(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`23 September 2010 (23.09.2010)
`
`PCT
`
`1111111111111111 IIIIII IIIII 111111111111111 II Ill lllll 111111111111111 IIIII IIII IIIIIII IIII IIII IIII
`
`(10) International Publication Number
`WO 2010/108125 A2
`
`(51) International Patent Classification:
`C07D 257/00 (2006.01)
`A61K 49/00 (2006.01)
`A61K 31/41 (2006.01)
`A61P 35/00 (2006.01)
`
`(21) International Application Number:
`PCT /US20 I 0/028020
`
`(22) International Filing Date:
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`19 March 2010 (19.03.2010)
`
`English
`
`English
`
`(30) Priority Data:
`61/161,485
`61/161,484
`61/248,067
`61/248,934
`
`19 March 2009 (19.03.2009)
`19 March 2009 (19.03.2009)
`2 October 2009 (02.10.2009)
`6 October 2009 (06.10.2009)
`
`us
`us
`us
`us
`Applicant (for all designated States except US): THE
`JOHNS HOPKINS UNIVERSITY
`[US/US]; 3400
`North Charles Street, Baltimore, Maryland 21218 (US).
`
`(71)
`
`(72)
`(75)
`
`Inventors; and
`Inventors/Applicants (for US only): POMPER, Martin
`G.
`[US/US];
`IOI Churchwarden's Road, Baltimore,
`Maryland 21212 (US). MEASE, Ronnie C. [US/US];
`12320 Cannonball Road, Fairfax, Virginia 22030 (US).
`RAY, Sangeeta [IN/US]; 3358 North Chatham Road,
`Apartment L, Ellicott City, Maryland 21042 (US).
`
`CHEN, Ying [CN/US]; 14 Breezy Tree Court, Apartment
`I, Timonium, Maryland 21093 (US).
`
`(74) Agent: HADDAWAY, Keith G.; VENABLE LLP, P.O.
`Box 34385, Washington, DC20043-9998 (US).
`
`(81)
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA,CH,CL,CN,CO,CR,CU,CZ,DE,DK,DM,DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, VA, VG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`ML, MR, NE, SN, TD, TG).
`
`Declarations under Rule 4.17:
`
`[Continued on next page]
`
`(54) Title: PSMA-TARGETING COMPOUNDS AND USES THEREOF
`
`--------------------------------------------
`
`;;;;;;;;;;;;;; ----;;;;;;;;;;;;;;
`;;;;;;;;;;;;;; ----
`-;;;;;;;;;;;;;; -;;;;;;;;;;;;;; -
`;;;;;;;;;;;;;; -
`-;;;;;;;;;;;;;; --;;;;;;;;;;;;;;
`;;;;;;;;;;;;;; ----;;;;;;;;;;;;;; -
`
`M <
`"' M
`
`30MIN
`
`3.5h
`
`FIG.1
`
`(57) Abstract: Prostate-specific membrane antigen (PSMA) targeting compounds are described. Uses of the compounds for imag(cid:173)
`ing, therapy, cell sorting, and tumor mapping are also described.
`
`,-.-1
`Q0
`0
`,-.-1
`.........
`0
`,-.-1
`0
`M
`0
`~
`
`Petitioner GE Healthcare – Ex. 1048, p. Front page-1
`
`

`

`w o 2010/108125 A2 1111111111111111 IIIIII IIIII 111111111111111 II Ill lllll 111111111111111 IIIII IIII IIIIIII IIII IIII IIII
`
`as to applicant's entitlement to apply for and be granted
`a patent (Rule 4.17 (ii))
`as to the applicant's entitlement to claim the priority of
`the earlier application (Rule 4.17 (iii))
`
`of inventors hip (Rule 4.17(iv))
`
`Published:
`
`without international search report and to be republished
`upon receipt of that report (Rule 48.2(g))
`
`Petitioner GE Healthcare – Ex. 1048, p. Front page-2
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`PSMA-TARGETING COMPOUNDS AND USES THEREOF
`
`CROSS-REFERENCE TO RELATED APPLICATION
`
`[0001]
`This application claims priority to U.S. Provisional Application Nos.
`61/161,484 filed March 19, 2009, 61/161,485, filed March 19, 2009, 61/248,067, filed October
`2, 2009, and 61 /248,934, filed October 6, 2009. The entire content of each Provisional
`Application is hereby incorporated by reference in their entirety. This invention was made using
`U.S. Government support under NIH grant NIH U24 CA92871. The government has certain
`rights in this invention.
`
`BACKGROUND
`
`Field of the Invention
`[0002]
`
`The present invention relates to prostate specific membrane antigen (PSMA)
`binding compounds, chemical precursors of PSMA binding compounds and imaging methods of
`using the compounds.
