals and used without further purifi~ation.
`Solvents we re reagent grade unless otherwise spec ified and were
`dried and di stilled by standard methods shortl y before use. All
`reactions were carried out under inert atmosphere conditions using
`e ither argon or nitrogen. Grav ity and fl as h column chromatography
`empl oyed silica gel 60 GEDURAN (40 -63 ,u m; EM Sc ience).
`Routine NMR spectra (' H and 13C) were measured using a Vari an
`400 MHz spectrometer in solution as specified. Elemental analyses
`were performed by the Microanal ytical Laboratory in the Depart(cid:173)
`ment of Chemistry at UC Berkeley.
`Preparation of [(Pr)4N)[Bi zH11 NH3]. The sodium salt of the
`amine borane was prepared from Na2B 12H 12 by the published
`proceclure. 11 It was necessary to convert thi s salt to one more
`compatible with the solubility of the porph yrin acid chloride starting
`material. After ex perimenting with a variety of tetraalkyl ammonium
`salts, the CH2Cl2 solubility of the tetrapropylammonium salt was
`fo und to be ideal. Treatment of an aqueous solution of the sodium
`tetrapropylammonium
`iod ide followed by filtrati on
`salt with
`produced a white precipitate, which was recrys tallized from hot
`water to give an essentially quantitati ve yield of the desired material.
`Preparation of Tetrakis(tetrapropylammonium) Salt of TABP.
`meso-Tetra(4-carboxyphenyl) porphine (0.0975 g; 0. 123 111!1101) was
`suspended in anhydrous benzene (6 mL) in a I 00 mL pear-shaped
`fl ask under argon. To this was aclclecl oxalyl chloride (6 mL; 68 .8
`mmol), and the reaction mi xture was re flu x eel for 4 h. After cooling
`to rt, the benzene anclunreactecl oxalyl chloride were removed under
`vacuum . The resulting green solid was di ssolved in anhydrous
`methylene chloride (72 mL). Tetrapropylamine borane [(Pr)4N](cid:173)
`[B 12H 11 NH3) (0.495 g; 1.44 mmol), p-climethylaminopyricline (0.048
`g; 0.39 mmol), and trimeth ylamine (0.6 mL; 4.30 mmo l) were
`placed in a I L three- necked fl ask prev iously flu shed with argon.
`Meth ylene chloride (300 mL) was aclclecl, and the mi xtu re was
`brought to reflu x. To this was aclclecl clropwise over the course of
`I h the solution of the porphyrin acid chloride. This reaction mixture
`was reflu xecl overnight and cooled to roo m temperature, and the
`sol vents were removed under vac uum . The remaining solid was
`completely dissolved in 15% aqueous acetone (30 mL). Thi s
`solution was evaporated, and the process was repeated two more
`times. W ater (650 mL) was aclclecl to the remaining solids, and the
`suspension was stirred for 1.5 h and then filtered. The filtered brown
`solid was washed with water (3 x I 0 mL) and dried on the filter.
`This solid was pl aced in a 250 mL round-bottom fl as k and
`tetrapropylammonium iodide (0.070 g; 0.22 mmol) and water ( 120
`mL) were aclclecl . The mi xture was re flu xecl for 1.5 h, cooled to
`room temperature, and filtered. The violet-colored solid was washed
`with warm water (60 oc ; 8 x 2 mL) and air-dried. The product
`was dried under vacuum overnight to yield 0.2 14 g (0. I 02 mmo l;
`82.9%) of the desired product. This material produced a single spot
`(Rr = 0.50) on thin layer chromatography on DEAE cellulose in
`SO% aqueous acetone. 1H NMR (400 MHz, DMSO) o 9.11-7.63
`(m, 24H, aromatic and pyrrole), 3.07 (m, 32H, propyl CH), 1.56
`(m, 32H, propyl C H), 0. 82 (m, 48H , propyl CH), 1.80 - 0.80 (br,
`44H, BH), -2.95 (s, 2H, ring NH); 13C NMR (400 MHz, DMSO)
`0 168. 13, 146. 10, 139.77, 135 .1 3, 13 1. 16, 128 .62, 127.30, 11 9 .00,
`59.98, 15.48, 11.20 ; IR (KBr) 3420.9, 2972. 8, 2880.2,2487.5 (BH),
`171 8.5, 1637 .6, 1606.8 cm- 1; UV-v is (CH3CN) 4 18 nm (logE=
`5.6 1 ), 5 14 ( 4.27), 548 (3 .99), 590 (3.79), 644 (3.68) . Elem. anal.
