Pyrrolobenzodiazepines and conjugates thereof

PYRROLOBENZODIAZEPINES AND CONJUGATES THEREOF

The present invention relates to pyrrolobenzodiazepines (PBDs), in particular pyrrolobenzodiazepines having a linker group connected to a cell binding agent.

Background to the invention

Pyrrolobenzodiazepines

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev. 1994,433-465 (1994); Antonow, D. and Thurston, D.E., Chem. Rev. 2011 111 (4), 2815-2864). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40,145-148 (1987)), chicamycin (Konishi. et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, etal„ Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et ai., J. Antibiotics. 41,1366-1373 (1988)), prothracarcin (Shimizu, etai, J. Antibiotics, 29,24922503 (1982); Langley and Thurston. J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC102)(Hara, eta!., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41,1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110. 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure:

<img file="CA2901941C_D0001.tif" />

They differ in the number, type and position of substituents, in both their aromatic A rings and pyrroio C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N=C), a carbinolamine(NH-CH(OH)), or a carbinolamine methyl ether (NHCH(OMe)) at the N10-C11 position which is the electrophilic centre responsible for alkylating

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics HI. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res.. 19, 230-237 (1986)). Their ability to form an adduct in the minor groove, enables them to interfere with DNA processing, hence their use as antitumour agents.

A particularly advantageous pyrrolobenzodiazepine compound is described by Gregson el al. (Chem. Commun. 1999, 797-798) as compound 1, and by Gregson et al. (J. Med. Chem. 2001, 44, 1161-1174) as compound 4a. This compound, also known as SJG-136, is shown below:

<img file="CA2901941C_D0002.tif" />

Other dimeric PBD compounds, such as those bearing C2 aryl substituents in WO

2005/085251, have been disclosed, an example being:

<img file="CA2901941C_D0003.tif" />

These compounds have been shown to be highly useful cytotoxic agents.

Antibody-drug conjugates

Antibody therapy has been established for the targeted treatment of patients with cancer, immunological and angiogenic disorders (Carter, P. (2006) Nature Reviews Immunology 6:343-357). The use of antibody-drug conjugates (ADC), i.e. immunoconjugates, for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer, targets delivery of the drug moiety to tumors, and intracellular accumulation therein, whereas systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Xie et a/(2006) Expert. Opin. Biol. Ther. 6(3):281-291: Kovtun et a/(2006)

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564

Cancer Res. 66(6):3214-3121 ; Law et a/(2006) Cancer Res. 66(4):2328-2337; Wu et al (2005) Nature Biotech. 23(9):1137-1145; Lambert J. (2005) Current Opin. in Pharmacol. 5:543-549; Hamann P. (2005) Expert Opin. Ther. Patents 15(9):1087-1103: Payne, G. (2003) Cancer Ce//3:207-212; Trail et a/(2003) Cancer Immunol. Immunother. 52:328-337; Syrigosand Epenetos (1999) Anticancer Research 19:605-614).

Maximal efficacy with minimal toxicity is sought thereby. Efforts to design and refine ADC have focused on the selectivity of monoclonal antibodies (mAbs) as well as drug mechanism of action, drug-linking, drug/antibody ratio (loading), and drug-releasing properties (Junutula, e/a/., 2008b Nature Biotech., 26(8):925-932; Doman et al (2009) Blood 114(13):2721-2729: US 7521541; US 7723485; W02009/052249; McDonagh (2006) Protein Eng. Design &amp; Sei. 19(7): 299-307; Doronina et al (2006) Bioconj. Chem. 17:114-124; Erickson et al (2006) Cancer Res. 66(8):1-8: Sanderson et al (2005) Clin. Cancer Res. 11:843-852; Jeffrey et al (2005) J. Med. Chem. 48:1344-1358; Hambletteta/(2004) Clin. Cancer Res. 10:70637070). Drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

PBDs in ADCs

Dimeric PBDs have been disclosed as the drugs in drug conjugates. For example, in WO 2011/130598, dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody, are disclosed where the linker group is attached to one of the available N10 positions, and are generally cleaved by action of an enzyme on the linker group.

By contrast, in WO 2011/130613 and WO 2011/130616, dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody, are disclosed where the linker group is attached via an aromatic group at one of the C2 postions, and are generally cleaved by action of an enzyme on the linker group. Such antibody drug conjugates are also described in Flygare, J., et al, Chem. Biol. Drug Des. 81:113-121 (2013), which also describes other types of antibody drug conjugates.

A further approach is described in WO 2007/085930, wherein tomamycin-like dimers have a linker group for connection to a cell binding agent, such as an antibody, where the linker group is attached to the tether between the tomamycin units, and are generally cleaved by action of an enzyme on the linker group.

The present inventors have developed a novel approach to forming PBD conjugates with cell 5 binding agents, and in particular PBD antibody conjugates.

Summary

Certain exemplary embodiments provide a conjugate of formula (A):

<img file="CA2901941C_D0004.tif" />

wherein:

R²is

<img file="CA2901941C_D0005.tif" />

, where R^Ma and R³⁶⁶ are independently selected from H, F, Cu saturated alkyl, C₂._s alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from Cm alkyl amido and Cm alkyl ester; or, when one of R^36a and R³⁶” is H, the other is selected from nitrile and a Cm alkyl ester;

R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NHz, NHR, NRR’, NOz, MesSn and halo;

R⁷ is independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR’, NO2, MeaSn and halo;

Y is selected from formulae A1, A2, A3, A4, A5 and A6:

<img file="CA2901941C_D0006.tif" />

(A1)

<img file="CA2901941C_D0007.tif" />

(A2)

CA 2901941 2019-03-08

4a

<img file="CA2901941C_D0008.tif" />

<img file="CA2901941C_D0009.tif" />

<img file="CA2901941C_D0010.tif" />

L is a linker connected to a cell binding agent:

CBA is the cell binding agent;

n is an integer selected in the range of 0 to 48;

R^A4 is a Ci_6 alkylene group;

either (a) R¹⁰ is H, and R¹¹ is OH or OR^A, where R^A is C1.4 alkyl; or (b) R’° and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R'° is H and R¹¹ is OSO_ZM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;

R and R’ are each independently selected from optionally substituted C1.12 alkyl, C3.20 heterocyclyl and C5.20 aryl groups, and optionally in relation to the group NRR’, R and R’ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring;

wherein R¹⁶, R¹⁷, R¹⁹, R²⁰, R²’ and R²² are as defined for R⁶, R⁷, R⁹, R¹⁰, R¹¹ and R²respectively;

wherein Z is CH or N;

wherein T and V are independently selected from a single bond or a C1.9 alkylene, which chain may be interrupted by one or more heteroatoms selected from O, S, N(H), and

CA

2901941 2019-06-11

4b

NMe, provided that the number of atoms in the shortest chain of atoms between X and X* is no more than 12 atoms; and

X and X' are independently selected from O, S and N(H).

In a general aspect the present invention provides a conjugate comprising a PBD dimer compound with a linker for connecting to a cell binding agent, wherein the linker has a triazole, piperazine, propargylene or oxime group attached to a phenylene or pyriydylene in the bridge linking the two PBD monomers. The cell binding agent is preferably an antibody.

In a first aspect, the present invention provides novel conjugate compounds of formula (A):

wherein:

R^36a

<img file="CA2901941C_D0011.tif" />

, where R³⁶⁸ and R³⁶¹* are independently selected from H, F, C_M saturated alkyl, C2.3 alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from Cm alkyl amido and C-m alkyl ester; or, when one of R^36a and R³⁶⁶ is H, the other is selected from nitrile and a C1-4 alkyl ester;

R⁸ and R® are independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR*, NO2, MejSn and halo;

R⁷ is independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR’, NO2, MeaSn and halo;

CA

2901941 2019-06-11

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

Y is selected from formulae A1, A2. A3, A4, A5 and A6:

<img file="CA2901941C_D0012.tif" />

(A1)

<img file="CA2901941C_D0013.tif" />

<img file="CA2901941C_D0014.tif" />

(A3)

<img file="CA2901941C_D0015.tif" />

<img file="CA2901941C_D0016.tif" />

L is a linker connected to a ceil binding agent;

CBA is the œl! binding agent;

n is an integer selected in the range of 0 to 48:

R^A4 is a C.„_Galkylene group;

either (a) R¹⁰ is H, and R¹¹ is OH, OR^A, where R^A is C_M alkyl; or (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R¹⁰ is H and R¹¹ is OSO_ZM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation;

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

R and R’ are each independently selected from optionally substituted Cm₂ alkyl, C3.20 heterocyclyl and C5.20 aryl groups, and optionally in relation to the group NRR’, R and R’ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring;

wherein R¹⁶, R¹⁷, R¹⁹, R²⁰, R^Z1 and R^2Z are as defined for R⁶, R⁷, R⁹, R¹⁰, R¹¹ and R²respectively;

wherein Z is CH or N;

wherein T and T are independently selected from a single bond or a C&lt;.₉ alkylene, which chain may be interrupted by one or more heteroatoms e.g. 0, S, N(H), NMe, provided that the number of atoms in the shortest chain of atoms between X and X’ is 3 to 12 atoms; and

X and X' are independently selected from O, S and N(H).

Thus formula A is selected from the following formulae A-l, A-ll, A-lll, A-iV, A-V and

A-VI depending on Y:

<img file="CA2901941C_D0017.tif" />

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

<img file="CA2901941C_D0018.tif" />

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

<img file="CA2901941C_D0019.tif" />

A second aspect of the present invention provides novei drug-linker compounds of formula (B):

<img file="CA2901941C_D0020.tif" />

Where all the groups are as defined in the first aspect of the invention; and

Y^L is selected from a group of formulae 81, B2, 83, B4, 85 and 86:

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

<img file="CA2901941C_D0021.tif" />

<img file="CA2901941C_D0022.tif" />

(B1) (B2)

<img file="CA2901941C_D0023.tif" />

<img file="CA2901941C_D0024.tif" />

<img file="CA2901941C_D0025.tif" />

(B5)

<img file="CA2901941C_D0026.tif" />

where G is a reactive group for connecting to a cel! binding agent.

A third aspect of the present invention provides compounds of formula (C) which may be used in the preparation of the compounds and conjugate compounds of the invention:

<img file="CA2901941C_D0027.tif" />

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564 where Y^c is selected from a group of formulae 01, C2. C3, C4 and C5:

<img file="CA2901941C_D0028.tif" />

<img file="CA2901941C_D0029.tif" />

<img file="CA2901941C_D0030.tif" />

<img file="CA2901941C_D0031.tif" />

<img file="CA2901941C_D0032.tif" />

<img file="CA2901941C_D0033.tif" />

(05) (06) either (a) R³⁰ is H, and R³¹ is OH, OR^A, where R^A is C_M alkyl; or (b) R³⁰ and R³’ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R³⁰ is H and R³¹ is OSO_ZM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; or (d) R³⁰ is a nitrogen protecting group and R³¹ is OProt°, where Prot° is a hydroxy protecting group; and

R⁴⁰ and R⁴¹ are as defined for R³⁰ and R³¹ respectively; and all the remaining groups are as defined in the first aspect of the invention.

A fourth aspect of the present invention provides compounds of formula (D) which may be used in the preparation of the compounds of the second and third aspects of the invention:

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

<img file="CA2901941C_D0034.tif" />

Y° is selected from a group of formulae D2, D3, D4 and D6:

<img file="CA2901941C_D0035.tif" />

(D2) (D3) h₂n—r^A4

<img file="CA2901941C_D0036.tif" />

(D4) (D6) and all the remaining groups are as defined in the third aspect of the invention.

A fifth aspect of the present invention provides compounds of formula (E) which may be used in the preparation of the compounds of the second, third and fourth aspects of the invention:

<img file="CA2901941C_D0037.tif" />

V^e is seiected from a group of formulae E1, E2 and E5:

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 r^e1 r^E2

II 1 (E1) (E2)

<img file="CA2901941C_D0038.tif" />

(E5) where

R^F1 is selected from H and TMS:

R⁶² is selected from Br, Cl and I; and all the remaining groups are as defined in the third aspect of the invention.

A sixth aspect of the present invention provides the use of a compound of the first aspect of the invention in a method of medical treatment. The fourth aspect also provides a pharmaceutical composition comprising a compound of the first aspect, and a pharmaceutically acceptable excipient.

A seventh aspect of the present invention provides a compound of the first aspect of the invention or a pharmaceutical composition of the fourth aspect of the invention for use in a method of treatment of a proliferative disease. The fifth aspect also provides the use of a compound of the first aspect in a method of manufacture of a medicament for the treatment of a proliferative disease, and a method of treating a mammai having a proliferative disease, comprising administering an effective amount of a compound of the first aspect or a pharmaceutical composition of the fourth aspect.

An eight aspect of the present invention provides a method of synthesis of a compound of the first aspect of the present invention, comprising the step of conjugating a drug-linker of the second aspect with a cell-binding agent.

The present invention also provides the synthesis of compounds of the second aspect of the invention from compounds of the third, foruth or fifth aspect of the invention by reacting them with suitable reagents.

12a

Description of the Figures

Figure 1 shows a plot of the in vivo mean tumor volume change overtime in breast cancermodel MMTV-HER2 Fo5 mammary allograft tumors inoculated into CRL nu/nu mice after single iv dosing on day 0 with: (1) Vehicle 20mM Histidine acetate, pH 5.5, 240mM sucrose, (2) XCD22-7 (115) at 6 mg/kg, (3) trastuzumab-7 (110) at 1 mg/kg, (4) trastuzumab-7 (110) at 3 mg/kg, and (5) trastuzumab-7 (110) at 6 mg/kg. The lines in the figure are indicated with the following symbols:

Vehicle —θ... ADC110 Tmab-7 HC A118C, 1 mg/kg

A ADC110 Tmab-7 HC A118C, 3 mg/kg -□ ADC110 Tmab-7 HC A118C, 6 mg/kg —▼—ADC115 CD22-7 HC A118C, 6 mg/kg

Figure 2 shows a plot of the in vivo mean tumor volume change over time in breast cancer-model MMTV-HER2 Fo5 mammary allograft tumors inoculated into CRL nu/nu mice after single IV dosing on day 0 with: (1) Vehicle 20 mM Histidine acetate, pH 5.5, 240 mM sucrose, (2) xCD22-10 (125) at 3 mg/kg, (3) trastuzumab-10 (120) at 0.3 mg/kg, (4) trastuzumab-10 (120) at 1 mg/kg, and (5) trastuzumab-10 (120) at 3 mg/kg. The lines in the figure are indicated with the following symbols:

Vehicle

ADC120 Tmab-10 HC A118C, 0.3 mg/kg

ADC120 Tmab-10 HC A118C, 1 mg/kg -B—ADC 120 Tmab-10 HC A118C, 3 mg/kg ΛΤ—ADC125 CD22 HC A118C, 3 mg/kg

Figure 3 shows a plot of the in vivo mean tumor volume change over time in breast cancermodel MMTV-HER2 Fo5 mammary allograft tumors inoculated into CRL nu/nu mice after single iv dosing on day 0 with: (1) Vehicle 20 mM Histidine acetate, pH 5.5, 240 mM sucrose, (2) XCD22-17 (135) at 3 mg/kg, (3) trastuzumab-17 (130) at 0.3 mg/kg, (4) trastuzumab-17 (130) at 1 mg/kg, and (5) trastuzumab-17 (130) at 3 mg/kg. The lines in the figure are indicated with the following symbols:

CA

2901941 2019-06-11

12b )( Vehicle —ta- ADC130 Tmab-17 HC A118C, 0.3 mg/kg “O— ADC130 Tmab-17 HC Al 18C, 1 mg/kg —S—ADC130 Tmab-17 HC Al 18C, 3 mg/kg -<u>-ADC135 CD22 HC A118C, 3 mg/kg

CA 2901941 2019-06-11

Detailed Description of the Invention

The present invention provides a conjugate comprising a PBD dimer connected through the dimer bridging portion via a specified linker to a cell binding agent.

The present invention is suitable for use in providing a PBD conjugate to a preferred site in a subject.

Nitrogen protecting groups

Nitrogen protecting groups are well known in the art. Preferred nitrogen protecting groups for use in the present invention are carbamate protecting groups that have the general formula:

<img file="CA2901941C_D0039.tif" />

wherein R’³⁰ is an optionally substituted alkyl (e.g. C1.20 alkyl), aryl (e g. C5-20 aryl) or heteroaryl (e.g. C3.20 heterocyclyl) group.

A large number of possible carbamate nitrogen protecting groups are listed on pages 706 to 772 of Greene’s Protective Groups in Organic Synthesis, 4^,h Edition, John Wiley &amp; Sons, Inc., 2007 (ISBN 978-0-471-69754-1).

Particularly preferred protecting groups include Alloc, Troc, Teoc, BOC, TcBOC, Fmoc, 1Adoc and 2-Adoc.

Hydroxyl protecting groups

Hydroxyl protecting groups are well known in the art. A large number of suitable groups are described on pages 24 to 298 of of Greene’s Protective Groups in Organic Synthesis, 4^ihEdition, John Wiley &amp; Sons, Inc., 2007 (ISBN 978-0-471-69754-1).

Classes of particular interest include silyl ethers, methyl ethers, alkyl ethers, benzyl ethers, esters, benzoates, carbonates, and sulfonates. Particularly preferred hydroxyl protecting groups include THP.

CA

2901941 2019-06-11

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

Preferences

The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.