`
`Background
`
`[0003]
`Prostate cancer (PCa) is the most commonly diagnosed malignancy and the
`second leading cause of cancer-related death in men in the United States (Cancer Facts &
`Figures; American Cancer Society: Atlanta, GA, 2009). In 2009, it is estimated that 192,000
`men will be diagnosed with prostate cancer and 27,000 men will die of the disease. Only one
`half of tumors due to PCa are clinically localized at diagnosis and one half of those represent
`extracapsular spread. Localization of that spread as well as determination of the total body
`burden of PCa have important implications for therapy, particularly as new combination and
`focal therapies become available.
`[0004]
`The prostate-specific membrane antigen (PSMA), while expressed in prostate
`tumor epithelium, has a curious property in that it is expressed in the neovasculature of many
`solid tumors but not in that of prostate cancer (Chang et al., Cancer Res., vol. 59, pp. 3192-3198,
`1999; Chang et al., Clin. Cancer Res., vol. 5, pp. 2674-2681, 1999; Gong et al., Cancer
`Metastasis Rev., vol. 18, pp. 483-490, 1999; Chang et al., Mol. Urol., vol. 3, pp. 313-320, 1999;
`Baccala et al., Urology, vol. 70, pp. 385-390, 2007; Chang et al., Urology, vol. 57, pp. 801-805,
`
`1
`
`Petitioner GE Healthcare – Ex. 1048, p. 1
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`2001Milowsky et al., J. Clin. Oncol., vol. 25, pp. 540-547, 2007). Because of that property, an
`111In-labeled monoclonal antibody to an extracellular epitope of PSMA, 111In-J591, was capable
`of identifying renal, bladder, lung, breast, colorectal and pancreatic tumors in a Phase I clinical
`imaging study (Milowsky et al., J. Clin. Oncol., vol. 25, pp. 540-547, 2007). That study
`validated 111In-J591 as a vascular targeting agent in human subjects. Since then other reports
`have further studied PSMA expression in certain tumor types. Baccala et al. noted that clear cell
`renal cell carcinoma expresses significantly more PSMA in its neovasculature than does the
`papillary variety (Baccala et al., Urology, vol. 70, pp. 385-390, 2007). Furthermore,
`angiomyolipoma, a benign renal lesion, did not express PSMA. As an enzyme with an
`extracellular active site, PSMA represents an excellent target for imaging and therapy directed
`toward solid tumor neovasculature in addition to prostate cancer itself. PSMA-based agents can
`report on the presence of this marker, which is increasingly recognized as an important
`prognostic determinate in PCa (Murphy et al., Urology, vol. 51, pp. 89-97, 1998). It is also the
`target for a variety of new PCa therapies (Galsky et al., J Clin Oneal, vol. 26, pp. 2147-2154,
`
`2008).
`
`[0005)
`
`ProstaScint™ is an 111In-labeled monoclonal antibody against PSMA that is
`clinically available for imaging PCa. Radioimmunotherapy based on ProstaScint™ and
`
`radio labeled variations of this antibody are fraught with similar difficulties to the use of
`radiolabeled antibodies for imaging, including prolonged circulation times, poor target to
`nontarget tissue contrast, unpredictable biological effects and the occasional need for pre(cid:173)
`targeting strategies, limiting the utility of these agents (Lange, P.H., Urology, vol. 57, pp. 402-
`406, 2001; Haseman et al., Cancer Biother Radiopharm, vol. 15, pp. 131-140, 2000; Rosenthal
`et al., Tech Ural, vol. 7, pp. 27-37, 2001). Furthermore, antibodies may have less access to
`tumor than low molecular weight agents, which can be manipulated pharmacologically.
`
`[0007)
`
`[0006)
`
`The development of low molecular weight radiotherapeutic agents is much
`different from developing radiopharmaceuticals for imaging in that longer tumor residence times
`can often be important for the former.
`Complete detection and eradication of primary tumor and metastatic foci are
`required to effect a cure in patients with cancer; however, current preoperative assessment often
`misses small metastatic deposits. More sensitive imaging techniques than computed
`tomography, magnetic resonance imaging and even positron emission tomography (PET), which
`can be used easily in the operating suite, are required. An old technique, recently revisited
`
`2
`
`Petitioner GE Healthcare – Ex. 1048, p. 2
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`because of improved optics and fluorescent dye chemistry, is intraoperative photodiagnosis
`(PDD) (Toda, Keio J. Med., vol. 57, pp. 155-161, 2008). Fluorescein dyes have been used
`intraoperatively to identify brain tumors and verify the clarity of tumor margins since 1948
`(Toda, Keio J. Med., vol. 57, pp. 155-161, 2008). A recent report describes its utility in
`identifying brain metastases (Okuda et al., Minim. Invasive Neurosurg., vol. 50, pp. 382-384,
`2007). A long history of the use of 5-aminolevulinic acid (5-ALA) for brain tumor resection is
`also evident, and its use has been associated with improvement in progression-free survival
`(Stummer et al., Lancet Oncol., vol. 7, pp. 392-401, 2006). PDD can be performed easily during
`surgery due to the lack of a need for complex imaging equipment. All that is needed is a light(cid:173)
`emitting diode to excite the fluorophore, which can be administered systemically or "painted" on
`the tissue directly. More recent incarnations of PDD have used quantum dots (Amdt-Jovin et
`al., IEEE Trans Nanobioscience, 2009), and more advanced dyes, such as indocyanine green
`(ICG) (Gotoh et al., J. Surg. Oncol., 2009), which emit in the near-infrared (NIR) region of the
`spectrum, enabling reasonable tissue penetration of emitted (and detected) light. Applications
`have included nontargeted approaches, such as preoperative evaluation of the vascular integrity
`of surgical flaps or identification of nodules of hepatocellular carcinoma (Matsui et al., Plast.