`Calc. for C96H1 s6N 120 4B4s: C, 55. 13; H, 8.96; N, 8.04. Found : C,
`55.37 ; H, 7. 14; N , 7.90.
`
`Ion Exchange to Tetrasodium Sa lt of TABP. The above
`tetrakis(tetrapropylammonium) porphyrin borane salt (0.030 g) was
`I 5% aq ueous aceto ne (v/v) and passed through a
`dissolved in
`Dowex SOW x 2-200 resin column ( I x 3 em), prev iously
`generated in the Na+ form. T he e luted so lution was evaporated in
`vacuo. The solid remnant was redissolved in 50% aqueous ace tone
`(v/v) and passed th rough a fresh ion exchange column ( I x 4.8
`em). The e luted so luti on band containing the prod uct was evapo(cid:173)
`rated in vacuo to y ie ld 0.030 g of the des ired sodium salt product
`as a reel powde r, which was we ll so luble in water. 1 H NMR (400
`MHz, D20) o 8.92-8.17 (br m, 24H, aro matic and pyrrole), 2.05 -
`0.40 (br, 44H, borane BH ; there was no ev idence of any res idual
`propyl CH in this region, ring NH disappears clue to exchange
`w/solvent).
`Preparation of the Octasodium Salt of T E BP. meso-Tetra(4-
`carboxyphenyl) porphine (0.402 g; 0 .508 mmol) was suspended in
`anhydrous benzene (32 mL) in a 500 mL round-bottom fl ask under
`argon. To this was aclclecl oxalyl chloride (24 mL; 275 .2 mmol),
`and the reaction mi xture was reflu xecl for 4 h. After cooling to rt,
`the benzene and unreactecl oxal yl chloride were removed under
`vac uum . T he resulting green solid was dissolved in anhyd rous
`methylene chloride (405 mL). Na2ll3 12H11SH] ( 1.280 g; 1.44 mmol),
`p-climeth ylaminopyricline (0.2 12 g; 1.72 mmol), and trimethylamine
`( 1.4 mL; I 0.0 mmol) were placed in a I L three- necked fl ask
`previously flu shed with argon. Anh ydrous acetonitrile (270 mL)
`was aclclecl, and the mixture was brought to re flu x. To thi s was
`aclclecl clropwise over the course of 70 min the so lution of the
`porphyrin acid chlo ride. Thi s reaction mi xture was re flu xecl fo r 8
`min, cooled to room te mperature, and filtered. The produc t was
`washed with water (250 mL) and air-dried on the filter. The filtered
`solid was di ssolved in acetonit rile/water (7:3 v/v; 30 mL) and then
`stirred overnight w ith Dowex 50W x 2-200 resin (70 g) prev iously
`generated in the Na+ fo rm . The reel solution was filtered, and the
`resin was was hed with 70% acetonitrile/water un til the fi ltrate was
`ve ry pale pink. The filtrate was concentrated under vacuum and
`filtered through a 0.2 micron filter. This filtrate was then completely
`evaporated under vacuum to prov ide a deep reel crystalline powder
`(0.407 g; 50.2% yield), which was stored in a vac uum desiccator
`overnight. For e lemental analys is, the sodium salt was con verted
`to the octaki s(tetrapropylammonium) salt by treatment o f an
`aqueous solution with tetrapropylammonium bromide. The reel
`precipitate was was hed exte nsively with water and air-dried. 1 H
`NMR ( 400 M Hz, DMSO) o 8.82 (s, 8H, pyrrole CH), 8.3 1 (m,
`8H, aro matic), 8.25 (m, 8H, aromatic) . 1.90 - 0.40 (br, 44H, BH),
`-2.95 (s, 2H, ring NH); 13C NMR (400 M Hz, DMSO) o 168 .50,
`145.75 , 141.1 2, J 35.00, J 3 1.93 , 128 .54, 125.98, 120.02; IR (KBr)
`3389.8, 2487.6, 1702.3, 1605.1 cm- 1; UV - vis (H20 ) 4 16 nm (log
`E = 5.46), 5 18 (4. J 2), 556 (3.90), 580 (3 .76), 636 (3 .57). E lem.
`anal. for octapropyl ammonium salt. Calc. fo r C 144H2ssN 120 4B4sS4:
`C, 59.66; H, 10.01 ; H, 5.80. Found : C, 59.55; H, 10.09; N, 5 .60.