R²

In some embodiments, R³⁶® and R³⁶” are both H.

In other embodiments, R^3fia and R^36b are both methyl.

in further embodiments, one of R³⁶® and R³⁶¹’ is H, and the other is selected from C_Msaturated alkyl, 0?.₃ alkenyl, which alkyl and alkenyl groups are optionally substituted. In some of these further embodiments, the group which is not H may be selected from methyi and ethyl.

R²²

The above preferences for R² apply equally to R²².

R^e

In one embodiment, R^e is independently selected from H, R, OH, OR, SH, SR, NH₂. NHR, NRR’, NO2, Me₃Sn- and Halo.

In one embodiment, R^b is independently selected from H, OH, OR, SH, NH₂, NO₂ and Halo. In one embodiment, R⁵ is independently selected from H and Halo.

In one embodiment, R^b is independently H.

In one embodiment, R⁶ and R⁷ together form a group -O-(CH₂)_?-O-, where p is 1 or 2.

These embodiments also apply to R¹⁶.

R⁷

R⁷ is independently selected from H, R, OH, OR, SH, SR. NH₂₎ NHR, NRR’, NO?, Me₃Sn and haio.

In one embodiment, R⁷ is independently OR.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

In one embodiment. R⁷ is independently 0R^7A. where R^?A is independently optionally substituted Cm alkyl.

In one embodiment. R^7A is independently optionally substituted saturated Cm alkyl.

In one embodiment, R^7A is independently optionally substituted C2-4 alkenyl.

In one embodiment, R^7A is independently Me.

In one embodiment, R^ZA is independently CH₂Ph.

In one embodiment, R^7A is independently allyl.

These embodiments also apply to R¹'.

R⁹

In one embodiment, R⁹ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR’, NO2, MeaSn- and Halo.

In one embodiment, R⁹ is independently H.

In one embodiment, R⁹ is independently R or OR.

These embodiments also apply to R¹⁹.

N10-C11

In some embodiments. R^1C is H, and R¹¹ is OH, 0R^A, where R^A is Cm alkyl. In some of these embodiments, R¹¹ is OH. In others of these embodiments, R¹¹ is 0R^A, where R^A is Cm alkyl. In some of these embodiments. R^A is methyl.

In some embodiments, R^1C and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.

In some embodiments, R^u is H and R¹¹ is OSO_ZM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation. In some of these embodiments. M is a monovalent pharmaceutically acceptable cation, and may be Na*. Furthermore, in some embodiments z is 3.

The above preferences apply equally to R²⁰ and R²¹.

In some embodiments, R^ac is H, and R³¹ is OH, OR^A, where R^A is Cm alkyl. In some of these embodiments, R³¹ is OH. In others of these embodiments, R³¹ is 0R^A, where R^A is Cm alkyl. In some of these embodiments, R^A is methyl.

CA 02901941 2015-00-19

WO 2014/159981 PCT/US2014/025564

In some embodiments, R^3C and R³¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.

In some embodiments, R^3C is H and R³¹ is OSO_ZM, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation. In some of these embodiments, M is a monovalent pharmaceutically acceptable cation, and may be Na⁺. Furthermore, in some embodiments z is 3.

In some embodiments, R^3C is a nitrogen protecting group and R³¹ is OProt°, where Prot° is a hydroxy protecting group.

In some of these embodiments, the nitrogen protecting group may be selected from Alloc, Troc, Teoc, BOC, TcBOC, Fmoc, 1-Adoc and 2-Adoc, and more preferably be Boc.

In some of these embodiments, the nitrogen protecting group may be THP.

For compounds of formula D, it may be preferred that R³⁰ and R³¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.

For compounds of formula E, it may be preferred that R³⁰ is a nitrogen protecting group and R³¹ is OProt°, where Prot° is a hydroxy protecting group.

For compounds of formula C, where Y^c is of formula C2, C3 or C4, it may be preferred that R³⁰ and R³¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.

For compounds of formula C, where Y^c is of formula 01 or 05, it may be preferred that R³⁰ is a nitrogen protecting group and R³¹ is OProt⁰, where Prot° is a hydroxy protecting group.

The above preferences apply equally to R⁴⁰ and R⁴¹.

T and Τ'

Each of T and T’ is independently selected from a single bond or a C_?.₃ alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, N(H) and/or NMe,

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564 provided (hat the number of atoms in the shortest chain of atoms between X and X’ is 3 to 12 atoms.

in one embodiment, each alkylene group of T and T is optionally interrupted by one or more heteroatoms selected from O, S, and NMe.

In one embodiment, each of T and T” is independently selected from a single bond and a Ci-9 alkylene group.

In one embodiment, T is selected from a single bond, Ci, C₂. C3 and a C₄ alkylene group and Γ is selected from a single bond, C_1s C₂, C3 and a C₄ alkylene group.

In one embodiment, T is selected from a single bond, Ci, and a C₂ alkylene group and T is selected from a single bond, Ci, and a C₂ alkylene group.

In one embodiment, T is selected from a single bond and a C_f alkylene group and Τ' is selected from a single bond and a Ci alkylene group.

In one embodiment, T is a single bond and T’ is a single bond.

In one embodiment, T is a Ci aikyiene group and T’ is a Ci alkylene group.

In some embodiments, T and T are the same.

The alkylene groups listed above may be optionally interrupted by one or more heteroatoms. The alkylene groups listed above may be unsubstituted linear aliphatic aikyiene groups.

X

In one embodiment, X is selected from O, S, or N(H).

Preferably, X is 0.

Dimers

In some embodiments, the groups R²², R¹⁶, R¹⁷, R¹⁹, R²⁰ and R²¹ are the same as the groups R², R'^J, R^s, R⁷, R¹'³ and R¹¹ respectively. In these embodiments, the PBD monomer units have the same substituents.

Particularly preferred compounds of the first aspect of the present invention may be of formula la:

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

<img file="CA2901941C_D0040.tif" />

la where

R¹⁰, R¹¹, R²⁰, R^Z1 and Y are as defined above;

h and t₂ are an independently selected from 0,1 and 2

R^7a and R^17a are independently selected from methyl and phenyl.

Particularly preferred compounds of the second aspect of the present invention may be of formula Ila:

<img file="CA2901941C_D0041.tif" />

Ila where

R^1C, R”, R²⁰, R²¹ and Y¹ are as defined above:

ti and t₂ are an independently seiected from 0.1 and 2 R^7a and R^1/a are independently selected from methyl and phenyl.

Particularly preferred compounds of the third aspect of the present invention may be of formula Illa:

<img file="CA2901941C_D0042.tif" />

Illa where

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

R^1C, R¹¹, R²⁰. R²¹ and V^e are as defined above;

ti and t₂ are an independently selected from 0,1 and 2

R^7a and R^17a are independently selected from methyl and phenyl.

n (Y, Y)

In some embodiments, n (in Y or Y^lj is an integer between 0 and 24.

In some embodiments, n (in Y or Y^L) is an integer between 0 and 12.

In some embodiments, n (in Y or Y' ) is an integer between 0 and 8.

In some embodiments, n (in Y or Y¹-) is an integer between 0 and 6.

In some embodiments, n (in Y or Y^lj is 0.

In some embodiments, n (in Y or Y^L) is 1.

In some embodiments, n (in Y or Y¹) is 2.

In some embodiments, n (in Y or Y¹·) is 3.

In some embodiments, n (in Y or Y¹) is 4.

In some embodiments, n (in Y or Y^L) is 5.

In some embodiments, n (in Y or Y^L) is 6.

In some embodiments, n (in Y or Y^L) is 7.

In some embodiments, n (in Y or Y^lj is 8.

In some embodiments, n (in Y or Y^L) is 9.

In some embodiments, n (in Y or Y^L) is 10.

In some embodiments, n (in Y or Y^L) is 11.

In some embodiments, n (in Y or Y¹) is 12.

In some embodiments, n (in Y or Y^L) is 13.

In some embodiments, n (in Y or Y^L) is 14.

In some embodiments, n (in Y or Y^L) is 15.

In some embodiments when Y is A1, or Y^L is B1, n may be selected from 3 and 6.

In some embodiments when Y is A2, or Y^L is B2, n may be selected from 4 and 6.

In some embodiments when Y is A3, or Y^L is B3, n may be 4.

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

In some embodiments when Y Is A4, or Y^L is B4, n may be 4.

In some embodiments when Y is A5, or Y^L is B5, n may be 11.

In some embodiments when Y is A6, or Y^l is B6, n may be 2.

L and G

L is a linker connected to the cell binding agent in the conjugate conmpound. G is a reactive group for connecting the PBD dimer to the cell binding agent to form the conjugate compound.

Preferably, the linker/reactive group contains an electrophiiic functional group for reaction with a nucleophilic functional group on the cell binding agent. Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. iysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) maleimide groups (ii) activated disulfides, (iii) active esters such as NHS (N-hydroxysuccinimide) esters, HOBt (N-hydroxybenzotriazole) esters, haloformates, and acid halides; (iv) alkyl and benzyl halides such as haloacetamides; and (v) aldehydes, ketones, carboxyl, and, some of which are exemplified as follows:

<img file="CA2901941C_D0043.tif" />

O

<img file="CA2901941C_D0044.tif" />

<img file="CA2901941C_D0045.tif" />

Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thio! nucleophiles. Additional nucleophilic groups can be introduced into antibodies through

CA 02901941 2015-09-19

WO 2014/159981

PCT7US2014/025564 the reaction of lysines with 2-iminothiolane (Traut’s reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by introducing one, two. three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or mere non-native cysteine amino acid residues). US 7521541 teaches engineering antibodies by introduction of reactive cysteine amino acids. In some embodiments, a Linker has a reactive nucleophilic group which is reactive with an electrophilic group present on an antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Useful nucleophilic groups on a Linker include, but are not limited to. hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group on an antibody provides a convenient site for attachment to a Linker.

In one embodiment, the group L is:

where the asterisk indicates the point of attachment to the rest of group Y, the wavy line indicates the point of attachment to the cell binding agent, and m is an integer selected from the range 0 to 6. In one embodiment, m is selected from 2, 3,4 and 5.

In one embodiment, the connection between the cell binding agent and L is through a thiol residue of the cell binding agent and a maleimide group of L.

In one embodiment, the connection between the cell binding agent and L is:

where the asterisk indicates the point of attachment to the remaining portion of the L group or the remaining portion of the Y group and the wavy line indicates the point of attachment to the remaining portion of the cell binding agent. In this embodiment, the S atom is typically derived from the cell binding agent.

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

In each of the embodiments above, an alternative functionality may be used in place of the maleimide-derived group shown below:

<img file="CA2901941C_D0046.tif" />

where the wavy line indicates the point of attachment to the cell binding agent as before, and the asterisk indicates the bond to the remaining portion of the L group or the remaining portion of the Y group.

In one embodiment, the maieimide-derived group is replaced with the group:

O

<img file="CA2901941C_D0047.tif" />

where the wavy fine indicates point of attachment to (he cell binding agent, and the asterisk indicates the bond to the remaining portion of the L group or the remaining portion of the Y group.

In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with the cell binding agent, is selected from:

-C(=0)NH-,

-C(=O)O-,

-NHC(=O)-,

-OC(=O)-,

-00(=0)0-,

-NHC(=O)O-,

-OC(=O)NH-,

-NHC(=O)NH-,

-NHC(=O)NH,

-0(=0 )NHC(=0)-,

-S-,

-S-S-,

-CH₂C(=O)-C(=0)CH₂-,

CA 02901941 2015-09-19

PCT/US2014/025564

WO 2014/159981 =N-NH-, and -NH-N=.

in one embodiment, the maleimide-derived group is replaced with a group, which optionally together with the ceil binding agent, is selected from:

<img file="CA2901941C_D0048.tif" />

<img file="CA2901941C_D0049.tif" />

where the wavy line indicates either the point of attachment to the cell binding agent or the bond to the remaining portion of the L group or the remaining portion of the Y group, and the asterisk indicates the other of the point of attachment to the ceil binding agent or the bond to the remaining portion of the L group or the remaining portion of the Y group.

Other groups that can be used as L for connecting the remaining portion of the Y group to the cell binding agent are described in WO 2005/082023.

Thus, in embodiments of the present invention, L is of formula: -L^A-(CH_?)_m- (L1)

Where m is from 0 to 6: and

L^A is selected from:

<td> (L^w'j</td><td> O F\! O</td><td> (I?⁶)</td><td> 0 C6Asft</td>

<td> (L^a«)</td><td> O U- .Ar y CBA{ / N 7 O</td><td> (L^a/)</td><td> CBA$ ^ry</td>

<td> (L^a?)</td><td> O 1 -O- À CBAj / Χ—ζ, o o</td><td></td><td> C6A</td>

CA 02901941 2015-00-19

WO 2014/159981

PCT/US2014/025564

<img file="CA2901941C_D0050.tif" />

in some embodiments where L is L1, m may be 2, 3 or 5.

in some embodiments where L is L1, L^A may be L^A1'¹.

In embodiments of the present invention, L is of formula: -L^A-(CH₂)_m-O- (L2)

Where m is from 0 to 6: and

L^a is selected from the groups above.

Without wishing to be bound by theory, such a group may be cleaved from the antibody such that the carbamate group yields a terminai amine.

in some embodiments where L is 12, L^A may be L^².

In some embodiments where L is L2, m may be 1.

In embodiments of the present invention, L is of formula:

-L^A-(CH₂)_q-O-C(=O)-NH-(CH₂)p- (L3)

Where q is from 1 to 3, and p is from 1 to 3: and

L^a is selected from the groups above.

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

Without wishing to be bound by theory, such a group may be cleaved from the antibody such that the carbamate group yields the group: H₂N-(CH₂)_P- (L3’).

In some embodiments where L is L3, q may be 1, and p may be 2.

In some embodiments where L is L3. L^A may be selected from l_^A7, L^A8’¹ and L^A8².

In embodiments of the present invention, L is of formula:

(L4)

Where m is from 0 to 6:

X¹ and X² are amino acid groups, selected from natural amino acids, which may be modified; L^a is selected from the groups above.

The natural amino acids may be selected such that the dipeptide group is calhpesin labile.

In one embodiment, the group -Χ,-Χς- is selected from:

-Phe-Lys-,

-Val-Ala-,

-Val-Lys-,

-Ala-Lys-,

-Val-Cit-,

-Phe-Cit-,

-Leu-Cit-,

-Île-Cit-,

-Phe-Arg-,

-Trp-Citwhere Cit is citrulline.

Preferably, the group -Xi-X₂- is selected from:

-Phe-Lys-,

-Val-Ala-,

-Val-Lys-,

-Ala-Lys-,

-Val-Crt-,

Most preferably, the group -X1-X2- is -Phe-Lys- or -Vai-Ala-.

In some embodiments where L is L4, m may be 1.

Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002,13,855-869.

In one embodiment, the amino acid side chain is derivatised, where appropriate. For example, an amino group or carboxy group of an amino acid side chain may be derivatised. In one embodiment, an amino group NH? of a side chain amino acid, such as lysine, is a derivatised form selected from the group consisting of NHR and NRR’.

In one embodiment, a carboxy group COOH of a side chain amino acid, such as aspartic acid, is a derivatised form selected from the group consisting of COOR, CONH2, CONHR and CONRR·.

In one embodiment, the amino add side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed below in relation to the group R^L. The present inventors have established that protected amino acid sequences are cleavable by enzymes. For example, it has been established that a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out in Protective Groups in Organic Synthesis, Greene and Wuts.

Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality:

Arg: Z, Mtr, Tos;

Asn: Trt, Xan;

Asp: Bzl, t-Bu;

Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;

Glu: Bzl, t-Bu;

Gin: Trt, Xan;

CA

2901941 2019-06-11

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

His: Boc. Dnp, Tos, Trt;

Lys: Bog, Z-CI, Fmoc, Z, Alioc;

Ser: Bzl, TBDMS, TBDPS;

Thr: Bz;

Trp: Bog:

Tyr: Bzl, Z, Z-Br.

Thus, in embodiments of the present invention, G is of formula: G^A-(CH₂)_ni- (Θ1)

Where m is from 0 to 6; and

G^a is selected from:

<td> (G^aj</td><td> 0 A'hA C ¹ 0</td><td> (G^A4)</td><td> o Hal N—1 H ⁵ Where Hal = 1, Br, Cl</td>

<td> (G^a1-²)</td><td> o VI ^so</td><td> (G^AS)</td><td> o 'M' . o—|</td>

<td> (G**)</td><td> 0 Λ^°γ^ V-L o 0</td><td> (G^AB)</td><td> 0 o /°“4 &gt;·</td>

<td> (G^)</td><td> s— (NO,) where the NO? group is optional</td><td> (G^a?)</td><td>^Br—V</td>

<td> (θΑ3-2)</td><td> s—s (NO₂) where the N0₂ group is optional</td><td> (G^A8)</td><td> Y</td>

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

<td> (G^)</td><td> o₂n-\^/ where the N0₂ group is optional</td><td> (G^ao)</td><td> n₃</td>

<td> (_GA3-4)</td><td> s~s^</td><td></td><td></td>

<td></td><td> o₂»0 where the N0₂ group is optional</td><td></td><td></td>

where Ar represents a C_s^ arylene group, e.g. phenylene.

In some embodiments where G is G1, m may be 2, 3 or 5.

In some embodiments where G is G1, G^A may be G^A1'¹.