`Reconstr. Surg., vol. 123, pp. 125e-127e, 2009). Targeted approaches are also emerging, such
`as use of a fluorophore-conjugated anti-CEA antibody to identify colon or pancreatic cancer
`(Kaushal et al., J. Gastrointest. Surg., vol. 12, pp. 1938-1950, 2008), or the use ofNIR
`activatable probes that emit light only when cleaved by a tumor-associated protease (Sheth et al.,
`Gynecol. Oncol., vol. 112, pp. 616-622, 2009).
`Recently, the application of 68Ga-labeled peptides has attracted considerable
`[0008]
`interest for cancer imaging because of the physical characteristics of Ga-68 (Reubi et al., J Nucl
`Med, vol. 49, pp. 1735-1738, 2008). Ga-68 is available from an in-house 68Ge/68Ga generator
`68Ge, 1112 = 270.8 day), which renders it independent of an onsite cyclotron. Therefore, 68Ga(cid:173)
`(
`based PET agents possess significant commercial potential and serve as a convenient alternative
`to cyclotron-based isotopes for positron emission tomography (PET), such as 18F or 1241. 68Ga
`has a high positron-emitting fraction (89% of its total decay). The maximum positron energy of
`68Ga (max. energy= 1.92 MeV, mean= 0.89 MeV) is higher than that of 18F (max= 0.63 MeV,
`mean= 0.25 MeV). However, a study of spatial resolution using Monte Carlo analysis revealed
`that under the assumption of 3 mm spatial resolution for most PET detectors, the full-width-at(cid:173)
`half-maximum (FWHM) of 18F and 68Ga are indistinguishable in soft tissue (3.01 mm vs. 3.09
`
`3
`
`Petitioner GE Healthcare – Ex. 1048, p. 3
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`mm) (Sanchez-Crespo et al., Eur J Nucl Med Mo! Imaging, vol. 31, pp. 44-51, 2004). That
`finding implies that with the standard spatial resolution of 5 to 7 mm for current clinical
`scanners, image quality using 68Ga-based radiotracers will likely be indistinguishable from that
`of 18F-based agents, stimulating interest in the development of 68Ga-labeled compounds for
`medical imaging (Sanchez-Crespo et al., Eur J Nucl Med Mo! Imaging, vol. 31, pp. 44-51, 2004;
`Khan et al., Eur J Surg Oneal, vol. 35, pp. 561-567, 2009; Fani et al., Contrast Media Mot
`Imaging, vol. 3, pp. 67-77, 2008). With a physical half-life of 68 min, 68Ga is also matched
`nicely to the pharmacokinetics of many peptides used for imaging. Few 68Ga-labeled,
`mechanism-based radiotracers for prostate cancer have been reported previously, and none for
`PSMA. Furthermore, 68Ga is introduced to biomolecules through macrocyclic chelators, which
`allows possible kit formulation and wide availability of the corresponding imaging agents.
`
`SUMMARY OF THE INVENTION
`The present invention satisfies the long standing and unmet need for new
`[0009)
`imaging and therapeutic compounds for targeting prostate cancer and cancer angiogenesis. The
`present invention, in particular, provides therapeutic compounds and imaging agents which
`differ from the prior art in modifications which were not previously known or suggested.
`Furthermore, the invention provides imaging agents that offer better contrast between target
`tissues and non-target tissues. The invention also provides compounds with greater cellular
`retention and low molecular weight.
`Embodiments of the invention include compounds having the structure
`
`[0010)
`
`wherein the subunits associated with elements p, q, r, ands may be in any
`[0011)
`order. Z is tetrazole or CO2Q; each Q is independently selected from hydrogen or a protecting
`group, a is 1, 2, 3, or 4, and R is each independently Hor C1-C4 alkyl.