`Preparation of the Octakis(methyltriphenylphosphonium)
`Salt of TOBP. meso-Tetra(4-ca rbo xy phenyl) porphine (0.2 1 5 g;
`0.272 mmol) was suspended in anh ydrous benzene ( 16 mL) in a
`500 mL round-bottom fl ask under argon. To thi s was aclclecl oxalyl
`chloride ( 12 mL; 137.6 mmol), and the reaction mixture was
`reflu xecl fo r 4 h. After cooling to rt, the benzene and unreactecl
`oxalyl chloride were removed under vacuum. T he resul ting green
`solid was dissolved in anh ydrous meth ylene chloride (345 mL).
`[PPh3CH3)z[B 12H1 10H] ( 1.20 g; 1.69 mmol) and proton sponge (500
`mg) were dissolved in methylene chloride (600 mL) in a 2 L three(cid:173)
`neck round-bottom flask under argon and brought to reflu x. To
`thi s was aclclecl clropwise over I h the green acid chloride solution
`in meth ylene chl oride. This mi xture was then refl uxecl for 3 clays,
`cooled to room temperature, and poured into .a 4 L beaker, to which
`was immediately aclclecl ethyl alcohol (3 L) with sti rring. This slurry
`was stirred for an additional 4 h and fi ltered through a medium
`coarse filter funnel (600 mL). T he reel solid was then isolated and
`di ssol ved in acetonitrile/wate r (220: I 00) . The acetonitrile was
`removed under a water aspirator until most had been removed. T he
`reel product was fi ltered and washed with water until the filtrate
`was completely colorless. The reel solid was dissolved in acetonitrile
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2042 - 4/10
`
`

`

`822
`
`lou mal of Medicinal Chemi.wy, 2007, Vol. 50, No. 4
`
`(I 00 mL) at 20 °C, and ethanol was added (300 mL) with stirring.
`The crystallized red solid was then isolated by filtration to yield
`0.678 g (0.190 mmol ; 69.8% yield). 1H NMR (400 MHz, DMSO)
`o 8.80 (s, 8H, pyrrole CH), 8.21 (m, 16H, porphyrin aromatic),
`7 .89-7.66 (m, 120H, phosphonium aromatic), 3.07 (s, 24H,
`phosphonium methyl), 1.80- 0.40 (br, 44H, BH), -3.00 (s, 2H,
`ring NH); 13C NMR (400 MHz, DMSO) o 165 .69, 145.2 1, 145.37,
`135.40, 134.72, 134.24, 133.55 , 13 1.20, 130.50, 129.61 , 128.08,
`127.30, 127.05, 122.54, 119.50, 45 .83; IR (KBr) 3591.0, 3056.6,
`2483.8, 1681.5, 1604.6 cm- 1; UV -vis (CH3CN) 416 nm (log E =
`5.69), 514 (4 .29), 548 (4.03), 590 (3.81), 644 (3.69); Elem. anal.
`Calc. for C2ooH214N40sB4sPs: C, 67.31; H, 6.04; N, 1.57. Found:
`C, 67.52; H, 6. 13; N, 1.67.
`Ion Exchange to the Octasodium Salt of TOBP. The above
`octakis(methyltriphenylphosphonium) porphyrin borane salt ( 161
`mg) was dissolved in 25% aqueous acetonitrile (v/v) and passed
`through a Dowex SOW x 2-200 resin column (I x 3 em). The
`eluted solution was evaporated in vacuo. The solid remnant was
`redissolved in 50% aqueous acetonitrile (v/v) and passed through
`a fresh ion exchange column (I x 4.8 em). The eluted solution
`band containing the product was evaporated in vacuo to yield 112
`mg of the desired sodium salt product as a red powder, which was
`well soluble in water and quite hygroscopic. 1H NMR (400 MHz,
`DMSO) o 8.82 (s, 8H, pyrrole CH), 8.21 (m, 16H, aromatic), 1.80-
`0.40 (br, 44H, BH; there is no evidence of additional aromatic or
`methyl protons indicative of the phosphonium cation), -2.95 (s,
`2H, ring NH); 13C NMR (400 MHz, DMSO) o 169.62, 149.21 ,
`144.76, 135.99, 132.36, 131.50, 128.37, 119.92; UV-vis (H20)
`416 nm (log E = 5.44), 518 (3.95), 554 (3.82), 582 (3 .67), 636
`(3.49).