In embodiments of the present invention, G is of formula:

G^A-(CH₂)_ffl-O- (G2)

Where m is from 0 to 6; and

G^A is selected from the groups above.

In some embodiments where G is G2, G^A may be G^A3'².

In some embodiments where G is G2, m may be 1.

In embodiments of the present invention, G is of formula:

G^A-(CH₂)_Q-O-C(=O)-NH-(CH₂)_P- (G3)

Where q is from 1 to 3, and p is from 1 to 3: and

G^a is selected from the groups above.

In some embodiments where G is G3, q may be 1, and p may be 2.

In some embodiments where G is G3, G^A may be selected from G^A7 and G^A8.

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

In embodiments of the present invention. G is of formula:

<img file="CA2901941C_D0051.tif" />

Where m is from 0 to 6;

X¹ and X² are as defined above for L4; G^a is selected from the groups above.

R and R‘

In one embodiment, R is independently selected from optionally substituted C1.12 alkyl.

C3.20 heterocyciyl and C5.20 aryl groups. These groups are each defined in the substituents section below.

In one embodiment. R is independently optionally substituted C1.12 alkyl.

In one embodiment, R is independently optionally substituted C3.20 heterocyclyl.

In one embodiment, R is independently optionally substituted C5.20 aryl.

In one embodiment, R is independently optionally substituted Cm₂ alkyl.

The preferences for R apply also to R’.

In some embodiments of the invention there is provided a compound having a substituent group -NRR’. In one embodiment, R and R’ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring. The ring may contain a further heteroatom, for example N, 0 or S.

In one embodiment, the heterocyclic ring is itself substituted with a group R. Where a further N heteroatom is present, the substituent may be on the N heteroatom.

rA4

In one embodiment, R^A4 is a C24 alkylene group.

In one embodiment, R^A4 is a C₂ alkylene group.

In one embodiment, R^A4 is a C3 alkylene group.

In one embodiment, R^A4 is an unsubstituted Ci-e alkylene group.

In one embodiment, R*⁴ is a linear C&lt;.e aikyiene group.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

In one embodiment. R^M is selected from the group consisting of -Ch^CH^·. -CH₂CH₂CH₂and -CH₂CH₂CH₂CH₂Celi Binding Agent

A cel! binding agent may be of any kind, and include peptides and non-peptides. These can include antibodies or a fragment of an antibody that contains at least one binding site, lymphokines, hormones, growth factors, nutrient-transport molecules, or any other cell binding molecule or substance.

The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller ef al (2003) Jour, of Immunology 170:4854-4861 ). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C., Travers, P.. Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a lull-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to. cancer cell or ceils that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. lgG1, lgG2, lgG3, lgG4. lgA1 and lgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin.

Antibody fragments comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')₂, and Fv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler ef al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, US 4816567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597.

The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US 4816567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies include “primatized antibodies comprising variable domain antigen-binding sequences derived from a nonhuman primate (e.g. Old World Monkey or Ape) and human constant region sequences.

An “intact antibody” herein is one comprising a VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding: complement dependent cytotoxicity; Fc receptor binding; antibody-dependent celt-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “classes There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGl, lgG2, lgG3, lgG4, IgA, and lgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

Examples of cell binding agents include those agents described for use in WO 2007/085930.

The cell binding agent may be, or comprise, a polypeptide. The polypeptide may be a cyclic polypeptide. The cell binding agent may be antibody. Thus, in one embodiment, the present invention provides an antibody-drug conjugate (ADC).

Drug loading

The drug loading is the average number of PBD drugs per antibody. Drug loading may range from 1 to 8 drugs (D) per antibody (Ab), i.e. where 1, 2,3,4, 5,6,7, and 8 drug moieties are covalently attached to the antibody. Compositions of ADC include collections of antibodies conjugated with a range of drugs, from 1 to 8. The average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, electrophoresis, and HPLC. The quantitative distribution of ADC in terms of p may also be determined. By ELISA, the averaged value of p in a particular preparation of ADC may be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843852). However, the distribution of p (drug) values is not discernible by the antibody-antigen binding and detection limitation of ELISA Also, ELISA assay for detection of antibody-drug conjugates does not determine where the drug moieties are attached to the antibody, such as the heavy chain or light chain fragments, or the particular amino acid residues. In some instances, separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.

CA

2901941 2019-06-11

CA 02901941 2015-09-19

WO 2014/159981

PCT7US2014/025564

For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached. Higher drug loading, e.g. p >5, may cause aggregation, insolubility, toxicity, or loss of ceilular permeability of certain antibody-drug conjugates.

Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with the drug-linker intermediate (D-L) or linker reagent. Only the most reactive lysine groups may react with an amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety. Most cysteine thiol residues in the antibodies of the compounds exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP, under partial or total reducing conditions. The loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug-linker intermediate (D-L) or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thioi modification.

Cysteine amino acids may be engineered at reactive sites in an antibody and which do not form intrachain or intermoiecular disulfide linkages (Junutula, et ai., 2008b Nature Biotech., 26(8):925-932; Doman et al (2009) Blood 114(13):2721-2729: US 7521541; US 7723485; W02009/052249, Shen et al (2012) Nature Biotech., 30(2):184-191: Junutula et al (2008) Jour of Immun. Methods 332:41-52). The engineered cysteine thiois may react with linker reagents or the drug-linker reagents of the present invention which have thiol-reactive, electrophilic groups such as maleimide or aipha-halo amides to form ADC with cysteine engineered antibodies (ThioMabs) and the PBD drug moieties. The location of the drug moiety can thus be designed, controlled, and known. The drug loading can be controlled since the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or drug-linker reagents in high yield. Engineering an IgG antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain gives two new cysteines on the symmetrical antibody. A drug loading near 2 can be achieved and near homogeneity of the conjugation product ADC.

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564

Where more than one nucleophilic or electrophilic group of the antibody reacts with a druglinker intermediate, or linker reagent followed by drug moiety reagent, then the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to an antibody, e.g. 1, 2. 3, etc. Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate compounds in the mixture by drug loading value. Preparations of ADC with a single drug loading value (p) may be isolated, however, these single loading value ADCs may still be heterogeneous mixtures because the drug moieties may be attached, via the linker, at different sites on the antibody.

Thus the antibody-drug conjugate compositions of the invention include mixtures of antibody-drug conjugate compounds where the antibody has one or more PBD drug moieties and where the drug moieties may be attached to the antibody at various amino acid residues.

In one embodiment, the average number of dimer pyrrolobenzodiazepine groups per cell binding agent is in the range 1 to 20. In some embodiments the range is selected from 1 to 8, 2 to 8,2 to 6, 2 to 4, and 4 to 8.

In some embodiments, there is one dimer pyrrolobenzodiazepine groups per cell binding agent.

Peptides

In one embodiment, the cell binding agent is a linear or cyclic peptide comprising 4-20, preferably 6-20, contiguous amino acid residues. In this embodiment, it is preferred that one cell binding agent is linked to one monomer or dimer pyrrolobenzodiazepine compound.

In one embodiment the cell binding agent comprises a peptide that binds integrin α_νββ- The peptide may be selective for α_νβ® over XYS.

In one embodiment the ceil binding agent comprises the A20FMDV-Cys polypeptide. The A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC. Alternatively, a variant of the A20FMDV-Cys sequence may be used wherein one, two, three, four, five, six, seven, eight, nine or ten amino acid residues is substituted with another amino acid residue.

In one embodiment the antibody is a monoclonal antibody; chimeric antibody; humanized antibody; folly human antibody: or a single chain antibody. One embodiment the antibody is a fragment of one of these antibodies having biological activity. Examples of such fragments include Fab, Fab', F(ab')₂ and Fv fragments.

In these embodiments, each antibody may be linked to one or several dimer pyrrolobenzodiazepine groups. The preferred ratios of pyrrolobenzodiazepine to cell binding agent are given above.

The antibody may be a domain antibody (DAB).

In one embodiment, the antibody is a monoclonal antibody.

Antibodies for use in the present invention include those antibodies described in WO 2005/082023. Particularly preferred are those antibodies for tumour-associated antigens. Examples of those antigens known in the art include, but are not limited to, those tumourassociated antigens set out in WO 2005/082023. See, for instance, pages 41-55.

The conjugates of the invention are designed to target tumour cells via their cell surface antigens. The antigens are usually normal cell surface antigens which are either overexpressed or expressed at abnormal times. Ideally the target antigen is expressed only on proliferative cells (preferably tumour cells), however this is rarely observed in practice. As a result, target antigens are usually selected on the basis of differential expression between proliferative and healthy tissue.

Tumor-associated antigens (TAA) are known in the art, and can prepared for use in generating antibodies using methods and information which are well known in the art. In attempts to discover effective cellular targets for cancer diagnosis and therapy, researchers have sought to identify transmembrane or otherwise tumor-associated polypeptides that are specifically expressed on the surface of one or more particular type(s) of cancer cell as compared to on one or more normal non-cancerous celi(s). Often, such tumor-associated polypeptides are more abundantly expressed on the surface of the cancer cells as compared to on the surface of the non-cancerous cells. The identification of such tumor-associated cell surface antigen polypeptides has given rise to the ability to specifically target cancer cells for destruction via antibody-based therapies.

CA

2901941 2019-06-11

Examples of TAA include, but are not limited to, TAA (1 )-(36) listed below. For convenience, information relating to these antigens, all of which are known in the art. is listed below and includes names, alternative names, Genbank accession numbers and primary reference(s), following nucleic acid and protein sequence Identification conventions of the National Center for Biotechnology Information (NCBI). Nucleic acid and protein sequences corresponding to TAA (1)-(36) are available in public databases such as GenBank. Tumor-associated antigens targeted by antibodies include all amino acid sequence variants and isoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequence identity relative to the sequences identified in the cited references, or which exhibit substantially the same biological properties or characteristics as a TAA having a sequence found in the cited references. For example, a TAA having a variant sequence generally is able to bind specifically to an antibody that binds specifically to the TAA with the corresponding sequence listed.

TUMOR-ASSOCIATED ANTIGENS (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank accession no. NM_001203) ten Dijke.P., et al Science 264 (5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997)); W02004/063362 (Claim 2); W02003/042661 (Claim 12); US2003/134790-A1 (Page 38-39); W02002/102235 (Claim 13; Page 296); W02003/055443 (Page 91-92); WO2002/99122 (Example 2; Page 528-530); W02003/029421 (Claim 6); W02003/024392 (Claim 2; Fig 112); WO2002/98358 (Claim 1; Page 183); W02002/54940 (Page 100-101); WO2002/59377(Page 349-350); W02002/30268 (Claim 27; Page 376); W02001/48204 (Example; Fig 4); NP_001194 bone morphogenetic protein receptor, type IB/pid=NP_001194.1. Cross-references: MIM:603248; NP_001194.1; AY065994 (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486) Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291 (1998), Gaugitsch, H.W., et a/(1992) J. Biol. Chem. 267 (16):11267-11273); W02004/048938 (Example 2); W02004/032842 (Example IV); W02003/042661 (Claim 12); W02003/016475 (Claim 1); WO2002/78524 (Example 2); W02002/99074 (Claim 19; Page 127-129); W02002/86443 (Claim 27; Pages 222, 393); W02003/003906 (Claim 10; Page 293); WO2002/64798 (Claim 33; Page 93-95); W02000/14228 (Claim 5; Page 133-136); US2003/224454 (Fig 3); W02003/025138 (Claim 12; Page 150); NP_003477 solute canier family 7 (cationic amino acid transporter, y+system), member 5/pid=NP_003477.3 - Homo sapiens; Crossreferences: MIM:600182; NP_003477.3; NM_015923; NM 003486 1

CA

2901941 2019-06-11

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564 (3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbank accession no. NM_012449); Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R.S., et al (1999) Proc. Natl. Acad. Sei. U.S.A. 96 (25):14523-14528): W02004/065577 (Ciaim 6); W02004/027049 (Fig 1L); EP1394274 (Example 11); W02004/016225 (Claim 2); W02003/042661 (Claim 12): US2003/157089 (Example 5); US2003/185830 (Example 5); US2003/064397 (Fig 2); WQ2002/89747 (Example 5; Page 618-619): W02003/022995 (Example 9; Fig 13A, Exampie 53; Page 173, Exampie 2; Fig 2A); NP_03658l six transmembrane epithelial antigen of the prostate; Cross-references: MIM:604415; NP_036581.1; NM_012449„1 (4) 0772P (CA125, MUC16, Genbank accession no. AF361486); J. Biol. Chem. 276 (29):27371-27375 (2001)); W02004/045553 (Claim 14); WO2002/92836 (Claim 6; Fig 12); WO2002/83866 (Claim 15: Page 116-121); US2003/124140 (Example 16); Crossreferences: GI:34501467; AAK74120.3; AF361486_1 (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin, Genbank accession no. NM_005823) Yamaguchi, N., eta!Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96 (20):11531-11536 (1999), Proc. Natl. Acad. Sei. U.S.A. 93 (1) :136-140 (1996), J. Biol. Chem. 270 (37):21984-21990 (1995)): W02003/101283 (Claim 14); (WQ2002/102235 (Claim 13; Page 287-288); W02002/101075 (Claim 4; Page 308309): WO2002/71928 (Page 320-321); WO94/10312 (Page 52-57); Cross-references: MIM:601051; NP_005814.2; NM_005823_1 (6) Napi3b (NAPI-3B, NaPi2B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2. type II sodium-dependent phosphate transporter 3b, Genbank accession no. NM_006424) J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2) :281-284 (1999), Feild, J.A., et al (1999) Biochem. Biophys. Res. Commun. 258 (3):578582); W02004/022778 (Claim 2); EP1394274 (Example 11); W02002/102235 (Claim 13; Page 326); EP0875569 (Claim 1; Page 17-19); W02001/57188 (Claim 20; Page 329): W02004/032842 (Example IV); W02001/75177 (Claim 24; Page 139-140): Crossreferences: MIM:604217; NP_006415.1; NM_006424_1. In certain embodiments, conjugate compounds of the invention comprise anti-NaPi2B antibodies. In one embodiment of the invention, an anti-NaPi2B antibody of an ADC of the invention comprise (a) CDR L1 of SEQ ID NO:1; (b) CDR L2 of SEQ ID NO:2; (c) CDR L3 of SEQ iD NO:3; (d) CDR H1 of SEQ ID NO:4; (e) CDR H2 of SEQ ID NO:5; (f) CDR H3 of SEQ ID NO:6. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:8 and SEQ ID NO:7,

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 respectively, including post-translational modifcations of those sequences. In one embodiment, the antibody comprises the heavy chain and light chain sequences in SEQ ID NO:10 and SEQ ID N0:9, respectively, including post-translational modifcations of those sequences.

<td> SEQ ID N0:1</td><td> I RSSETLVHSSGNTYLE</td>

<td> SEQ ID N0:2</td><td> |RVSNRFS</td>

<td> SEQ ID N0:3</td><td> I FQGSFNPLT Ï</td>

<td> SEQ ID N0:4</td><td> I GFSFSDFAMS I</td>

<td> SEQ ID N0:5</td><td> I ATIGRVAFHTYYPDSMKG</td>

<td> SEQ ID N0:6</td><td> | ARHRGFDVGHFDF</td>

<td> SEQ ID NO:7</td><td> I DIQMTQSPSSLSASVGDRVTITCRSSETLVHSSGNTYLEWYQQ | KPGKAPKLLIYRVSNRFSGVPSRFSGSGSGTDFTLTISSLQPED I FATYYCFQGSFNPLTFGQGTKVEIKR I</td>

<td> SEQ ID NO:8</td><td> I EVQLVESGGGLVQPGGSLRLSCAASGFSFSDFAMSWVRQAP | GKGLEWVATIGRVAFHTYYPDSMKGRFTISRDNSKNTLYLQMN | SLRAEDTAVYYCARHRGFDVGHFDFWGQGTLVTVSS</td>

<td> SEQ ID NO:9</td><td> | DIQMTQSPSSLSASVGDRVTITCRSSETLVHSSGNTYLEWYQQ I KPGKAPKLLIYRVSNRFSGVPSRFSGSGSGTDFTLTISSLQPED I Î FATYYCFQGSFNPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLK ! SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS | KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR | GEC I</td>

<td> SEQ ID NO:10</td><td> j EVQLVESGGGLVQPGGSLRLSCAASGFSFSDFAMSWVRQAP | GKGLEWVATIGRVAFHTYYPDSMKGRFTISRDNSKNTLYLQMN | SLRAEDTAVYYCARHRGFDVGHFDFWGQGTLVTVSSCSTKGP | SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG | VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYÎCNVNHKPSNTK | VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI | SRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ I YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA ! i KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW | ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF | SCSVMHEALHNHYTQKSLSLSPGK -J__________________________________________________________________________________________________________</td>