`Variable r is O or 1. Tz is a triazole group selected from the group consisting of
`[0012)
`
`4
`
`Petitioner GE Healthcare – Ex. 1048, p. 4
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`Rs
`)-(CH2)d-~-
`where LI is -~-(CHz)d-~- or -~-X2
`
`, L 2 is -!-(CHz)b-~- or
`
`Rs
`-!-(CH2)b---( 1 ~
`X -~-, X1 is -NRC(O)-, -NRC(O)NR-, -NRC(S)NR-, or -NRC(O)O-; X2 is
`-C(O)NR-, -NRC(O)NR-, -NRC(S)NR-, or -OC(O)NR-; R5 is H, CO2H, or CO2R6, where R6 is
`a C1-C6 alkyl, C2-C12 aryl, or C4-C16 alkylaryl; bis 1, 2, 3, or 4; and dis 1, 2, 3, or 4.
`Variable q is O or 1. Wis -NRC(O)-, -NRC(O)NR-, NRC(S)NR-, -NRC(O)O-,
`[0013]
`-OC(O)NR-, -OC(O)-,-C(O)NR-, or -C(O)O-; R2 and R3 are independently H, CO2H, or CO2R4,
`where R4 is a C1-C6 alkyl, C2-C12 aryl, or C4-C16 alkylaryl, wherein if one of R2 and R3 is CO2H
`or CO2R2, then the other is H; n is 1, 2, 3, 4, 5 or 6.
`Variables is 0 or 1. Y is -C(O)-, -NRC(O)-, -NRC(S)-, -OC(O); and mis 1, 2,
`[0014]
`
`3, 4, 5, or 6.
`Variable pis 0, 1, 2, or 3, and when pis 2 or 3, each R1 may be the same or
`[0015]
`different. R1 is H, C1-C6 alkyl, C2-C12 aryl, or C4-C16 alkylaryl.
`G is a moiety selected from the group consisting of
`[0016]
`
`-~-N3 -~ ==
`
`'
`
`Ch_,,.V,N'\:_ FG_,,V,N'\:_
`R
`R
`
`'
`
`'
`
`'
`
`where Ch is a metal chelating moiety, optionally including a chelated metal; FG is a fluorescent
`dye moiety which emits in the visible or near infrared spectrum; one of A and A' is Ch and the
`other is FG; V and V' are independently -C(O)- , -NRC(O)- , -NRC(S)-, or -OC(O)-; and g is
`1, 2, 3, 4, 5, or 6. The following conditions also apply:
`
`5
`
`Petitioner GE Healthcare – Ex. 1048, p. 5
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`A'-V' "'
`NH R
`R
`)\J-(CH2)g-l .. r\t
`If
`A-V
`O
`
`Ch_.,,V,N\_
`I
`R
`
`, or
`
`and r is 0, then q ands are
`
`1)
`
`when G is
`
`both 1;
`
`2)
`
`when G is
`
`FG_.-V,N\_
`I
`R
`
`0
`
`and r is 0, then q and s are both 0 or both 1;
`
`HO)(,:,
`
`3)
`
`when G is
`
`~N-~-
`N.::N'
`
`then pis 0 and R2 is H, and the structure optionally
`
`includes a chelated metal ion.
`
`Ch_.,,V,N\_
`I
`when G is •
`R
`
`4)
`
`and the other is H; and
`
`and r is 0, then if pis 0, then one of R2 and R3 is CO2R2,
`
`5)
`
`when g is -~-N3 or -~ ==, then r is 0.
`
`[0017]
`
`Embodiments include compounds having the structure
`
`wherein Z is tetrazole or CO2Q; each Q is independently selected from hydrogen or a protecting
`group, a is 1, 2, 3, or 4, and R is each independently Hor C1-C4 alkyl. Ch is a metal chelating
`' moiety optionally including a chelated metal. Wis -NRC(O)-, -NRC(O)NR-, NRC(S)NR-,
`-NRC(O)O-, -OC(O)NR-, -OC(O)-, -C(O)NR-, or -C(O)O-. Y is -C(O)-, -NRC(O)-, -NRC(S)-,
`-OC(O). V is-C(O)-, -NRC(O)-, -NRC(S)-, or-OC(O)-. In exemplary embodiments mis 1,
`2, 3, 4, 5, or 6; n is 1, 2, 3, 4, 5 or 6; and pis 0, 1, 2, or 3, and when pis 2 or 3, each R1 may be
`the same or different. R1 is H, C1-C6 alkyl, C2-C12 aryl, or C4-Ci6 alkylaryl. R2 and R3 are
`independently H, CO2H, or CO2R4, where R4 is a C1-C6 alkyl, C2-C12 aryl, or C4-C16 alkylaryl,
`6
`
`Petitioner GE Healthcare – Ex. 1048, p. 6
`
`

`

`HO\:Ni(CH) __J w ~1
`
`N.:::N'
`
`2
`n W
`
`(CH2)m-Y-N
`
`WO 2010/108125
`
`PCT /0S2010/028020
`
`wherein when one ofR2 and R3 is CO2H or CO2R2, the other is H, and when pis 0, one ofR2
`and R3 is CO2R4, and the other is H.