`Animals. Six-week-old male Fischer 344 rats ( ~200 g body
`weight) were purchased from Simonsen Laboratories (Gilroy, CA)
`and used in all studies. Rats were housed and cared for in
`accordance with the United States Department of Health and Human
`Services Guide for the Care and Use of Laboratory Animals. All
`protocols were approved by the UCSF Institutional Animal Care
`and Use Committee. Rats were maintained in a temperature and
`li ght-controlled environment with an alternating 12-hour light/dark
`cycle and provided food and water ad Libitum. Rats were anesthe(cid:173)
`tized by an intraperitoneal· injection of ketamine (60 mg/kg) and
`xylazine (7.5 mg/kg) for drug injection and blood collection.
`Determination of Maximum Tolerated Dose. Anesthetized
`animals were injected i.v. via tail vein with graded concentrations
`of aqueous solutions of the boronated compounds adjusted to pH
`7.4 with NaHC03 and were monitored closely up to 28 days.
`Dosage groups consisted of 2 to 4 animals per dose. When animals
`showed systemic symptoms such as loss of activity or hemorrhaging
`fro m body orifices, animals were euthanized. Dosages at which
`50% or more of the animals died at any point during the 28-day
`period were considered to be above the MTD. Organs were removed
`and fixed with 10% buffered formalin for 24 to 48 hand then cut
`with a razor blade and embedded in paraffin. The paraffin-fixed
`tissue was cut into 5 pm-thick sections, stained with hematoxylin
`and eosin using standard staining methodology, and used for
`histopathologic investigations.
`Toxicology at the Maximum Tolerated Dose. After determining
`the MTD for each compound, an investigation was made of the
`effects of injecting the three boronated porphyrins at their respective
`MTDs on various blood chemistry values, including: platelets,
`hematocrit (Hct), low-density lipoprotein (LDL), alanine amino(cid:173)
`transferase (ALT), aspartate aminotransferase (AST), albumin (alb),
`and serum potassium (K+). Porphyrins were administered .at the
`MTD by i. v. bolus into the tail veins of two groups of four animals
`each. One group was used to provide blood samples for platelet
`and hematocrit determinations and the other for the remaining
`parameters. Blood samples were taken 24 h prior to compound
`administration to provide baseline comparative values. Blood
`samples were then collected l , 7, and 28 days postinjection via
`tail arteries, and samples were analyzed using the Roche COBAS
`Mira Classic automated chemistry analyzer (Roche Diagnostic
`System, Branchburg, NJ) and the Serano-Baker 9010 hematology
`
`Koo eta/.
`
`n
`
`TABP X= NH2 n = 4
`n = B
`TEBP X= S
`
`n =B
`TOBP X= 0
`Figure 1. Structures of the three borane anion porphyrin derivatives
`prepared. Open circles represent BH units except at the point of linkage
`to the phenyl rings where they represent a boron atom.
`
`analyzer (Serano Baker Co., Allentown, PA) in the Comparative
`Pathology Laboratory at
`the University of California, Davis.
`Animals were also weighed at the time of each blood draw to assess
`overall health.
`
`Results
`
`Chemistry. One of the goals of our synthe tic work in this
`area was the successful preparation and characterization of a
`structurally related series of water-soluble borane anion deriva(cid:173)
`tives of tetraphenyl porphine. The para-carboxylic acid group
`was chosen for the linkage chemistry largely as a result of the
`breadth of stable derivatives that could potentially be formed
`2- polyhedral anion derivatives. As a result,
`from known B 12H 12
`amide (TABP), thioester (TEBP), and oxoester (TOBP) deri va(cid:173)
`tives were successfully prepared and characterized (Figure I ).