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG. Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1 -like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B, Genbank accession no. AB040878); Nagase T., et al (2000) DNA Res. 7 (2):143-150): W02004/000997 (Claim 1): W02003/003984 (Claim 1): W02002/06339 (Claim 1; Page 50); W02001/88133 (Claim 1; Page 41-43. 48-58): W02003/054152 (Claim 20): W02003/101400 (Claim 11); Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC:10737 (8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no. AY358628); Ross et ai (2002) Cancer Res. 62:2546-2553; US2003/129192 (Claim 2); US2004/044180 (Claim 12); US2004/044179 (Claim 11); US2003/096961 (Claim 11); US2003/232056 (Example 5); WG2003/105758 (Claim 12): US2003/206918 (Example 5): EP1347046 (Claim 1): W02003/025148 (Claim 20); Cross-references: Gl:37182378; AAQ88991.1; AY358628_1 (9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463); Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39,1991; Ogawa Y., et al Biochem. Biophys. Res. Commun. 178,248-255,1991; Arai H., etalJpn. Cire. J. 56, 1303-1307,1992; Arai H.. et al J. Biol. Chem. 268, 3463-3470,1993; Sakamoto A., Yanagisawa M., et al Biochem. Biophys. Res. Commun. 178, 656-663.1991: Elshourbagy N.A.. etalJ. Biol. Chem. 268. 3873-3879,1993; Haendler B., et al J. Cardiovasc. Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al Gene 228,43-49,1999; Strausberg R.L., et al Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903. 2002: Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123,1997; Okamoto Y., et al Biol. Chem. 272, 21589-21596,1997; Verheij J.B., et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R.M.W.. etalEur. J. Hum. Genet. 5. 180-185, 1997; Poffenberger E.G., et al Cell 79,1257-1266,1994; Attie T., et al, Hum. Moi. Genet. 4, 24072409,1995; Auricchio A., et al Hum. Mol. Genet. 5:351-354,1996: Amiel J., et al Hum. Mol. Genet. 5, 355-357,1996: Hofstra R.M.W., et al Nat. Genet. 12, 445-447, 1996; Svensson P.J., et al Hum. Genet. 103, 145-148, 1998: Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingauit V., et al (2002) Hum. Genet. 111, 198-206; W02004/045516 (Claim 1): W02004/048938 (Example 2); W02004/040000 (Claim 151); W02003/087768 (Claim 1); W02003/016475 (Claim 1); W02003/016475 (Claim 1); W02002/61087 (Fig 1); W02003/016494 (Fig 6); W02003/025138 (Claim 12; Page 144): W02001/98351 (Claim 1; Page 124-125): EP0522868 (Claim 8: Fig 2); W02001/77172 (Claim 1; Page 297-299); US2003/109676; US6518404 (Fig 3); US5773223 (Claim 1a; Col 31-34); W02004/001004

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 (10) MSG783 (RNF124, hypothetical protein FLJ20315. Genbank accession no. NM-017763); W02003/104275 (Claim 1); W02004/046342 (Example 2); W02003/042661 (Claim 12); W02003/083074 (Claim 14; Page 61); W02003/018621 (Claim 1); W02003/024392 (Claim 2; Fig 93); WO2001/66689 (Example 6); Cross-references:

Locus!D:54894: NP.060233.2; NM_017763_1 (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancer associated protein 1. six transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein, Genbank accession no. AF455138); Lab. Invest. 82 (11):1573-1582 (2002)); W02003/087306; US2003/064397 (Claim 1; Fig 1); WO2002/72596 (Claim 13; Page 54-55); WO2001/72962 (Claim 1; Fig 4B); W02003/104270 (Claim 11); W02003/104270 (Claim 16); US2004/005598 (Claim 22); W02003/042661 (Claim 12); US2003/060612 (Claim 12; Fig 10); WO2002/26822 (Claim 23; Fig 2); WO2002/16429 (Claim 12; Fig 10); Cross-references: Gl:22655488; AAN04080.1; AF455138_1 (12) TrpM4 (BR22450. FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4, Genbank accession no. NM_017636); Xu, X.Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278 (33):30813-30820 (2003)); US2003/143557 (Claim 4); W02000/40614 (Claim 14: Page 100-103): W02002/10382 (Claim 1; Fig 9A); W02003/042661 (Claim 12): W02002/30268 (Claim 27; Page 391); US2003/219806 (Claim 4); WO2001/62794 (Claim 14; Fig 1A-D); Cross-references: MIM:606936; NP_060106.2; NM_017636_1 (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor. Genbank accession no. NP_003203 or NM_003212); Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum. Genet. 49 (3):555-565 (1991)); US2003/224411 (Claim

1) ; W02003/083041 (Example 1); W02003/034984 (Claim 12): W02002/88170 (Claim 2: Page 52-53); W02003/024392 (Claim 2; Fig 58); W02002/16413 (Claim 1; Page 94-95, 105); W02002/22808 (Claim 2; Fig 1): US5854399 (Example 2: Col 17-18); US5792616 (Fig

2) ; Cross-references: MIM:187395; NP_003203.1; NM_003212_1 (14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs.73792 Genbank accession no. M26004); Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125): Weis J.J., et al J. Exp. Med. 167, 1047-1066,1988; Moore M., el al Proc. Natl. Acad. Sci. U.S.A. 84, 9194-9198,1987; Barel M, et al Mol. Immunol. 35, 1025-1031,

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

1998; Weis J.J., et al Proc. Natl. Acad. Set. U.S.A. 83. 5639-5643,1986; Sinha S.K., et al (1993) J. Immunol. 150, 5311-5320; W02004/045520 (Example 4); US2004/005538 (Example 1); W02003/062401 (Claim 9); W02004/045520 (Example 4); WO91/02536 (Fig 9.1-9.9); W02004/020595 (Claim 1); Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

(15) CD79b (CD79B, CD79p, IGb (immunoglobulin-associated beta), B29, Genbank accession no. NM_000626 or 11038674); Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):41264131, Blood (2002) 100 (9):3068-3076, Muller et al (1992) Eur. J. Immunol. 22 (6):16211625); W02004/016225 (claim 2, Fig 140); W02003/087768, US2004/101874 (claim 1, page 102); W02003/062401 (claim 9); WO2002/78524 (Example 2); US2002/150573 (claim 5, page 15); US5644033; W02003/048202 (ciaim 1, pages 306 and 309); WO 99/58658, US6534482 (claim 13, Fig 17A/B); W02000/55351 (claim 11, pages 1145-1146); Crossreferences: MIM:147245; NP_000617.1; NM_000626_1 (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764, AY358130); Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54 (2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci. U.S.A. 98 (17):9772-9777 (2001), Xu, M.J., et al (2001) Biochem. Biophys. Res. Commun. 280 (3):768-775: W02004/016225 (Claim 2);

W02003/077836; W02001/38490 (Ciaim 5; Fig 18D-1-18D-2); W02003/097803 (Claim 12); W02003/089624 (Claim 25); Cross-references: MIM:606509; NP_110391.2; NM_030764_1 (17) HER2 (ErbB2, Genbank accession no. M11730); Coussens L, et al Science (1985) 230(4730):1132-1139); Yamamoto T„ et al Nature 319, 230-234, 1986; Semba K., et al Proc. Natl. Acad. Sci. U.S.A. 82, 6497-6501,1985; Swiercz J.M., eta! J. Cell Biol. 165, 869880, 2004; Kuhns JJ., et al J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., et al Nature 421, 756-760, 2003; Ehsani A., et al (1993) Genomics 15, 426-429; W02004/048938 (Example 2); W02004/027049 (Fig 11); W02004/009622; W02003/081210;

W02003/089904 (Claim 9); W02003/016475 (Claim 1); US2003/118592; W02003/008537 (Claim 1); W02003/055439 (Ciaim 29; Fig 1A-B); W02003/025228 (Claim 37; Fig 5C); WO2002/22636 (Example 13; Page 95-107); W02002/12341 (Claim 68; Fig 7); WO2002/13847 (Page 71-74); W02002/14503 (Page 114-117); WO2001/53463 (Ciaim 2; Page 41-46); W02001/41787 (Page 15); W02000/44899 (Claim 52; Fig 7); W02000/20579 (Claim 3; Fig 2); US5869445 (Claim 3; Col 31-38); WO9630514 (Claim 2; Page 56-61 ); EP1439393 (Claim 7); W02004/043361 (Claim 7); W02004/022709; W02001/00244

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 (Example 3; Fig 4); Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1. In certain embodiments, conjugate compounds of the invention comprise antiHER2 antibodies. In one embodiment of the invention, an anti-HER2 antibody of an ADC of the invention comprises a humanized anti-HER2 antibody, e.g., huMAb4D5-1, huMAb4D5-2. huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8, as described in Table 3 of US 5821337. Those antibodies contain human framework regions with the complementarity-determining regions of a murine antibody (4D5) that binds to HER2. The humanized antibody huMAb4D5-8 is also referred to as trastuzumab, commercially available under the tradename HERCEPTIN. In another embodiment of the invention, an anti-HER2 antibody of an ADC of the invention comprises a humanized antiHER2 antibody, e.g., humanized 2C4, as described in US7862817. An exemplary humanized 2C4 antibody is pertuzumab, commercially available under the tradename PERJETA.

(18) NCA (CEACAM6, Genbank accession no. M18728); Barnett T., etal Genomics 3, 5966,1988; Tawaragi Y., et al Biochem. Biophys. Res. Commun. 150, 89-96,1988; Strausberg R.L., etalProc. Natl. Acad. Sci. U.S.A. 99:16899-16903, 2002; W02004/063709;

EP1439393 (Claim 7); W02004/044178 (Example 4): W02004/031238; W02003/042661 (Claim 12); WO2002/78524 (Example 2); WO2002/86443 (Claim 27; Page 427); W02002/60317 (Claim 2); Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728 (19) MDP (DPEP1, Genbank accession no. BC017023); Proc. Natl. Acad. Sci. U.S.A. 99 (26):16899-16903 (2002)): WQ2003/016475 (Claim 1); WO2002/64798 (Claim 33; Page 8587); JP05003790 (Fig 6-8); WO99/46284 (Fig 9); Cross-references: MIM:179780; AAH17023.1; BC017023J (20) IL20Ra (IL20Ra, ZCYTOR7, Genbank accession no. AF184971); Clark H.F., etai Genome Res. 13, 2265-2270, 2003; Mungall A.J., et al Nature 425, 805-811, 2003; Blumberg H., et al Cell 104,9-19,2001; Dumoutier L., etai J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J., etal J. Biol. Chem. 277. 47517-47523, 2002: Pletnev S., etal(2003) Biochemistry 42:12617-12624: Sheikh F., et al (2004) J. Immunol. 172, 2006-2010;

EP1394274 (Example 11); US2004/005320 (Example 5); WC2003/029262 (Page 74-75); W02003/002717 (Claim 2; Page 63); WO2002/22153 (Page 45-47); US2002/042366 (Page 20-21); W02001/46261 (Page 57-59); WO2001/46232 (Page 63-65): WO98/37193 (Claim 1; Page 55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564 (21) Brevican (BCAN, BEHAB. Genbank accession no. AF229053); Gary S.C., et al Gene 256, 139-147, 2000; Clark H.F., et al Genome Res. 13, 2265-2270, 2003; Strausberg R.L., et al Proc. Natl. Acad. Sci. U.S.A. 99,16899-16903, 2002; US2003/186372 (Claim 11); US2003/186373 (Claim 11): US2003/119131 (Claim 1; Fig 52); US2003/119122 (Claim 1; Fig 52); US2003/119126 (Claim 1); US2003/119121 (Claim 1; Fig 52); US2003/119129 (Claim 1); US2003/119130 (Claim 1); US2003/119128 (Claim 1; Fig 52); US2003/119125 (Claim 1); W02003/016475 (Claim 1); W02002/02634 (Claim 1) (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no. NM_004442): Chan, J. and Watt, V.M., Oncogene 6 (6), 1057-1061 (1991) Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int. Rev. Cytol. 196:177-244 (2000)); W02003042661 (Claim 12); W0200053216 (Claim 1; Page 41); W02004065576 (Claim 1); W02004020583 (Claim 9): W02003004529 (Page 128-132); W0200053216 (Claim 1: Page 42); Cross-references: MIM:600997; NP_004433.2; NM_004442_1 (23) ASLG659 (B7h, Genbank accession no. AX092328); US2004/0101899 (Claim 2); W02003104399 (Claim 11): W02004000221 (Fig 3): US2003/165504 (Claim 1): US2003/124140 (Example 2); US2003/065143 (Fig 60); W02002/102235 (Claim 13; Page 299); US2003/091580 (Example 2); W02002/10187 (Claim 6; Fig 10); W02001/94641 (Claim 12; Fig 7b); W02002/02624 (Claim 13; Fig 1A-1B); US2002/O34749 (Claim 54: Page 45-46): W02002/06317 (Example 2: Page 320-321, Claim 34; Page 321-322); W02002/71928 (Page 468-469); W02002/02587 (Example 1; Fig 1); WO2001/40269 (Example 3; Pages 190-192); W02000/36107 (Example 2: Page 205-207); W02004/053079 (Claim 12); W02003/004989 (Claim 1); WO2002/71928 (Page 233-234, 452-453); WO 01/16318 (24) PSCA (Prostate stem ceil antigen precursor, Genbank accession no. AJ297436); Reiter R.E., et al Proc. Natl. Acad. Sci. U.S.A. 95,1735-1740, 1998; Gu Z., et al Oncogene 19, 1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000) 275(3):783-788; W02004/022709: EP1394274 (Example 11); US2004/018553 (Claim 17); W02003/008537 (Claim 1); W02002/81646 (Claim 1; Page 164); W02003/003906 (Claim 10; Page 288); W02001/40309 (Example 1; Fig 17); US2001/055751 (Example 1; Fig 1b); W02000/32752 (Claim 18; Fig 1); WO98/51805 (Claim 17; Page 97); WO98/51824 (Claim 10; Page 94); W098/40403 (Claim 2; Fig 1B); Accession: 043653; EMBL; AF043498; AAC39607.1

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 (25) GEDA (Genbank accession No. AY260763); AAP14954 lipoma HMGÎC fusion-partnerlike protein /pid=AAP14954.1 - Homo sapiens (human); W02003/054152 (Claim 20); W02003/000842 (Claim 1); W02003/023013 (Example 3, Claim 20); US2003/194704 (Claim 45); Cross-references: &lt;31:30102449; AAP14954.1; AY260763_1 (26) BAFF-R (B ceil -activating factor receptor, BLyS receptor 3, BR3, Genbank accession No. AF116456); BAFF receptor /pid=NP_443177.1 - Homo sapiens: Thompson, J.S., et al Science 293 (5537). 2108-2111 (2001 ); W02004/058309; W02004/011611;

W02003/045422 (Example: Page 32-33); WO2003/C14294 (Claim 35; Fig 68);

W02003/035846 (Claim 70; Page 615-616); WO2002/94852 (Col 136-137); WO2002/38766 (Claim 3; Page 133); W02002/24909 (Example 3; Fig 3); Cross-references: M1M:6O6269; NP_443177.1; NM_052945_1; AF132600 (27) CD22 (B-ceil receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8, SiGLEC-2, FU22814. Genbank accession No. AK026467); Wilson et al (1991) J. Exp. Med. 173:137-146; W02003/072036 (Claim 1; Fig 1); Cross-references: MIM:107266; NP_001762.1; NM_OO1771_1. In certain embodiments, conjugate compounds of the invention comprise anti-CD22 antibodies. In one embodiment of the invention, an anti-CD22 antibody of an ADC of the invention comprise comprise three light chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3) and three heavy chain hypervariable regions (HVR-H1, HVR-H2 and

HVR-H3), according to US 8226945:

<td> HVR-L1</td><td> RSSQSIVHSVGNTFLE</td>

<td> HVR-L2</td><td> KVSNRFS</td>

<td> HVR-L3</td><td> FQGSQFPYT</td>

<td> HVR-H1</td><td> GYEFSRSWMN</td>

<td> HVR-H2</td><td> GRIYPGDGDTNYSGKFKG</td>

<td> HVR-H3</td><td> DGSSWDWYFDV</td>

(SEQIDNO:11) (SEQIDNO:12) (SEQIDNO:13) (SEQ ID NO:14) (SEQ ID NO: 15) (SEQ ID NO:16) (28) CD79a (CD79A. CD79a. immunoglobulin-assocsated alpha, a B cell-specific protein that covalently interacts with Ig beta (CD79B) and forms a complex on the surface with Ig M molecules, transduces a signal involved in B-cell differentiation), pl: 4.84, MW: 25028 TM: 2 [P] Gene Chromosome: 19q13.2, Genbank accession No. NP_OO1774.1O);

W02003/088808, US2003/0228319; W02003/062401 (claim 9); US2002/150573 (claim 4, pages 13-14); WO99/58658 (claim 13, Fig 16); WO92/07574 (Fig 1); US5644033; Ha et al (1992) J. Immunol. 148(5):1526-1531: Müller et al (1992) Eur. J. Immunol.. 22:1621-1625; Hashimoto et al (1994) Immunogenetics 40(4):287-295; Preud’homme et a/(1992) Clin. Exp.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

Immunol. 90(1 ): 141-146; Yu et al (1992) J. Immunol. 148(2) 633-637: Sakaguchi et al (1988) EMBOJ. 7(11):3457-3464 (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupied receptor that is activated by the CXCL13 chemokine, functions in lymphocyte migration and humoral defense, plays a role in HIV-2 infection and perhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa, pl: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accession No. NP_001707.1); W02004/040000; W02004/015426; US2003/105292 (Example 2); US6555339 (Example 2); W02002/61087 (Fig 1); W02001/57188 (Claim 20, page 269); W02001/72830 (pages 12-13); W02000/22129 (Example 1, pages 152-153, Example 2, pages 254-256); WO99/28468 (claim 1, page 38); US5440021 (Example 2, col 49-52); WO94/28931 (pages 56-58); WO92/17497 (claim 7, Fig 5); Dobner et a/(1992) Eur. J. Immunol. 22:2795-2799; Bareila et a/(1995) Biochem. J. 309:773-779 (30) HLA-DOB (Beta subunit of MHC class II molecule (la antigen) that binds peptides and presents them to CD4+ T lymphocytes); 273 aa, pl: 6.56, MW: 30820.TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No. NP_002111.1 ); Tonnelle et al (1985) EMBO J. 4(11 ):2839-2847: Jonsson et al (1989) Immunogenetics 29(6):411-413: Beck et al (1992) J. Mol. Biol. 228:433-441; Strausberg et a/(2002) Proc. Natl. Acad. Sri USA 99:1689916903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766; Beck et al (1996) J. Mol. Biol. 255:1-13; Naruse eta/ (2002) Tissue Antigens 59:512-519; WO99/58658 (claim 13, Fig 15); US6153408 (Col 35-38): US5976551 (col 168-170); US60W46 (col 145-146); Kasahara et al (1989) Immunogenetics 30(1 ):66-68: Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119 (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, deficiency may contribute to the pathophysiology of idiopathic detrusor instability); 422 aa), pl: 7.63, MW: 47206 TM: 1 [P] Gene Chromosome: I7p13.3. Genbank accession No. NP_002552.2); Le et al (1997) FEBS Lett. 418(1-2):195-199; W02004/047749; W02003/072035 (claim 10); Touchman et al (2000) Genome Res. 10:165-173; W02002/22660 (claim 20); W02003/093444 (claim 1); W02003/087768 (claim 1); W02003/029277 (page 82) (32) CD72 (B-cell differentiation antigen CD72, Lyb-2); 359 aa, pl: 8.66, MW: 40225, TM: 1 [Pj Gene Chromosome: 9p13.3, Genbank accession No. NP__001773.1); W02004042346

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564 (claim 65); W02003/026493 (pages 51-52, 57-58); W02000/75655 (pages 105-106); Von Hoegen et a! (1990) J. Immunol. 144(12):4870-4877; Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99:16899-16903.