`[0018]
`Some embodiments further include a chelated metal. In some embodiments,
`
`the chelated metal is Tc, In, Ga, Y, Lu, Re, Cu, Ac, Bi, Pb, Sm, Sc, Co, Ho, Gd, Eu, Tb, or Dy.
`
`In some embodiments, the chelated metal an isotope, for example. In some embodiments, the
`
`isotope is Tc-94m, Tc-99m, In-111, Ga-67, Ga-68, Y-86, Y-90, Lu-177, Re-186, Re-188, Cu-64,
`
`Cu-67, Co-55, Co-57, Sc-47, Ac-225, Bi-213, Bi-212, Pb-212, Sm-153, Ho-166, or Dy-166.
`
`Embodiments include compounds having the structure
`
`0
`
`s (CH2)a O ~ z
`002C~N)l_N C020
`H
`H
`optionally including a chelated metal ion. Z is tetrazole or CO2Q; each Q is independently
`selected from hydrogen or a protecting group, and a is 1, 2, 3, or 4. R is each independently H
`
`q
`
`or C1-C4 alkyl. Wis -NRC(O)-, -NRC(O)NR-, NRC(S)NR-, -NRC(O)O-, -OC(O)NR-,
`
`-OC(O)-, -C(O)NR-, or -C(O)O-. Y is -C(O)-, -NRC(O)-, -NRC(S)-, -OC(O)-;
`
`[0019]
`In exemplary embodiments mis 1, 2, 3, 4, 5, or 6; n is 1, 2, 3, 4, 5 or 6; q is O or
`1; ands is O or 1. R3 is H, CO2H, or C02R4, where R4 is a C1-C6 alkyl, C2-C12 aryl, or C4-C16
`alkylaryl. Some embodiments further include a chelated metal ion. In some embodiments, the
`
`metal ion is Tc, Re, Cu, or Ga. In some embodiments, the metal ion is Tc-99m, Re-186, Re-188,
`
`Cu-64, or Ga-68. In some embodiments, the metal ion is Tc-99m.
`[0020]
`
`Embodiments include compounds having the structure
`
`(CH,) 1 ~
`FG,v'N r [R' ~
`I r1 R'
`
`R
`
`P
`
`~l
`" w (CH2)m-Y~Nt----
`s (CH2)a O ~ z
`002C~N)l_N C020
`H
`H
`where p, q, and s are in the order drawn, and q and s are either both O or both 1. Z is tetrazole or
`
`q
`
`CO2Q; each Q is independently selected from hydrogen or a protecting group, and a is 1, 2, 3, or
`
`7
`
`Petitioner GE Healthcare – Ex. 1048, p. 7
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`4. FG is a fluorescent dye moiety which emits in the visible or near infrared spectrum. R is
`each independently Hor C1-C4 alkyl.Vis -C(O)- or-NRC(O)- or-NRC(S)-. Wis -NRC(O)-,
`-NRC(O)NR-, NRC(S)NR-, -NRC(O)O-, -OC(O)NR-, -OC(O)-, -C(O)NR-, or -C(O)O-. Y is
`
`-C(O)-, -NRC(O)-, -NRC(S)-, -OC(O). In exemplary embodiments mis 1, 2, 3, 4, 5, or 6; n is 1,
`2, 3, 4, 5 or 6; pis 0, 1, 2, or 3, and when pis 2 or 3, each R1 may be the same or different. R1 is
`H, C1-C6 alkyl, C2-C 12 aryl, or C4-C16 alkylaryl. R2 and R3 are independently H, CO2H, or
`CO2R2, where R2 is a C1-C6 alkyl, C2-C12 aryl, or C4-C16 alkylaryl, wherein when one of R2 and
`R3 is CO2H or CO2R2, the other is H. In some embodiments, the fluorescent dye moiety emits in
`the near infrared spectrum.