`For synthesis of the first compound (TABP), we felt it
`necessary to initially convert the sodium salt of B 12H 1 1NH3- to
`a salt more appropriate to the solvent conditions to be used for
`coupling to the acid chloride (methylene chloride). Afte r
`experimenting with tetraalkylammonium cations of various
`carbon chain lengths, it was found that the propyl side chain
`presented the optimum solubilizing characteristics. After com(cid:173)
`bining the reactants and refluxing overnight, complete conver(cid:173)
`sion into the tetrapropylammonium salt was insured by refluxing
`the crude material in water containing tetrapropylammoniu m
`iodide. The final product, obtained in 83% overall yield, was
`characterized in this form because the sodium salt is difficult
`to purify and is hygroscopic. The proton NMR of this compound
`showed no evidence of the protonated imino! form of the amide
`tautomer reported by Gabel et al. for acyl derivati ves
`[B 12H 10NH3r ion. 13 For administration to animals, the tetra(cid:173)
`propylammonium salt was ion exchanged several times in an
`aqueous acetone solution until 1H NMR showed no evidence
`of saturated CH signals. In the preparation of TEBP, we tri ed
`to avoid such an elongated procedure and found that addition
`of some acetonitrile to the borane anion solution was sufficient
`to keep the sod ium salt in so lution while adding the methyl ene
`chloride solution of the porphyrin acid chloride. This made it
`possible to isolate the sodium salt directly, although a s ma ll
`amount of the product was then converted to the tetrapropyl(cid:173)
`ammonium · salt for elemental analysis. The oxoester TOBP
`followed the original pattern and was isolated initially as the
`water insoluble methyltriphenylphosphonium salt and then
`converted into the water so luble sodium salt by ion exchange
`prior to administration. All three porphyrins have UV -visible
`absorption spectra of the elio-type that is typical for the
`
`CFAD v. Anacor, IPR2015-01776 ANACOR EX. 2042 - 5/10
`
`

`

`Borane Anion-Subslilu/ed Telrapll enyl P01phyrins
`
`Joumal of Medicinal Che111is1ry, 2007, \I of. 50, No.4 823
`
`Table 1. Liver Enzy me Leve ls in Norma l Fischer 344 Rats G iven Each Porph yrin at the MTD
`
`rat
`
`day -
`
`I
`
`day I
`
`day 7
`
`AL T (uni t/L)
`
`I'
`2'
`3'
`4'
`
`5'
`6'
`7'
`8'
`
`13'
`14'
`15'
`16'
`
`54
`66
`54
`52
`
`51
`51
`48
`52
`
`49
`45
`47
`38
`
`65
`50
`67
`79
`
`90
`(dead)
`(dead)
`576
`
`81
`81
`65
`48
`
`48
`45
`45
`48
`
`150
`
`170
`
`96
`137
`218
`83
`
`. 48
`47
`43
`56
`
`day 28
`rat
`TABP 15 mg/kg
`I'
`2'
`3'
`4'
`TEBP 100 mg/kg
`5'
`6'
`7'
`8'
`TOBP 40 mg/kg
`13'
`(hemolyzed)
`14'
`(hemolyzed)
`15'
`60
`16'
`45
`
`58
`
`71
`
`AST (uni t/L)
`
`day -
`
`I
`
`day I
`
`day 7
`
`day 28
`
`11 4
`244
`127
`133
`
`99
`104
`90
`128
`
`86
`74
`71
`69
`
`160
`126
`137
`199
`
`183
`(dead)
`(dead)
`687
`
`11 7
`156
`165
`84
`
`138
`75
`120
`90
`
`87
`
`47
`
`43
`51
`105
`47
`
`84
`87
`69
`73
`
`11 2
`
`11 8
`
`(hemolyzed)
`(he molyzed)
`104
`88
`
`Table 2. Platelet Counts and Hematocrit in Norma l Fischer 344 Rats
`Given Each Porphyrin at the MTD
`
`plate let ( x I Q3fmm3)
`
`hematocrit (%)
`rat day - I day I day 7 day 28
`rat day - I day I day 7 day 28
`TABP 15 mg/kg
`626
`36.7
`I
`420
`2
`34.3
`673
`3
`34.8
`4
`44.4
`669
`TEBP 100 mg/kg
`611
`5
`35.3
`62 1
`6
`3 1.2
`7 18
`7
`34.5
`33.1
`730
`8
`TOBP 40 mg/kg
`13
`769
`30.7
`14
`28.6
`650
`15
`624
`33. 1
`16
`19.3
`600
`
`tetraphenyl porphine framework. Their infrared spectra have
`carbonyl stretching frequencies that suggest substantial electron
`donation fro m the borane anion into the linkage group. For
`example, the carbonyl stretch ofTOBP is found at 168 1 cm- 1,
`which is almost identical to that of the benzoyl deri vati ve of
`[B 12H 11 0H] - 2 reported to be 1683 cm- 1 by Gabel and co(cid:173)
`workers.14 Like them, we also found that the BH stretching
`frequency was insensiti ve to the nature of the deri vative and
`was invari abl y observed to fall within a narrow range of 2484-
`2488 cm- 1. Thus, any electron donation fro m the polyhedral
`anion across the linker into the aromatic ring is not re flected
`by changes in the B- H bond strength .
`Toxicology. For the amide TABP, a MTD in normal