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of the leucine rich repeat (LRR) family, regulates B-cell activation and apoptosis, loss of function is associated with increased disease activity in patients with systemic lupus erythematosis); 661 aa, pl: 6.20, MW: 74147 TM: 1 [Pl Gene Chromosome: 5q12, Genbank accession No. NP_005573.1); US2002/193567; WO97/07198 (claim 11, pages 39-42); Miura et al (1996) Genomics 38(31:299-304; Miura et al (1998) Blood 92:2815-2822; W02003/083047; WO97/44452 (claim 8, pages 57-61); WG2000/12130 (pages 24-26) (34) FcRH1 (Fc receptor-like protein 1, a putative receptor for the immunoglobulin Fc domain that contains C2 type lg-like and ITAM domains, may have a role in B-lymphocyte differentiation); 429 aa, pl: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession No. NP_443170.1); W02003/077836; W02001/38490 (claim 6, Fig 18E-1-18-E-2); Davis et a/(2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777: W02003/089624 (claim 8); EP1347046 (claim 1); W02003/089624 (claim 7) (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated 2, a putative immunoreceptor with possible roles in B cell development and lymphomagenesis; deregulation of the gene by translocation occurs in some B cell malignancies); 977 aa, pl: 6.88, MW: 106468, TM: 1 [P] Gene Chromosome: 1q21, Genbank accession No. Human:AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475. AL834187, AY358085; Mouse:AK089756. AY158090, AY506558; NP_112571.1; W02003/024392 (claim 2, Fig 97); Nakayama et al (2000) Biochem. Biophys. Res. Commun. 277(1 ):124-127; W02003/077836; W02001/38490 (claim 3, Fig 18B-1-18B-2) (36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembrane proteoglycan, related to the EGF/hereguiin family of growth factors and foliistatin); 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq: NP_057276; NCBI Gene: 23671; OMiM: 605734; SwissProt Q9UIK5; Genbank accession No. AF179274; AY358907, CAF85723, CQ782436; W02004/074320: JP2004113151; W02003/042661; W02003/009814; EP1295944 (pages 69-70); W02002/30268 (page 329); W02001/90304; US2004/249130; US2004/022727; W02004/063355; US2004/197325; US2003/232350; US2004/005563; US2003/124579; Horie et al (2000) Genomics 67:146-152; Uchida et al

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564 (1999) Biochem. Biophys. Res. Commun. 266:593-602: Liang et al (2000) Cancer Res. 60:4907-12; Glynne-Jones et ai (2001) Int J Cancer. Oct 15; 94(2):178-84.

(37) CD33 (CD33 molecule, SIGLEC-3, SIGLEC3, p67; CD33 antigen (gp67); gp67; myeloid cell surface antigen CD33; sialic acid binding lg-!ike lectin 3; sialic acid-binding lg-iike lectin); Nucleotide : Genbank accession no. MJ23197; Genbank version no. NM_23197.1 GI:180097;Genbank record update date: Jun 23, 2010 08:47 AM; Polypeptide: Genbank accession no. AAA51948; Genbank version no. AAA51948.1 Ol: 188098: Genbank record update date: Jun 23,2010 08:47 AM; Simmons D., et al J. Immunol. 141 (8), 2797-2800 (IQStylAntibodies : H195 (Lintuzumab)- Raza A., et al Leuk Lymphoma. 2009 Aug;50(8): 1336-44; US6,759.045 (Seattle Genetics/lmmunomedics); mAb OKT9: Sutherland, D.R. et al. Proc Natl Acad Sci USA 78(7): 4515-4519 1981, Schneider,C.. et al J Biol Chem 257, 8516-8522 (1982); rnAb E6: Hoogenboom.H.R., et al J Immunol 144, 32113217 (1990); US6,590,088 (Human Genome Sciences) -for example, SEQ ID NOs: 1 and 2 and ATCC accession no. 97521; US7,557,189 (Immunogen) -fFor example, an antibody or fragment thereof comprising a heavy chain variable region which comprises three CDRs having the amino acid sequences of SEQ ID NOs:1-3 and a light chain variabie region comprising three CDRs having the amino acid sequences of SEQ ID NOs:4-6.

In some embodiments, the anti-CD33 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:20; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:21; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:22; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:17; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:18; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the anti-CD33 antibody comprises the VH and VL sequences in SEQ ID NO:24 and SEQ ID NO:23, respectively, including post-translational modifications of those sequences.

In some embodiments, the anti-CD33 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:28; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:29; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:30: (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:25; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:26; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:27.

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564 in some embodiments, the anti-CD33 antibody comprises the VH and VL sequences in SEQ iD NO:32 and SEQ iD N0:31, respectively, inciuding post-transiationai modifications of those sequences, in some embodiments, the anti-CD33 antibody comprises tine VH and VL sequences in SEQ ID NO:34 and SEQ ID NO:33, respectively, including post-translational modifications of those sequences. In some embodiments, the anti-CD33 antibody comprises the VH and VL sequences in SEQ ID NO:36 and SEQ ID NO:35, respectively, including post-translational modifications of those sequences. In one embodiment, In some embodiments, the anti-CD33 antibody comprises the VH and VL sequences in SEQ ID NO:38 and SEQ ID NO:37, respectiveiy, including post-translational modifications of those sequences.

<td> 15G15.33 HVR-L1</td><td> RSSQSLLHSNGYNYLD</td><td> SEQ iD NO: 17 | ! Ï I</td>

<td> 15G15.33 HVR-L2</td><td> LGVNSVS</td><td> I SEQ !D NO: 18 | I i i</td>

<td> 15G15.33 HVR-L3</td><td> MQALQTPWT</td><td> SEQ ID NO:19 I 1 | i</td>

<td> 15G15.33 HVR-H1</td><td> NHAIS</td><td> SEQ ID NQ:20 1 i i i</td>

<td> 15G15.33 HVR-H2</td><td> GIIPIFGTANYAQKFQG</td><td> SEQ ID NO:21 | t i !</td>

<td> 15G15.33 HVR-H3</td><td> EWADVFD</td><td> SEQ ID NO:22 | i ï i</td>

<td> 15G15.33</td><td> EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYL DWYLQKPGQSPQLLIYLGVNSVSGVPDRFSGSGSGT</td><td> .................................................Ï SEQ ID NO:23 |</td>

<td> VL</td><td> DFTLKiSRVEAEDVGVYYCMQALQTPWTFGQGTKVEI K</td><td> i i ï j</td>

<td></td><td></td><td></td>

<td> 15G15.33</td><td> QVQLVQSGAEVKKPGSSVKVSCKASGGIFSNHAISWV</td><td> SEQ iD NO:24 |</td>

<td rowspan="2"> VH</td><td> RQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADES</td><td> 1 1</td>

<td> TSTAFMELSSLRSEDTAVYYCAREWADVFDIWGQGT MVTVSS</td><td> Ï Ï i 1</td>

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

<td> 9C3-HVR L1</td><td> RASQGIRNDLG</td><td> SEQ ID NO:25 | i</td>

<td> 9C3-HVR L2</td><td> AASSLQS</td><td> SEQ ID NO:26 | !</td>

<td> 9C3-HVR L3</td><td> LQHNSYPWT</td><td> SEQ ID NO:27 | I</td>

<td> 9C3-HVR H1</td><td> GNYMS</td><td> SEQ ID NO:28 | I</td>

<td> 9C3-HVR H2</td><td> LIYSGDSTYYADSVKG</td><td> SEQ ID NO:29 | |</td>

<td> 9C3-HVR H3</td><td> DGYYVSDMVV</td><td> SEQ !D NO:30 | j</td>

<td> 9C3 VL</td><td> DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQ QKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTI SSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK</td><td> SEQ ID N0:31 I I I ! i I</td>

<td> 9C3 VH</td><td> EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWV RQAPGKGLEWVSLIYSGDSTYYADSVKGRFNISRDISK NTVYLQMNSLRVEDTAVYYCVRDGYYVSDMWWGKG TTVTVSS</td><td> SEQ ID NO:32 | ! I | I</td>

<td> 9C3.2 VL</td><td> DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQ QKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTI SSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK</td><td rowspan="2"> SEQ ID NO:33 | I i I ------------j SEQ ID NO:34 I ! I I I ΐ</td>

<td> 9C3.2 VH</td><td> EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWV RQAPGKGLEWVSLIYSGDSTYYADSVKGRFTISRDÎSK NTVYLQMNSLRVEDTAVYYCVRDGYYVSDMWWGKG TTVTVSS</td>

<td> 9C3.3VL</td><td> DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQ QKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTI SSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK</td><td> SEQ ID NO:35 ΐ I I ! I</td>

<td> 9C3.3 VH</td><td> EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWV RQAPGKGLEWVSLIYSGDSTYYADSVKGRFSISRDISK NTVYLQMNSLRVEDTAVYYCVRDGYYVSDMWWGKG TTVTVSS</td><td> SEQ ID NO:36 | ! Ϊ I i</td>

<td> 9C3.4 VL</td><td> DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQ QKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTI SSLQPEDFATYYCLQHNSYPWTFGQGTKLEIK</td><td> SEQ ID NO:37 | I I I i</td>

<td> 9C3.4 VH</td><td> EVQLVESGGALIQPGGSLRLSCVASGFTISGNYMSWV RQAPGKGLEWVSLIYSGDSTYYADSVKGRFAISRDISK</td><td> SEQ ID NO:38 Ϊ 1 ! ! _________________________________________________________________________1</td>

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

NTVYLQMN$LRVEDTAVYYCVRDGYYV$DMWWGKG

TTVTVSS (38) LGR5/GPR49; Nucleotide: Genbank accession no. NM_003667; Genbank version no. NM_003667.2 01:24475886; Genbank record update date: Jul 22,2012 03:38 PM; Polypeptide: Genbank accession no. NP_003658; Genbank version no. NP_003658.1 GI:4504379; Genbank record update date: Jul 22,2012 03:38 PM.

In some embodiments, the anti-LGR5 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 46; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 47; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 48; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 43; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 40 and SEQ ID NO: 39, respectively, including post-translational modifications of those sequences.

In some embodiments, the anti-LGR5 comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 52; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 53; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 54; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 49; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 50: and (f) HVR-L3 comprising an amino acid sequence selected from SEQ ID NO: 51.

In some embodiments, the anti-LgR5 antibody comprises a VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO:42 and SEQ ID NO:41, respectively, including post-translational modifications of those sequences.

<td> 8E11 VL</td><td> DIVMTQSPDS LAVSLGERAT INCRASESVD NYGNSFMHWY QQKPGQPPKL LIYLASNLES GVPDRFSGSG SGTDFTLTÎS SLQAEDVAVY YCQQNYEDPF TFGQGTKVEI KR</td><td> SEQ ID NO:39 I 1 I I ! i 1 1 I</td>

<td> 8E11 VH</td><td> EVQLVQSGAE VKKPGASVKV SCKASGYTFS</td><td> SEQ ID NO:40|</td>

<td></td><td> AYWIEWVRQA PGQGLEW1GE ILPGSDSTDY NEKFKVRATF TSDTSTSTVY LELSSLRSED TAVYYCARGG HYGSLDYWGQ GTLVTVSS</td><td> I</td>

<td> YW353 VL</td><td> DIQMTQSPSS LSASVGDRVTITCRASQDVS TAVAWYQQKP GKAPKLLIYS ASFLYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYTTPPTFGQ GTKVEIKR</td><td> SEQ ID NO:41</td>

<td> YW353 VH</td><td> EVQLVESGGG LVQPGGSLRL SCAASGFTFT SYSISWVRQA PGKGLEWVAE IYPPGGYTDY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCAKAR LFFDYWGQGT LV1VSS</td><td> SEQ ID NO:42</td>

<td> 8E11-HVR L1</td><td> RASESVDNYG NSFMH</td><td> SEQ ID NO:43</td>

<td> 8E11-HVRL2</td><td> LASNLES</td><td> SEQ ID NO:44</td>

<td> 8E11-HVR L3</td><td> QQNYEDPFT</td><td> SEQ ID NO;45</td>

<td> 8E11-HVR H1</td><td> GYTFSAYWIE</td><td> SEQ ID NO:46</td>

<td> 8E11-HVRH2</td><td> EILPGSDSTD YNEKFKV</td><td> SEQ ID NO.47</td>

<td> 8E11-HVR H3</td><td> GGHYGSLDY</td><td> SEQ ID NO;48</td>

<td> YW353 HVR L1</td><td> RASQDVSTAVA</td><td> SEQ ID NO:49</td>

<td> YW353 HVR L2</td><td> SASFLYS</td><td> SEQ ID NO:50</td>

<td> YW353 HVR L3</td><td> QQSYTTPPT</td><td> SEQ ID NO:51</td>

<td> YW353 HVR H1</td><td> GFTFTSYSIS</td><td> SEQ ID NO:52</td>

<td> YW353 HVR H2</td><td> EIYPPGGYTD YADSVKG</td><td> SEQ ID NO:53</td>

<td> YW353 HVR H3</td><td> ARLFFDY</td><td> SEQ ID NO:54</td>

The parent antibody may also be a fusion protein comprising an albumin-binding peptide (ABP) sequence (Dennis et al. (2002) “Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins’ J Biol Chem. 277:35035-35043; WO

01/45746). Antibodies of the invention include fusion proteins with ABP sequences taught by: (i) Dennis et al (2002) J Biol Chem. 277:35035-35043 at Tables III and IV, page 35038; (ii) US 2004/0001827 at [0076]; and (iii) WO 01/45746 at pages 12-13.

In one embodiment, the antibody has been raised to target specific the tumour related 10 antigen α_νββ.

CA 2901941 2019-06-11

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

The cel! binding agent may be labelled, for example to aid detection or purification of the agent either prior to incorporation as a conjugate, or as part of the conjugate. The label may be a biotin label. In another embodiment, the cell binding agent may be labelled with a radioisotope.

Substituents

The phrase “optionally substituted” as used herein, pertains to a parent group which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substituents. The term “substituent” is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to. or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.

In a preferred embodiment, the substituents described herein (which include optional substituents) are limited to those groups that are not reactive to a cell binding agent. The link to the cell binding agent in the present case is formed from the bridge between the two PBD moieties through a linker group to the cell binding agent. Reactive functional groups located at other parts of the PBD structure may be capable of forming additional bonds to the cell binding agent (this may be referred to as crosslinking). These additional bonds may alter transport and biological activity of the conjugate. Therefore, in some embodiment, the additional substituents are limited to those lacking reactive functionality.

In one embodiment, the substituents are selected from the group consisting of R, OR, SR, NRR’, NO₂, halo, CO₂R, COR, CONH₂, CONHR, and CONRR’.

In one embodiment, the substituents are selected from the group consisting of R, OR, SR, NRR’, NO₂, COjR, COR, CONH₂, CONHR, and CONRR'.

In one embodiment, the substituents are selected from the group consisting of R, OR, SR, NRR’, NO₂, and halo.

In one embodiment, the substituents are selected from the group consisting of R. OR. SR, NRR’. and NO₂.

Any one of the embodiment mentioned above may be applied to any one of the substituents described herein. Alternatively, the substituents may be selected from one or more of the groups listed below.

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564

Examples of substituents are described in more detail below.

Ci-12 aikyi: The term “C1.12 alkyl as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, tolly unsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyi, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to, methyl (Ci), ethyl (C₂), propyl (C3), butyl (C₄), pentyl (C5), hexyl (Ce) and heptyl (0-).