`
`[0021)
`
`Embodiments include compounds having the structure
`
`\-:~H,:~o hR1 ~h
`
`-V/
`A
`
`~
`R
`
`O
`
`p R2
`
`RI
`~3
`( CH2)n ___,___
`W-(CH2)m-Y-N-(CH2)4 0 ~ z
`
`002C~N)lN C020
`H
`H
`wherein Z is tetrazole or CO2Q; each Q is independently selected from hydrogen or a protecting
`group, and a is 1, 2, 3, or 4. One of A and A' is Ch and the other is FG, where FG is a
`
`fluorescent dye moiety which emits in the visible or near infrared spectrum and Ch is metal
`
`chelating moiety optionally including a chelated metal. R is each independently Hor C1-C4
`
`alkyl. V or V' are independently -C(O)- , -NRC(O)- , or -NRC(S)-. Wis -NRC(O)-,
`
`-NRC(O)NR-, NRC(S)NR-, -NRC(O)O-, -OC(O)NR-, -OC(O)-, -C(O)NR-, or -C(O)O-. Y is
`
`-C(O)-, -NRC(O)-, -NRC(S)-, -OC(O). In exemplary embodiments mis 1, 2, 3, 4, 5, or 6; n is 1,
`2, 3, 4, 5 or 6; and g is 1, 2, 3, 4, 5, or 6; pis 0, 1, 2, or 3, and when pis 2 or 3, each R1 may be
`the same or different. R1 is H, C1-C6 alkyl, C2-C12 aryl, or C4-C16 alkylaryl. R2 and R3 are
`independently H, CO2H, or C02R4, where R4 is a C1-C6 alkyl, C2-C12 aryl, or C4-C16 alkylaryl,
`wherein when one ofR2 and R3 is CO2H or CO2R2, the other is H. In some embodiments, the
`fluorescent dye moiety emits in the near infrared spectrum. Some embodiments further include
`
`a chelated metal.
`
`8
`
`Petitioner GE Healthcare – Ex. 1048, p. 8
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`[0022)
`
`Embodiments include compounds having the structure
`1
`
`llR
`~
`G ' l )'N p
`
`3
`R
`(CH2k-l
`
`~
`
`2
`R
`
`q
`
`Tz 1 f(CH2)m-Y-~l
`0
`~ ;:
`
`(CH2)a O
`
`Z
`
`002C~N)l_N CO Q
`2
`H
`H
`wherein subunits associated with p, q, r, and s may be in any order. Z is tetrazole or CO2Q; each
`Q is independently selected from hydrogen or a protecting group, and a is 1, 2, 3, or 4. R is each
`independently Hor C1-C4 alkyl. In this exemplary embodiment r is 1. Tz is a triazole group
`having the structure
`
`1
`where L is
`
`-~-(CH2)d-~-
`~ or
`~
`
`Rs
`J-(CH2)d-~-
`-~-X2
`~
`
`2
`• L is
`'
`
`-~-(CH ) -~-
`s
`~ or
`2 b
`
`Rs
`-i-(CH2)b ~ 1 ~
`X -s-; X1 is -NRC(O)-, -NRC(O)NR-, NRC(S)NR-, or -NRC(O)O-; X2 is -
`C(O)NR-, -NRC(O)NR-, NRC(S)NR-, or-OC(O)NR-; R5 is H, CO2H, or CO2R6, where R6 is a
`C1-C6 alkyl, C2-C12 aryl, or C4-C 16 alkylaryl; bis 1, 2, 3, or 4; and dis 1, 2, 3, or 4. In
`exemplary embodiments q is O or 1, Wis -NRC(O)-, -NRC(O)NR-, NRC(S)NR-, -NRC(O)O-, -
`OC(O)NR-, -OC(O)-, -C(O)NR-, or -C(O)O-; n is 1, 2, 3, 4, 5 or 6; and R2 and R3 are
`independently H, CO2H, or CO2R4, where R4 is a C1-C6 alkyl, C2-C12 aryl, or C4-C16 alkylaryl,
`wherein if one ofR2 and R3 is CO2H or CO2R2, then the other is H. In exemplary embodiments
`sis 0 or 1; Y is -C(O)-, -NRC(O)-, -NRC(S)-, -OC(O); and mis 1, 2, 3, 4, 5, or 6. In exemplary
`embodiments pis 0, 1, 2, or 3, and when pis 2 or 3, each R1 may be the same or different; and
`R1 is H, C1-C6 alkyl, C2-C12 aryl, or C4-C16 alkylaryl. G1 is a moiety selected from the group
`consisting of
`
`9
`
`Petitioner GE Healthcare – Ex. 1048, p. 9
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`where Ch is a metal chelating moiety, optionally including a chelated metal; FG is a fluorescent
`dye moiety which emits in the visible or near infrared spectrum; one of A and A' is Ch and the
`other is FG; V and V' are each independently -C(O)- , -NRC(O)- , -NRC(S)-, or -OC(O)-;and g
`is 1, 2, 3, 4, 5, or 6. In some embodiments, the fluorescent dye moiety emits in the near infrared
`
`spectrum. Some embodiments include a chelated metal.
`Embodiments of the invention include methods of imaging one or more cells, organs
`[0023]
`or tissues by exposing the cell to or administering to a organism an effective amount of a
`compound discussed above, where the compound includes a fluorescent dye moiety, or a metal
`
`isotope suitable for imaging.
`
`Embodiments of the invention include methods of treating a tumor comprising
`[0024]
`administering a therapeutically effective amount of a compound discussed above, where the
`
`compound includes a therapeutically effective radioisotope.
`Embodiments of the invention include methods for sorting cells by exposing
`[0025]
`the cells to a compound discussed above, where the compound includes a fluorescent dye
`moiety, followed by separating cells which bind the compound from cells which do not bind the
`
`compound.
`
`Embodiments of the invention include methods of intraoperative tumor
`[0026]
`mapping comprising administering an effective amount of a compound discussed above to a
`subject, where the compound includes a fluorescent dye moiety.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`Figure 1 shows SPECT-CT images of a PSMA + LNCaP tumor-bearing mouse
`[0027]
`injected intravenously with exemplary compound [99mTc]SRV32.
`Figure 2. GE eXplore VISTA pseudodynamic PET image ( co-registered with
`[0028]
`the corresponding CT image) of a PSMA + LNCaP tumor-bearing mouse injected intravenously
`with 0.2 mCi (7.4 MBq) of exemplary compound [68Ga]SRV27.
`Figure 3. GE eXplore VISTA PET image ( co-registered with the
`[0029]
`corresponding CT image) of a PSMA+ PIP and PSMA- flu tumor-bearing mouse injected
`intravenously with 0.2 mCi (7.4 MBq) of exemplary compound [68Ga]SRV100.
`Figure 4 shows a synthetic scheme for exemplary compound SRVl00 and
`[0030]
`111In]SRV100.
`
`[
`
`Petitioner GE Healthcare – Ex. 1048, p. 10
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`Figure 5 shows SPECT-CT images of a PSMA + PC-3 PIP tumor-bearing
`[0031]
`mouse injected intravenously with exemplary compound [111 In]SRV27.
`Figure 6 shows SPECT-CT images of a PSMA+ PC-3 PIP tumor-bearing
`[0032]
`mouse injected intravenously with exemplary compound [111 In]SRV100.
`Figure 7 shows SPECT-CT images of a PSMA+ PC-3 PIP tumor-bearing
`[0033]
`mouse injected intravenously with exemplary dual modality compound [111In]SRV73.
`Figure 8 shows the absorbance and emission spectra, and quantum yield of
`[0034]
`exemplary compound YC-27.
`Figure 9 shows the fluorescence decay of exemplary compound YC-27.
`[0035]
`Figure 10 shows an IC50 curve of compound YC-27 using a fluorescence-based
`[0036]
`NAALADase assay
`Figure 11 shows in vivo imaging of a NOD/SCID mouse (mouse #1), bearing
`[0037]
`PC3-PIP (forward left flank) and PC3-flu (forward right flank) tumors. Mouse #1 received 10
`nmol ofYC-27 and dorsal (animal prone) and ventral (animal supine) views were obtained.
`Dorsal and ventral views at 40 min p.i. (A, B, respectively); 18.5 h (C, D); 23 h (E, F); 42.5 h
`(G, H); 68 h (I, J). Dorsal view of pre-injection image (K). Dorsal and ventral views 70.5 h p.i.
`(L, M). Images after midline laparotomy (N) and individually harvested organs (0) on a Petri
`dish at 70.5 h p.i .. Images were scaled to the same maximum (arbitrary units).
`Figure 12 shows in vivo imaging of a NOD/SCID mouse (mouse #2) (left
`[0038]
`panel), bearing PC3-PIP (forward left flank) and PC3-flu (forward right flank) tumors. Mouse
`#2 received 1 nmol ofYC-27 and dorsal (animal prone) and ventral (animal supine) views were
`obtained. Dorsal and ventral views of the pre-injection image (A, B, respectively); 10 min p.i.
`(C, D); 20.5 h (E, F); 24 h (G, H). Images after midline laparotomy (I) and individually
`harvested organs (J) on a Petri dish at 24 h p.i .. Right Panels: Mouse #3 in same orientation as
`mouse #2. Mouse #3 received 1 nmol ofYC-27 co-injected with 1 µmol ofDCIBzL, which
`served as a blocking agent to test binding specificity. Images were scaled to the same maximum
`(arbitrary units).
`Figure 13 shows SPECT-CT images of a PSMA+ LNCaP tumor-bearing
`[0039]
`mouse injected intravenously with exemplary compound [99mTc]SRVI34B.
`Figure 14 shows SPECT-CT images of a PSMA+ PC3-PIP tumor-bearing
`[0040]
`mouse injected intravenously with exemplary compound [99mTc]SRVI34B.
`
`11
`
`Petitioner GE Healthcare – Ex. 1048, p. 11
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`Figure 15 shows SPECT-CT images of a PSMA+ PC3-PIP (forward left flank)
`[0041]
`and PSMA- PC3-flu (forward right flank) tumor-bearing mouse injected intravenously with
`exemplary compound [99mTc]SRVl34A.