Examples of saturated linear alkyl groups include, but are not limited to, methyl (Ci), ethyl (C₂), π-propyl (C3), n-butyl (C₄), n-pentyl (amyl) (Cs), n-hexyl (C₆) and n-heptyl (C?).

Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), and neo-pentyl (C₅).

An aikyi group may optionally be interrupted by one or more heteroatoms selected from O, N(H) and S. Such groups may be referred to as “heteroalkyl”.

C₂_12 Heteroalkyl: The term “C2.12 heteroalkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 2 to 12 carbon atoms, and one or more heteroatoms selected from 0, N(H) and S, preferably 0 and S.

Examples of heteroalkyl groups include, but are not limited to those comprising one or more ethylene glycol units of the type -(OCH2CH2)-. The terminal of a heteroalkyl group may be the primary form of a heteroatom, e.g. -OH, -SH or -NH₂. In a preferred embodiment, the terminal is -CH3.

C2.12 Alkenyl: The term “C2.12 alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564

Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, -CH=CH₂), 1-propenyl (-CH=CH-CH₃), 2-propenyi (allyl, -CH-CH=CH₂), isopropenyl (1-methylvinyl, -C(CH₃)=CH₂), butenyl (C,·.), pentenyl (C₅), and hexenyl (C₆).

C2-12 alkynyl: The term “C2-12 alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C^CH) and 2-propynyl (propargyl, -CH₂-CsCH).

C₃.i₂ cycloalkyl: The term 'Ό₃.ι₂ cycloalkyl” as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, those derived from:

saturated monocyclic hydrocarbon compounds: cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅), cyclohexane (C₆), cycloheptane (C7), methylcyclopropane (C₄), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (Ce), methylcyclopentane (C₃), dimethylcyclopentane (C7) and methylcyclohexane (C7);

unsaturated monocyclic hydrocarbon compounds: cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅). cyclohexene (C_fi), methylcyclopropene (C₄), dimethylcyclopropene (C5), methylcyclobutene (C»), dimethylcyclobutene (C_s), methylcyclopentene (Ge), dimethylcyclopentene (C7) and methylcyclohexene (C7); and saturated polycyclic hydrocarbon compounds:

norcarane (C7), norpinane (G?), norbornane (C?).

C3-20 heterocyclyl: The term ‘^:C₃.₂o heterocyclyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.

In this context, the prefixes (e.g. C₃.₂o, C₃.7, Cs-β, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 term “Cg^heterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:

N, : aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole) (Cs), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (Ce), dihydropyridine (Ce), tetrahydropyridine (Ce), azepine (C7);

O, : oxirane (C3), oxetane (C₄), oxolane (tetrahydrofuran) (C&amp;), oxole (dihydrofuran) (Cg), oxane (tetrahydropyran) (C₆), dihydropyran (C_s), pyran (C_e), oxepin (C₇);

Sf thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (Cg), thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);

0?: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);

0₃: trioxane (Ce);

N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);

NiO,: tetrahydrooxazole (Cg), dihydrooxazole (C₅), tetrahydroisoxazole (Cg), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (Ce);

N, S,: thiazoline (Cs). thiazolidine (Cs), thiomorpholine (Cg);

N₂0,: oxadiazine (Ce);

O, S,·. oxathiole (Cg) and oxathiane (thioxane) (Ce); and,

Ν,Ο,δ,: oxathiazine (Ce).

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (Cg), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (Ce), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.

Cg.20 aryl: The term “C5.20 aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

In this context, the prefixes (e.g. C3.20, C5.7, C₅^, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “Cs-garyl” as used herein, pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be ail carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (Cg), naphthalene (C10), azuiene (C10), anthracene (Cm), phenanthrene (Cm), naphthacene (Cis), and pyrene (Cig).

Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1 H-indene) (C₈), indene (C₉), isoindene (C₉). tetraline (1,2.3,4-tetrahydronaphthalene (Ct₀), acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene (C₁₅), and aceanthrene (Cis).

Alternatively, the ring atoms may include one or more heteroatoms, as in “heteroaryl groups. Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:

N&lt;: pyrrole (azole) (Cs), pyridine (azine) (Ce);

Ον furan (oxole) (C₅);

Sv thiophene (thiole) (C5);

NiO&lt;: oxazole (C5), isoxazole (C_s), isoxazine (Cs);

N₂0v oxadiazole (furazan) (C₅);

N₃0v oxatriazole (Cs);

NiSv thiazole (C5), isothiazole (C5);

N₂: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (Ce) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (Ce): N₃: triazole (Cs), triazine (Cg); and,

N₄: tetrazole (C5).

Examples of heteroaryi which comprise fused rings, include, but are not limited to: Co (with 2 fused rings) derived from benzofuran (OO, isobenzofuran (OO, indole (NO, isoindole (NO, indolizine (NO, indoline (NO, isoindoline (NO, purine (NO (e.g.. adenine, guanine), benzimidazole (N₂), indazole (N₂), benzoxazole (N:O0, benzisoxazole (N1OO. benzodioxole (O₂), benzofurazan (N₂O0, benzotriazole (N₃), benzothiofuran (S0> benzothiazole (N1SO, benzothiadiazole (N₂S);

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564

Cio(with 2 fused rings) derived from chromene (Oi). isochromene (0₅), chroman (0^), isochroman (Oi), benzodioxan (0₂), quinoline (Ni), isoquinoline (hh), quinolizine (N,), benzoxazine (N,Oi), benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂), quinazoline (N₂), cinnoline (N₂), phthalazine (N₂), naphthyridine (N₂), pteridine (N₄);

C: ; (with 2 fused rings) derived from benzodiazepine (N₂);

C13 (with 3 fused rings) derived from carbazole (N,)_t dibenzofuran (Os), dibenzothiophene (Si), carboline (N₂), perimidine (N₂), pyridoindole (N₂); and,

C14 (with 3 fused rings) derived from acridine (Ni), xanthene (O1). thioxanthene (Si), oxanthrene (0₂), phenoxathiin (0&lt;SO, phenazine (N₂), phenoxazine (NiOi), phenothiazine (NiSi), thianthrene (S₂), phenanthridine (Nt), phenanthroline (N₂), phenazine (N₂).

The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.

Halo: -F, -Cl, -Br. and -I.

Hydroxy: -OH.

Ether -OR, wherein R is an ether substituent, for example, a C1.7 alkyl group (also referred to as a C1.7 alkoxy group, discussed below), a C3-20 heterocyclyl group (also referred to as a C3-20 heterocyclyloxy group), or a C₅.2caryl group (also referred to as a C5-20 aryloxy group), preferably a Cwalkyl group.

Alkoxy: -OR, wherein R is an alkyl group, for example, a Chalky! group. Examples of C1.7 alkoxy groups include, but are not limited to, -OMe (methoxy), -OEt (ethoxy), -O(nPr) (npropoxy), -O(iPr) (isopropoxy), -O(nBu) (n-butoxy), -O(sBu) (sec-butoxy), -O(iBu) (isobutoxy), and -O(tBu) (tert-butoxy).

Acetal: -CH(OR¹)(OR²), wherein R¹ and R² are independently acetal substituents, for example, a C1.7 alkyl group, a C3.2o heterocyclyl group, or a C5-20aryl group, preferably a Chalky I group, or, in the case of a “cyclic” acetal group, R¹ and R², taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, -CH(OMe)2, -CH(OEt)₂, and -CH(OMe)(OEt).

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

Hemiacetal: -CH(OH)(OR¹). wherein R¹ is a hemiacetal substituent, for example, a Cv? alkyl group, a C3.20 heterocyclyl group, or a C^oaryl group, preferably a Cm alkyl group.

Examples of hemiacetal groups include, but are not limited to, -CH(OH)(OMe) and CH(OH)(OEt).

Ketal: -CR(0R¹)(0R²), where R¹ and R² are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C1.7 alkyl group, a C3.20 heterocyclyl group, or a C5.20 aryl group, preferably a C1.7 alkyl group. Examples ketal groups include, but are not limited to, -C(MeXOMe)a, -C(Me)(OEt)₂, -C(Me)(OMe)(OEt), -C(Et)(OMe)2, -C(Et)(0Et)2, and -C(Et)(OMe)(OEt).

Hemiketal: -CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a C,.? alkyl group, a C3.20 heterocyclyl group, or a C5.2Ü aryl group, preferably a Cm alkyl group. Examples of hemiacetal groups include, but are not limited to, -C(Me)(0H)(0Me), -C(Et)(OH)(OMe), -C(Me)(OH)(OEt), and -C(Et)(OH)(OEt).

Oxo (keto, -one): =O.

Thione (thioketone): =S.

Imino (imine): =NR, wherein R is an imino substituent, for example, hydrogen, C1.7 alkyl group, a C.vxi heterocyclyl group, or a Cs-2oaryl group, preferably hydrogen or a C_w alkyl group. Examples of ester groups include, but are not limited to, =NH, =NMe, =NEt, and =NPh.

Formyl (carbaldehyde, carboxaldehyde): -C(=O)H.

Acyl (keto): -C(=O)R, wherein R is an acyl substituent, for example, a Cm alkyl group (also referred to as Cm alkylacyi or Cm alkanoyl), a C3-20 heterocyclyl group (also referred to as C3-20 heterocyclylacyl), or a Cs.2oaryl group (also referred to as C5.20 arylacyl), preferably a Cm alkyl group. Examples of acyl groups include, but are not limited to, -C(=O)CH3 (acetyl), -C(=O)CH2CH3 (propionyl), -C(=O)C(CH₃)₃ (t-butyryl), and -C(=O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): -C(=O)OH.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

Thiocarboxy (thiocarboxylic acid): -C(=S)SH.

Thiolocarboxy (thiolocarboxylic acid): -C(=O)SH.

Thionocarboxy (thionocarboxylic acid): -C(=S)OH.

Imidic acid: -C(=NH)OH.

Hydroxamic acid: -C(=NOH)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyi): -C(=O)OR, wherein R is an ester substituent, for example, a 0|.₇ alkyl group, a C₃.₂₀ heterocyclyl group, or a C₅.2oaryl group, preferably a Cv? alkyl group. Examples of ester groups include, but are not limited to, -C(=O)OCH_3; -C(=O)OCH2CH₃, -C(=O)OC(CH₃)₃, and -C(=O)OPh.

Acyloxy (reverse ester): -OC(=O)R, wherein R is an acyloxy substituent, for example, a alkyl group, a C3.20 heterocyclyl group, or a C^aryi group, preferably a C-., alkyl group. Examples of acyloxy groups include, but are not limited to, -OC(=O)CH₃ (acetoxy), -OC(=O)CH₂CH₃, -0C(=0)C(CH₃)3, -OC(=O)Ph, and -OC(=O)CH₂Ph.

Oxycarboyloxy: -OC(=O)OR. wherein R is an ester substituent, for example, a C1.7 alkyl group, a C3.20 heterocyclyl group, or a C₅.20 aryl group, preferably a C1-7 alkyl group. Examples of ester groups include, but are not limited to, -00(=0 )OCH₃. -OC(=O)OCH_?CH₃, -OC(=O)OC(CH₃)₃, and -OC(=O)OPh.

Amino: -NR¹R², wherein R¹ and R² are independently amino substituents, for example, hydrogen, a C1.7 alkyl group (also referred to as C1-? alkylamino or di-Cv? alkylamino), a C^c· heterocyclyl group, or a C;_20aryl group, preferably H or a C1.7 alkyl group, or, in the case of a “cyclic” amino group, R¹ and R², taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (-NH2), secondary (-NHR¹), or tertiary (-NHR¹R²), and in cationic form, may be quaternary (-‘NR¹R²R³). Examples of amino groups include, but are not limited to, -NH₂, -NHCH3, -NHC(CH₃)₂, -N(CH₃)2, ~N(CH₂CH₃)2, and -NHPh. Examples of cyclic amino groups indude, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

Amido (carbamoyl, carbamyl, aminocarbonyi, carboxamide): -C(=O)NR¹R², wherein R' and R² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to. -C(=O)NH₂, -C(=O)NHCH₃, -C(=O)N(CH3)2, -C(=O)NHCH₂CH₃. and -C(=O)N(CH₂CH₃)₂, as well as amido groups in which R' and R², together with the nitrogen atom to which they are attached, form a heterocyciic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorphoiinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): -0(=3)ΝΡΈ², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, -C(=S)NH₂, -C(=S)NHCH₃, -C(=S)N(CH₃)₂, and -C(=S)NHCH₂CH₃.

Acylamido (acylamino): -NR¹C(=O)R², wherein R¹ is an amide substituent, for example, hydrogen, a Gw alkyl group, a C3-20 heterocyciyl group, ora C3.20 aryl group, preferably hydrogen or a Cv? alkyl group, and R² is an acyl substituent, for example, a Ci.7 alkyl group, a 0:3.20 heterocyclyl group, or a C₅.₂oaryl group, preferably hydrogen or a C1.7 alkyl group. Examples of acylamide groups include, but are not limited to. -NHC^OJCHs. -NHC(=O)CH₂CH₃, and -NHC(=O)Ph. R¹ and R² may together form a cyclic structure, as in, for example, succinimidyi, maleimidyi, and phthalimidyl:

O.N.

maleimidyi succinimidyi phthalimidyl

Aminocarbonyloxy: -OC(=O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, -OC(=O)NH₂, -0C(=0)NHMe, -0C(=0)NMe₂, and -OC(=O)NEt₂.

Ureido: -N(R¹)CONR²R³ wherein R² and R³ are independently amino substituents, as defined for amino groups, and R¹ is a ureido substituent, for example, hydrogen, a Cw alkyl group, a C&amp;.20 heterocyclyl group, or a C^oaryi group, preferably hydrogen or a Gw alkyl group. Examples of ureido groups include, but are not limited to, -NHCONH₂, -NHCONHMe, -NHCONHEt. -NHCONMe?, -NHCONEt₂, -NMeC0NH₂, -NMeCONHMe, -NMeCONHEt. NMeCONMe₂, and -NMeCONEt₂.

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

Guanidino: -NH-C(=NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom,

<img file="CA2901941C_D0052.tif" />

Imino: =NR, wherein R is an imino substituent, for example, for example, hydrogen, a C1.7 alkyl group, a C^o heterocyclyl group, or a C^aryl group, preferably H or a C^alkyl group. Examples of imino groups include, but are not limited to. =NH, =NMe, and =NEt.

Amidine (amidino): -C(=NR)NR₂, wherein each R is an amidine substituent, for example, hydrogen, a Cm alkyl group, a C3.20 heterocyclyl group, or a C^aryl group, preferably H or a C1.7 alkyl group. Examples of amidine groups include, but are not limited to, -C(=NH)NH₂, -C(=NH)NMe₂, and -C(=NMe)NMe₂.

Nitro: -NO?.

Nitroso: -NO.

Azido: -N₃.

Cyano (nitrile, carbonitrile): -CN.

Isocyano: -NC.

Cyanato: -OCN.

Isocyanato: -NCO.

Thiocyano (thiocyanato): -SCN.

Isothiocyano (isothiocyanate): -NCS.

Sulfhydryl (thiol, mercapto): -SH.

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564

Thioether (sulfide): -SR, wherein R is a thioether substituent, for example, a Cg? alkyl group (also referred to as a Cwalkylthio group), a C3-20 heterocyclyl group, or a Cs^aryl group, preferably a C1.7 alkyl group. Examples of Gw alkylthio groups include, but are not limited to, -SCH3 and -SCH₂CH₃.

Disulfide: -SS-R, wherein R is a disulfide substituent for example, a Cm alkyl group, a C.^o heterocyclyl group, or a C&amp;.₂₃ aryl group, preferably a C1.7 alkyl group (also referred to herein as C1.7 alkyl disulfide). Examples of C1.7 alkyl disulfide groups include, but are not limited to, -SSCH₃ and -SSCH₂CH₃.

Sulfine (sulfinyl, sulfoxide): -S(=O)R, wherein R is a sulfine substituent, for example, a C1.7 alkyl group, a heterocyclyl group, or a C^aryl group, preferably a 0^.7 alkyl group. Examples of sulfine groups include, but are not limited to, -S(-O)CH₃ and -S(=O)CH₂CH₃.

Sulfone (sulfonyl): -S(=O)₂R, wherein R is a sulfone substituent, for example, a Cw alkyl group, a C^₂₀ heterocyclyl group, or a C₅.₂₀aryl group, preferably a C1.7 alkyl group, including, for example, a fluorinated or perfluorinated Ci.₇ alkyl group. Examples of sulfone groups include, but are not limited to, -S(=O)2CH₃ (methanesulfonyl, mesyl), -S(=O)₂CF₃ (triflyl), -S(=O)2CH₂CH₃ (esyl), -S(=O)₂C₄F₉ (nonafiyl), -S(=O)₂CH₂CF₃ (tresyl), -S(=O)₂CH₂CH₂NH₂(tauryl), -S(=O)₂Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl).

4-chiorophenylsulfonyl (closyl), 4-bromophenylsuffonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): -S(=O)OH, -S0₂H.

Sulfonic acid (sulfo): -S(=O)₂OH, -SO₃H.

Sulfinate (sulfinic acid ester): -S(=O)OR; wherein R is a sulfinate substituent, for example, a C-i-7 alkyl group, a C^o heterocyclyl group, or a C^o aryl group, preferably a C1.7 alkyl group. Examples of sulfinate groups include, but are not limited to, -S(=O)OCH₃ (methoxysulfinyl; methyl sulfinate) and -S(-OpCH₂CH₃ (ethoxysulfinyl; ethyl sulfinate).