`Figure 16 shows SPECT-CT images of a PSMA+ PC3-PIP (forward left flank)
`[0042]
`and PSMA- PC3-flu (forward right flank) tumor-bearing mouse injected intravenously with
`exemplary compound [99mTc]SRVI34B.
`Figure 17 shows PC3-PIP and PC3-flu cells treated with fluorescent compound
`[0043]
`YC-VIIl-36 (green, top left) and DAPI (blue), and PC3-PIP and PC3-flu cells treated with both
`YC-VIII-36 and PSMA inhibitor, PMPA.
`Figure 18 shows PC3-PIP cells treated with DAPI (blue) and varying
`(0044]
`concentrations of YC-VIII-36 (green).
`Figure 19 shows time dependent internalization of YC-VIII-36 into PC3-PIP
`[0045]
`cells treated with YC-VIII-36 (green) and DAPI (blue).
`Figure 20 shows titration and detection of varying amounts ofYC-VIII-36
`[0046]
`injected subcutaneously into a nude mouse. (IVIS spectrum with 10 second exposure followed
`by spectral unmixing)
`Figure 21 shows fluorescence images of a PSMA+ PC3-PIP and PSMA- PC3-
`[0047]
`flu tumor-bearing mouse injected intravenously with exemplary compound YC-VIII-36.
`Figure 22 shows fluorescence images of a PSMA+ PC3-PIP and PSMA- PC3-
`[0048]
`flu tumor-bearing mouse injected intravenously with exemplary compound YC-VIIl-36 180
`minutes after injection (top) and biodistribution of exemplary compound YC-VIII-36 180
`minutes after injection (bottom).
`Figure 23 shows F ACS analysis showing the percent subpopulation of PSMA
`[0049]
`positive cells in PC3-flu, PC3-PIP, and LNCaP cells.
`Figure 24 shows cell sorting results for PC3-PIP cells treated with exemplary
`[0050]
`compound YC-VIII-36, including initial percentage (top center), and after 3 passages of sorting
`(bottom).
`Figure 25 shows the number of spiked PIP-pos cells into 10 million of PC3-flu
`(0051]
`detectable by 100 nM compound YC-VIII-36 in flow cytometry (BD LSR-11). Gate Pl is total
`number of single cells counted; gate P2 at higher intensity is the number of Pip-pos cells
`detected and gate P3 at lower intensity.
`
`12
`
`Petitioner GE Healthcare – Ex. 1048, p. 12
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
`
`[0052]
`
`Some embodiments of the current invention are discussed in detail below. In
`
`describing embodiments, specific terminology is employed for the sake of clarity. However, the
`
`invention is not intended to be limited to the specific terminology so selected. A person skilled
`
`in the relevant art will recognize that other equivalent components can be employed and other
`
`methods developed without departing from the broad concepts of the current invention. All
`
`references cited anywhere in this specification are incorporated by reference as if each had been
`
`individually incorporated.
`
`[0053]
`
`Where a range of values is provided in the present application, it is understood
`
`that each intervening value, to the tenth of the unit of the lower limit unless the context clearly
`
`dictates otherwise, between the upper and lower limit of that range and any other stated or
`
`intervening value in that stated range, is encompassed within the invention. The end values of
`
`any range are included in the range.
`
`Definitions
`
`[0054]
`
`The following terms below generally have the meaning that would be readily
`
`understood by persons skilled in the art. The definitions are provided herein for clarity. Where
`
`a definition excludes an art-recognized meaning, the term should be taken to have the meaning
`
`set forth below. Where the art-recognized meaning and the meaning below differ but are not
`
`exclusive, the intended meaning is clear by the context in which it is used.
`
`[0055]
`
`[0056]
`
`As used herein, "agent" is a non-peptide, small molecule compound.
`
`By "cell substrate" is meant the cellular or acellular material ( e.g., extracellular
`
`matrix, polypeptides, peptides, or other molecular components) that is in contact with the cell.
`
`[0057]
`
`[0058]
`
`By "control" is meant a standard or reference condition.
`
`By "disease" is meant any condition or disorder that damages or interferes with
`
`the normal function of a cell, tissue, organ or subject.
`
`[0059]
`
`By "effective amount" is meant a quantity sufficient to produce a measurable
`
`difference, when compared with a control. For example, an amount sufficient to produce a
`
`measurable image, when the compound is used for imaging, or an amount sufficient to
`
`ameliorate the symptoms of a disease, when the compound is used for therapy. The effective
`
`amount of an active therapeutic agent for the treatment of a disease or injury varies depending
`
`13
`
`Petitioner GE Healthcare – Ex. 1048, p. 13
`
`

`

`WO 2010/108125
`
`PCT /0S2010/028020
`
`upon the manner of administration, the age, body weight, and general health of the subject.
`Ultimately, the attending clinician will decide the appropriate amount and dosage regimen.
`By "modifies" is meant alters. An agent that modifies a cell, substrate