Sulfonate (sulfonic acid ester): -S(=O}₂OR, wherein R is a sulfonate substituent, for example, a C1.7 alkyl group, a C3.20 heterocyclyl group, or a aryl group, preferably a C1.7 alkyl group. Examples of sulfonate groups include, but are not limited to, -S(=O)₂OCH₃(methoxysulfonyl; methyl sulfonate) and -S^O^OCHjCHs (ethoxysulfonyl; ethyl sulfonate).

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

Sulfinyloxy: -OS(=O)R, wherein R is a sulfinyloxy substituent for example, a C1.7 alkyl group, a C3-20 heterocyclyl group, or a C5.20 aryl group, preferably a Cm alkyl group. Examples of sulfinyioxy groups include, but are not limited to, -OS(=O)CH₃ and -OS(=O)CH₂CH₃.

Sulfonyloxy: -OS(-O)₂R, wherein R is a sulfonyloxy substituent for example, a C1-7 alkyl group, a C^o heterocyclyl group, or a Cs-₂oaryl group, preferably a C1.7 alkyl group. Examples of sulfonyloxy groups include, but are not limited to, -OS(=O)₂CH₃ (mesylate) and -OS(=O>₂CH₂CH₃ (esylate).

Sulfate: -OS(=O)₂OR; wherein R is a sulfate substituent, for example, a Chalky! group, a C₃.2c>heterocyclyl group, ora C₅.₂₀ aryl group, preferably a C_walkyl group. Examples of sulfate groups include, but are not limited to, -OS(=O)2OCH₃ and -SO^OkOCHzCHj.

Sulfamyl (sulfamoyl: sulfinic acid amide; suifinamide): -S(=O)NR¹R², wherein R’ and R² are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, -S(=0)NI-k -S(-O)NH(CH₃), -Sf-OJNiCHj}?, -S(=O)NH(CH₂CH₃), -S(=O)N(CH₂CH₃)₂, and -S(=O)NHPh.

Sulfonamide (sulfinamoyl; sulfonic acid amide; sulfonamide): -S(=O)₂NR¹R²> wherein R' and R^z are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, -S(=O)2NH₂, -S(=O)₂NH(CH₃), -S(=O)?N(CH₃)₂, -S(=O)₂NH(CH_?CH₃), -S(=O;hN(CH₂CH₃)₂, and -S(=O)?NHPh.

Sulfamino: -NR¹S(=O)2OH, wherein R¹ is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, -NHS(=O)sOH and -N(CH₃)S(=O)₂OH.

Sulfonamino: -NR¹S(=^rO)2R, wherein R¹ is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a Cs.20 aryl group, preferably a Cw alkyl group. Examples of sulfonamino groups include, but are not limited to, -NHS(=O)2CH₃ and -N(CH₃)S(=O)₂CeH₅.

Sulfinamino; -NR¹S(=^sO)R, wherein R¹ is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C1.7 alkyl group, a C2_₂o heterocyclyl group,

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 or a C^aryl group, preferably a Cw alkyl group. Examples of sulfinamino groups include, but are not limited to, -NHS(=O)CH₃ and -N(CH₃)S(=O)C6H₅.

Phosphino (phosphine): -PR₂₎ wherein R is a phosphino substituent, for example, -H, a C1.7 alkyl group, a C320 heterocyclyl group, ora O&amp;æaryl group, preferably-H, a C1.7 alkyl group, or a Cô-joaryl group. Examples of phosphino groups include, but are not limited to, -PH₂. -P(CH₃)₂, -P(CH₂CH₃)2, -P(t-Bu)₂, and -P(Ph)2.

Phospho: -P(=O)₂.

Phosphinyl (phosphine oxide): -P(=O)R₂, wherein R is a phosphinyl substituent, for example, a C1.7 alkyl group, a C₃.₂₀ heterocyclyl group, or a Cs-æaryl group, preferably a C_t.₇ alkyl group or a C^o aryl group. Examples of phosphinyl groups include, but are not limited to, -P(=O)(CH₃)_2> -P(=O)(CH₂CH₃h, -P(=O)(t-Bu)₂, and -P(=O)(Ph)₂.

Phosphonic acid (phosphono): -P(=O)(OH)₂.

Phosphonate (phosphono ester): -P(=O)(OR)₂, where R is a phosphonate substituent, for example, -H, a Ci ? alkyl group, a C_{3 2}o heterocyclyl group, or a C_{6 20}aryl group, preferably -H, a G1.7 alkyl group, or a C5-» aryl group. Examples of phosphonate groups include, but are not limited to, -P(=O)(OCH₃)2, -P(=O)(OCH₂CH₃)₂, -P(=O)(O-t-Bu)₂, and -P(=O)(OPh)₂.

Phosphoric acid (phosphonooxy): -OP(=O)(OH)₂.

Phosphate (phosphonooxy ester): -OP(=O)(OR)2, where R is a phosphate substituent, for example, -H, a Cv? alkyl group, a heterocyclyl group, or a C^aryl group, preferably -H, a Ci-? alkyl group, or a ¢5-20 aryl group. Examples of phosphate groups include, but are not limited to, -OP(=O)(OCH₃)_2t -OP(=O)(OCH₂CH₃)₂, -OP(=O)(O-t-Bu)₂, and -OP(=O)(OPh}₂.

Phosphorous acid: -OP(OH)₂.

Phosphite: -OP(OR)₂, where R is a phosphite substituent, for example, -H, a Chalky! group, a Cj.jo heterocyclyl group, or a C^oaryi group, preferably -H, a Ci.7 alkyl group, or a Cs-₂oaryl group. Examples of phosphite groups include, but are not limited to, -OP(OCH₃)₂, -OP(OCH₂CH₃)₂, -OP(O-t-8u)₂, and -OP(OPh)₂.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

Phosphoramidite: -OP(OR’)-NR²2. where R¹ and R² are phosphoramidite substituents, for exampie, -H, a (optionaliy substituted) C1-7 alkyl group, a C₃.₂₀ heterocyclyl group, or a C5-20 aryl group, preferably -H, a C1-7 alkyl group, or a C^aryl group. Examples of phosphoramidite groups include, but are not limited to, -OP(OCH₂CH₃)-N(CH₃)₂, -OP(OCH₂CH₃)~N(i-Pr)₂, and -OP(OCH₂CH₂CN)-N(i-Pr)₂.

Phosphoramidate: -OP(=O)(OR¹)-NR²2, where R¹ and R² are phosphoramidate substituents, for example, -H, a (optionally substituted) C1.7 alkyl group, a C3.20 heterocyclyl group, or a Cs.20 aryl group, preferably -H, a C,.x alkyl group, or a aryl group. Examples of phosphoramidate groups include, but are not limited to, -OP(=O)(OCH2CH₃)-N(CH₃)₂, -OP(=O)(OCH₂CH₃)-N(i-Pr)₂, and -OP(=O)(OCH₂CH₂CN)-N(i-Pr)₂.

Alkylene

C₃_i₂ alkylene: The term “0-3.12 alkylene”, as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term “alkylene” indudes the sub-classes alkenylene, alkynylene, cycloalkyiene, etc., discussed below.

Examples of linear saturated C3-12 alkylene groups include, but are not limited to, -(0Η₂)_ηwhere n is an integer from 3 to 12, for example, -CH₂CH₂CH₂- (propylene), -CH₂CH₂CH₂CH₂- (butyiene), -CH₂CH₂CH₂CH₂CH₂- (pentylene) and -CH_?CH₂CH₂CH₂CH₂CH₂CH2- (heptylene).

Examples of branched saturated C₃.₁₂ alkylene groups include, but are not limited to, -CH(CH₃)CH₂-, ~CH(CH₃)CH₂CH2-, -CH(CH₃)CH₂CH₂CH2-, -CH₂CH(CH₃)CH2-, -CH₂CH(CH₃)CH₂CH₂-. -CH(CH₂CH₃)-, -CH(CH₂CH₃)CH₂-, and -CH₂CH(CH₂CH₃)CH2-.

Examples of linear partially unsaturated Cs-i₂ alkylene groups (C3-₁₂ aikenylene. and alkynylene groups) include, but are not limited to, -CH-CH-CH₂-, -CH^CH^CH^, -CH=CH-CH_rCH2-, -CH=CH-CH₂-CH₂-CH_r. -CH=CH-CH=CH-, -CH=CH-CH=CH-CH₂-, CH=CH-CH=CH-CH₂-CH₂-, -CH=CH-CH₂-CH=CH-, -CH=CH-CHj-CH₂-CH=CH-, and -CH₂C=C-CH₂-.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

Examples of branched partially unsaturated C3.12 alkylene groups (C₃.₁₂ alkenylene and alkynylene groups) include, but are not limited to, -C(CH₃)=CH-, -C(CH₃)=CH-CH2-, -CH=CH-CH(CH₃}- and -CsC-CH(CH₃)-.

Examples of alicyclic saturated C3-12 alkylene groups (C3-12 cycloalkylenes) include, but are not limited to. cyciopentylene (e.g. cyclopent-1,3-ylene). and cyclohexyiene (e.g. cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated alkylene groups (C^ cycloalkylenes) indude, but are not limited to, cydopentenyiene (e.g. 4-cydopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyciohexen-1,4-ylene; 3-cyclohexen-1,2-yiene; 2,5-cyclohexadien1,4-ylene).

Includes Other Forms

Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-C00‘), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N*HR¹R²), a salt or soivate of the amino group, for example, a hydrochloride salt, as well as conventionai protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-O'), a salt or soivate thereof, as well as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, etai., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group which may be anionic (e.g. -COOH may be -COO’), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na* and K*, alkaline earth cations such as Ca²‘ and Mg²*, and other cations such as ΑΓ³. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH₄*) and substituted ammonium ions (e.g. NH3R*, NH2R2*, NHR₃*, NRZ). Exampies of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine,

CA 02901941 2015-09-19

WO 2014/159981 PCT7US2014/025564 dicyclohexylamine, triethylamine, butylamine, ethyienediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenyibenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is Ν(ΟΗ₃)Λ lithe compound is cationic, or has a functional group which may be cationic (e.g. -NH₂ may be -NH₃⁺), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonlc, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic acid and valeric.

Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

The invention includes compounds where a solvent adds across the imine bond of the PBD moiety, which is illustrated below where the solvent is water or an alcohol (R^AOH, where R^Ais C1.4 alkyl):

<img file="CA2901941C_D0053.tif" />

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

These forms can be called the carbinolamine and carbinolamine ether forms of the PBD (as described in the section relating to R¹⁰ above). The balance of these equilibria depend on the conditions in which die compounds are found, as well as the nature of the moiety itself.

These particular compounds may be isolated in solid form, for example, by lyophilisation.

isomers

Certain compounds of the invention may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis· and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal· and anticlinal-forms: o- and β-forms: axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).

The term “chiral” refers to molecules which have the property of non-superimposability of the mirror image partner, while the term “achiral refers to molecules which are superimposable on their mirror image partner.

The term “stereoisomers” refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

“Diastereomer” refers to a stereoisomer with two os’ more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g, melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography, “Enantiomers” refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.

Stereochemical definitionsand conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S,, “Stereochemistry of Organic Compounds, John Wiley &amp; Sons, Inc., New York. 1994. The compounds of the invention may contain asymmetric or chiral

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and i or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or I meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms “racemic mixture” and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group. -OCH₃, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, -CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, metachlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. Cm alkyl includes n-propyl and iso-propyl; butyl indudes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and paramethoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

„0 X ,OH H⁺ k O’ —-C~C' =s=s= C~C c-c'

I \ / X H* z \ keto enol enolate

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

The term tautomer” or tautomeric form'’ refers to structurai isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹H, ²H (D), and ³H (T); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ^1CO and ¹⁸O; and the like.

Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as, but not limited to ²H (deuterium, D), ³H (tritium), ”C, -³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S, ³⁶CI, and ¹²⁶l. Various isotopically labeled compounds of the present invention, for example those into which radioactive isotopes such as 3H, 13C, and 14C are incorporated. Such isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or singlephoton emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. Deuterium labelled or substituted therapeutic compounds of the invention may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism, and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements. An 18F labeled compound may be useful for PET or SPECT studies. Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopicaily labeled reagent. Further, substitution with heavier isotopes, particularly deuterium (i.e., 2H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index. It is understood that deuterium in this context is regarded as a substituent. The concentration of such a heavier isotope, specifically deuterium, may be defined by an isotopic enrichment factor. In the compounds of

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 this invention any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Biological Activity

In vitro cell proliferation assays

Generaily, the cytotoxic or cytostatic activity of an antibody-drug conjugate (ADC) is measured by: exposing mammalian cells having receptor proteins, e.g. HER2, to the antibody of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays are used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of an ADC of the invention.

The in vitro potency of antibody-drug conjugates can be measured by a cell proliferation assay. The Cell Titer-Gio® Luminescent Cell Viability Assay is a commercially available (Promega Corp., Madison. Wl), homogeneous assay method based on the recombinant expression of Coleoptera luciferase (US Patent Nos. 5583024; 5674713 and 5700670). This cell proliferation assay determines the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active ceils (Crouch ef at (1993) J- Immunol. Meth. 160:81-88; US 6602677). The CellTiter-Glo® Assay is conducted in 96 well format, making it amenable to automated high-throughput screening (HTS) (Cree eta! (1995) AntiCancer Drugs 6:398-404). The homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to ceils cultured in serum-supplemented medium. Celi washing, removal of medium and multiple pipetting steps are not required. The system detects as few as 15 ceils/weli in a 384-well format in 10 minutes after adding reagent and mixing. The cells may be treated continuously with ADC, or they may be treated and separated from ADC. Generally, cells treated briefly, i.e. 3 hours, showed the same potency effects as continuously treated cells.

The homogeneous “add-mix-measure” format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 proportional to the number of cells present in culture. The CellTiter-Glo® Assay generates a glow-type” luminescent signal, produced by the luciferase reaction, which has a half-life generally greater than five hours, depending on cell type and medium used. Viable cells are reflected in relative luminescence units (RLU). The substrate, Beetle Luciferin, is oxidatively decarboxylated by recombinant firefly luciferase with concomitant conversion of ATP to AMP and generation of photons.

In vivo efficacy

The in vivo efficacy of antibody-drug conjugates (ADC) of the invention can be measured by tumor xenograft studies in mice. For example, the in vivo efficacy of an anti-HER2 ADC of the invention can be measured by a high expressing HER2 transgenic expiant mouse model. An allograft is propagated from the Fo5 mmtv transgenic mouse which does not respond to, or responds poorly to, HERCEPTIN® therapy. Subjects were treated once with ADC al certain dose levels (mg/kg) and PBD drug exposure (pg/m²); and placebo buffer control (Vehicle) and monitored over two weeks or more to measure the time to tumor doubling, log cell kill, and tumor shrinkage.

Use

The conjugates of the invention may be used to provide a PBD conjugate at a target location.

The target location is preferably a proliferative cell population. The antibody is an antibody for an antigen present in a proliferative cell population.

In one embodiment the antigen is absent or present at a reduced level in a non-proliferative cell population compared to the amount of antigen present in the proliferative cell population, for example a tumour cell population.

The target location may be in vitro, in vivo or ex vivo.

The antibody-drug conjugate (ADC) compounds of the invention include those with utility for anticancer activity. In particular, the compounds indude an antibody conjugated, i.e. covalently attached by a linker, to a PBD moiety.

At the target location the linker may not be cleaved. The antibody-drug conjugate (ADC compounds of the invention may have a cytotoxic effect without the cleavage of the linker to

CA 02901941 2015-09-19

WO 2014/159981 PCT7US2014/025564 release a PBD drug moiety. The antibody-drug conjugates (ADC) of the invention selectively deliver cytotoxic agent to tumor tissue whereby greater selectivity, i.e. a lower efficacious dose, may be achieved.

Thus, in one aspect, the present invention provides a conjugate compound as described herein for use in therapy.

In a further aspect there is also provides a conjugate compound as described herein for use in the treatment of a proliferative disease. A second aspect of the present invention provides the use of a conjugate compound in the manufacture of a medicament for treating a proliferative disease.

One of ordinary skill in the art is readily able to determine whether cr not a candidate conjugate treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below.

The term “proliferative disease pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.

Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to. neoplasms and tumours (e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi’s sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Cancers of particular interest include, but are not limited to, leukemias and ovarian cancers.

Any type of cell may be treated, including but not limited to, lung, gastrointestinal (including, e.g. bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.

In one embodiment, the treatment is of a pancreatic cancer.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

In one embodiment, the treatment is of a tumour having a_vp_G integrln on the surface of the cell.

It is contempiated that the antibody-drug conjugates (ADC) of the present invention may be used to treat various diseases or disorders, e.g. characterized by the overexpression of a tumor antigen. Exemplary conditions or hyperproliferative disorders include benign or malignant tumors; leukemia, haematological, and lymphoid malignancies. Others include neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic, including autoimmune, disorders.

Generally, the disease or disorder to be treated is a hyperproliferative disease such as cancer. Examples of cancer to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, coion cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.

Autoimmune diseases for which the ADC compounds may be used in treatment include rheumatologic disorders (such as, for example, rheumatoid arthritis. Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, poiymyositis/dermatomyositis, cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriatic arthritis), osteoarthritis, autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel diseases (e.g. ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener’s granulomatosis, and polyarteriitis), autoimmune neurological disorders (such as, for example, multiple sclerosis, opsoclonus myoclonus syndrome, myasthenia gravis, neuromyeiitis optica, Parkinson’s disease, Alzheimer’s disease, and autoimmune polyneuropathies), renal disorders (such as, for example, glomerulonephritis, Goodpasture’s syndrome, and Berger’s disease), autoimmune

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564 dermatologic disorders (such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bulious pemphigoid, and cutaneous iupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, posttransfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders (such as, for example, diabetic-related autoimmune diseases such as insulindependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid disease (e.g. Graves’ disease and thyroiditis)). More preferred such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjogren's syndrome, Graves’ disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

Methods of Treatment

The conjugates of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate compound of the invention. The term “therapeutically effective amount is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.

A compound of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e g. drugs, such as chemotherapeutics); surgery; and radiation therapy.

A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Ciasses of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in “targeted therapy” and conventional chemotherapy.

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

Examples of chemotherapeutic agents include: erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(ll), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyi-5-oxo- 2.3,4,6,8pentazabicyclo [4,3.0} nona-2,7.9-triene- 9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1enyl)phenoxy]-MN-dimethyiethanamine. NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2, HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozoie (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exeiixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exeiixis), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®. AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson &amp; Johnson), ABRAXANE™ (Cremophor-free), albuminengineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg. Il), vandetanib (r!NN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Teiik), thiotepa and cyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including aitretamine, triethyienemeiamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; caliystatin; CC-1065 (including its adozelesin, carzeiesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8): dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin: nitrogen mustards such as chlorambucil, chiornaphazine, chlorophosphamide, estrarnusline, ifosfamide, mechiorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, calicheamicin gammall, calicheamicin omegall (Angew Chem. Inti. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazO’5OXO’L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin. tubercidin. ubenimex. zinostatln, zorubicin; anti-metabolites such as methotrexate and 5-fiuorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine anaiogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitïostanol. mepitiostane, testolactone: antl-adrenals such as aminoglutéthimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aidophosphamide glycoside: aminolevulinic acid: eniluracil: amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone: etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone: mitoxantrone: mopidanmol; nitraerine; pentostatin: phenamet; pirarubicin; losoxantrone; podophyilinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2’,2trichiorotriethyiamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); ureîhan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C ‘); cyclophosphamide; thiotepa; 6-thioguanine; mercapiopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO): retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

Also included in the definition of “chemotherapeutic agent are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestroi acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, Rl VISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARÎMIDEX® (anastrozole; AstraZeneca): (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, ieuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, for example, PKC-alpha, Raf and Η-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTlN®, LEUVECTIN®. and VAXID®; PROLEUKIN® rlL-2; topoisomerase 1 inhibitors such as LURTOTECAN®: ABARELIX® rmRH; (ix) antiangiogenic agents such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idee), pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents in combination with the conjugates of the invention include: alemtuzumab, apolizumab, aseiizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, peefusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab,

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tociiizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and visiiizumab.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a conjugate compound, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection at toe site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH. isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer’s Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Formulations

While it is possible for toe conjugate compound to be used (e.g., administered) atone, it is often preferable to present it as a composition or formulation.

In one embodiment, the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a conjugate compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

CA 02901941 2015-09-19

WO 2014/159981 PCT7US2014/025564

In one embodiment, the composition is a pharmaceutical composition comprising at least one conjugate compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to. pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.

In one embodiment, the composition further comprises other active agents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams &amp; Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

Another aspect of the present invention pertains to methods of making a pharmaceutical composition comprising admixing at least one [¹¹C]-radiolabelled conjugate or conjugate-like compound, as defined herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in foe art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound.

The term “pharmaceutically acceptable.’ as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with foe tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. In general, foe formulations are prepared by uniformly and intimately bringing into association the active compound with

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.

Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticuiate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active ingredient in the liquid is from about 1 ng/ml to about 10 pg/ml, for example from about 10 ng/ml to about 1 pg/ml. The formulations may be presented in unit-dose or muiti-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriate dosages of the conjugate compound, and compositions comprising the conjugate compound, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used tn combination, the severity of the condition, and the species, sex, age, weight, condition, genera! health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or ciinician.

In general, a suitable dose of the active compound is in the range of about 100 ng to about 25 mg (more typically about 1 pg to about 10 mg) per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 100 mg, 3 times daily.

In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 150 mg, 2 times daily.

In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 200 mg, 2 times daily.

However in one embodiment, the conjugate compound is administered to a human patient according to the following dosage regime: about 50 or about 75 mg, 3 or 4 times daily.

In one embodiment, the conjugate compound is administered to a human patient according to the following dosage regime: about 100 or about 125 mg, 2 times daily.

The dosage amounts described above may apply to the conjugate (including the PBD moiety and the linker to the antibody) or to the effective amount of PBD compound provided, for example the amount of compound that is releasable after cleavage of the linker.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

For the prevention or treatment of disease, the appropriate dosage of an ADC of the invention wilt depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinicai history and response to the antibody, and the discretion of the attending physician. The molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 gg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initiai candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 gg/kg to 100 mg/kg or more, depending on the factors mentioned above. An exemplary dosage of ADC to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. An exemplary dosing regimen comprises a course of administering an initial loading dose of about 4 mg/kg, followed by additional doses every week, two weeks, or three weeks of an ADC. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

Treatment

The term “treatment,” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included.

The term “therapeutically-effective amount,” as used herein, pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.

Similarly, the term “prophylactically-effective amount,” as used herein, pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired prophylactic effect,

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564 commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.

Preparation of Antibody drug conjugates

Antibody drug conjugates may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent to form antibody-linker intermediate Ab-L, via a covalent bond, followed by reaction with an activated drug moiety reagent ; and (2) reaction of a drug moiety reagent with a linker reagent, to form drug-linker reagent D-L, via a covalent bond, followed by reaction with the nucleophilic of an antibody. Conjugation methods (1) and (2) may be employed with a variety of antibodies, and linkers to prepare the antibody-drug conjugates of the invention.

Nucleophilic groups on antibodies include, but are not limited to side chain thiol groups, e.g. cysteine. Thiol groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties such as those of the present invention. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (Cleland's reagent, dithiothreitol) orTCEP (tris(2-carboxyethyl)phosphine hydrochloride: Getz et al (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, MA). Each cysteine disulfide bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut’s reagent) resulting in conversion of an amine into a thiol.

The Subject/Patient

The subject/patient may be an animal, mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilled platypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang, gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development, for example, a foetus. In one preferred embodiment, the subject/patient is a human.

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

In one embodiment, the patient is a population where each patient has a tumour having α_νβδ integrin on the surface of the cell.

Synthesis

One possible synthesis route to a dimer intermediate of formula IV is shown below:

<img file="CA2901941C_D0054.tif" />

w

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

Intermediate IV can be used to make intermediate VII

Hal

<img file="CA2901941C_D0055.tif" />

<img file="CA2901941C_D0056.tif" />

<img file="CA2901941C_D0057.tif" />

<img file="CA2901941C_D0058.tif" />

<img file="CA2901941C_D0059.tif" />

CA 02901941 2015-0Ô-19

<img file="CA2901941C_D0060.tif" />

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

<img file="CA2901941C_D0061.tif" />

Alternatively, intermediate IV can be coupled with intermediate X to make intermediate !X:

<img file="CA2901941C_D0062.tif" />

H

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

<img file="CA2901941C_D0063.tif" />

+ XII

<img file="CA2901941C_D0064.tif" />

<img file="CA2901941C_D0065.tif" />

1. Deprotection

<img file="CA2901941C_D0066.tif" />

O

<img file="CA2901941C_D0067.tif" />

<img file="CA2901941C_D0068.tif" />

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

Intermediate IV can be used to make intermediate XVI

<img file="CA2901941C_D0069.tif" />

<img file="CA2901941C_D0070.tif" />

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

Intermediate IV can be used to make intermediate XIX

Hal

<img file="CA2901941C_D0071.tif" />

<img file="CA2901941C_D0072.tif" />

<img file="CA2901941C_D0073.tif" />

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

One possible synthesis route to a dimer intermediate of formula XIV is shown below:

<img file="CA2901941C_D0074.tif" />

<img file="CA2901941C_D0075.tif" />

<img file="CA2901941C_D0076.tif" />

<img file="CA2901941C_D0077.tif" />

<img file="CA2901941C_D0078.tif" />

XXH

XIV

<img file="CA2901941C_D0079.tif" />

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

O'

XXf + XXtB o

<img file="CA2901941C_D0080.tif" />

<img file="CA2901941C_D0081.tif" />

XXIV intermediate XXi can be used to make intermediate XXVil:

<img file="CA2901941C_D0082.tif" />

XXf

<img file="CA2901941C_D0083.tif" />

xxv

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

<img file="CA2901941C_D0084.tif" />

XXVI

<img file="CA2901941C_D0085.tif" />

In the above schemes, R^N, R^N and R^N“ each independently represent a nitrogen protecting group. R^c and R^c each independently represent OH or OProt°, where Prot⁰ is a hydroxy protecting group. Protecting groups are well known in the art. R^N, R^N and R^r’ may be, for example. BOC. Prot⁰ may be THP. it may be the protection of the N10-C11 imine bonds is removed at a diffemet stage in the synthesis methods to that shown above, dependent on the chemistries employed.

In general, the compounds and conjugates can be prepared by first linking two PBD monomers with a phenylene or pyridylene dimer bridge to produce intermediate IV or XXI. The halogen group on the aryl ring in the dimer bridge of intermediate IV may then be used

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 to form the tether (including linker group G or L) to connect the PBD dimer to the cell binding agent.

In more detail, two PBD monomers with -XH and -ΧΉ groups at the C8 position of each PBD monomer (intermediates I and II, respectively) may be reefed with -T-Hal and -T’-Ha! groups on intermediate III or intermediate XX. Such a method of synthesis allows for the PBD monomers to be different and so the resulting PBD dimer is asymmetrical. Equally, the PBD monomers may be the same.

PBD dimer intermediate IV may be used to provide the compounds and conjugates of the present invention by reacting the aryl halogen group in the bridge in a number of ways.

First, intermediate IV can be used in a Sonogishira cross-coupling reaction to provide an acetylene group on the aryl group of the dimer bridge. Sonogishira cross-coupling reactions are well known in the art for coupling a terminal alkyne with an aryl halide in the presence of a palladium catalyst, such as Pd(Ph₃)4, a copper catalyst, such as Cui, and a base, such as diethylamine.

When acetylene is to be used as the terminal acetylene, one side of the acetylene molecule is typically protected with, for example, TMS in order to prevent cross-linking of the PBD dimers. Once the Sonogishira reaction is complete, the TMS group can be cleaved to provide alkyne intermediate V.

Intermediate V can be reacted with an azido compound to form a triazole derivative in an azide-alkyne Huisgen cycloaddition. Such a reaction may be catalysed by a copper catalyst. To form the compounds and conjugates of the present invention, the azide is bonded to an ethylene group and a variable number of PEG groups. The azide may be terminated with an amine group to react further. Reaction of intermediate V with an amino-azide compound will provide intermediate VI.

The free amine group of intermediate VI can then be reacted with a carboxylic acid group of a linker group for connecting to a cell binding unit to form the amido group linking the PBD dimer to the linker group G or L to provide compound VII.

The linker/reactive group, G, of intermediate VII can be conjugated to a cell binding agent to provide conjugates of the present invention.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

As an alternative Sonogishira reaction, intermediate iV can be coupled to an acetylamine, such as propargylamine in the presence of palladium and copper catalysts and base. Such a reaction provides part of a tether attached to the PBD dimer bridge where the aclyne group is preserved and a free terminal amine is available for further reaction. For example, the reaction of intermediate IV with propargylamine provides intermediate VIII.

The terminal amine of intermediate VIII can be reacted with, for example, a carboxylic acid group attached to a linker/reactive group G (for connecting to a cell binding agent) to provide intermediate IX.

As an alternative synthesis of intermediate IX, the carboxylic acid group of intermediate XI can be reacted with propargylamine to form intermediate Xli. Reaction of intermediate IV with intermediate XII in a Sonogoshira reaction yiedls intermediate Xill.

The protected amine group terminated the variable PEG chain can be deprotected and reacted with the carboxylic acid group of intermediate XIV in order to couple the linker/reactive group G onto the PBD dimer and produce intermediate XIV.

Intermediate IV may also used in a cross-coupling amination reaction, such as a Buchwald-Hartwig amination. A carbon-nitrogen bond is formed via a palladium-catalysed cross-coupling of an amine with an aryl halide. A number of palladium catalysts for use in such cross-coupling reactions are known, such as Pd(Ph₃)₄ or RuPhos/RuPhosPd.

Reaction of intermediate IV with a piperizine functionlised with a protected propan-1-amine provides intermediate XV. The protected amine of intermediate XV can be further reacted with, for example, a carboxylic acid group attached to a linker/reactive group, Θ, for connecting to a cell binding agent to provide intermediate XVI.

Cross-coupling amination reaction, such as a Buchwald-Hartwig amination, of intermediate IV with a partially protected piperazine followed by deprotection (for example with trifluoroacetic acid) provides intermediate XVII.

The deprotected piperazine amine group of intermediate XVII can be reacted with a carboxylic acid group in intermediate XVIII to provide intermediate XIX.

Intermediate XXI can be used to form the oxime intermediate XXIV. For example, a partially protected PEG-diamine, intermediate XXII, may be reacted with the carboxylic acid group of intermediate XIV. Deprotection yields intermediate XIII.

Reaction of intermediates XXI and XXIII yields oxime intermediate XXIV. The syn and anti oximes can be resolved using preparative HPLC.

Intermediate XXI can also be used to form the acrylamide intermediate XXVII. For example, the aldehyde intermediate XXI can be reacted with malonic acid in a Knoevenagel condensation to yield the acryclic add intermediate XXV. This can be reacted with a partially protected PEG-diamine to yield intermediate XXVI. Deprotectopm and coupling with intermediate XIV yield the acrylamide intermediate XXVII.

The synthesis of PBD compounds containing two imine moieties is extensively discussed in the following references:

a) WO 00/12508 (pages 14 to 30);

b) WO 2005/023814 (pages 3 to 10); and

c) WO 2005/085259 (pages 31 to 39).

Examples

General Experimental Methods

Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd.) and concentrations (c) are given in g/100mL. Melting points were measured using a digital melting point apparatus (Electrothermal). IR spectra were recorded on a Perkin-Elmer Spectrum 1000 FT IR Spectrometer. ¹H and ¹³C NMR spectra were acquired at 300 K using a Bruker Avance NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported relative to TMS (S = 0.0 ppm), and signals are designated as s (singlet), d (doublet), t (triplet), dt (double triplet), dd (doublet of doublets), ddd (double doublet of doublets) or m (multiplet), with coupling constants given in Hertz (Hz). Mass spectroscopy (MS) data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 HPLC with a Waters 2996 PDA. Waters Micromass ZQ parameters used were: Capillary (kV), 3.38; Cone (V), 35; Extractor (V), 3.0; Source temperature (’C), 100; Desolvation Temperature (°C), 200; Cone flow rate (L/h), 50; De-solvation flow rate (L/h), 250. High-resolution mass spectroscopy (HRMS) data were recorded on a Waters Micromass QTOF Global in positive

CA

2901941 2019-06-11

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564 '//-mode using metal-coated borosilicate glass tips to introduce the samples into the instrument. Thin Layer Chromatography (TLC) was performed on silica gel aluminium plates (Merck 60, F254), and flash chromatography utilised silica gel (Merck 60,230-400 mesh ASTM). All chemicals and solvents were purchased from Sigma-Aldrich and were used as supplied without further purification.

General LC/MS conditions: The HPLC (Waters Alliance 2695) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B over 1.0 min then 5% B to 95% B within 3 min. The composition was held for 0.5 min at 95% B, and then returned to 5% B in 0.3 minutes. Total gradient run time equals 5 min. Flow rate 3.0 mL/min, 400pL was split via a zero dead volume tee piece which passes into the mass spectrometer. Wavelength detection range: 220 to 400 nm. Function type: diode array (535 scans). Column: Phenomenex® Onyx Monolithic C18 50 x 4.60 mm.

The analytical LC/MS conditions for Example 5 to 11 were as follows: Positive mode electrospray mass spectrometry was performed using a Shimadzu Nexera®/Prominence® LCMS-2020. Mobile phases used were solvent A (H₂O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic acid). Gradient: Initial composition 5% B held over 0.25 min, then increased from 5% B to 100% B over a 2 min period. The composition was held for 0.50 min at 100% B, then returned to 5% B in 0.05 min and held there for 0.05 min. The total duration of the gradient run was 3.0 min. Flow rate was 0.8 mL/min. Detection was at 214 and 254 nm. Column: Waters Acquity UPLC® BEH Shield RP18 1.7pm 2.1 x 50 mm at 50 °C.

The preparative HPLC conditions for Example 5 to 11 were as follows: Reverse-phase ultrafast high-performance liquid chromatography (UFLC) was carried out on a Shimadzu Prominence® machine using Phenomenex® Gemini NX 5μ C18 columns (at 50 °C) of the following dimensions: 150 x 4.6 mm for analysis, and 150 x 21.2 mm for preparative work. Eluents used were solvent A (H₂O with 0.1% formic acid) and solvent B (CH3CN with 0.1% formic acid). All UFLC experiments were performed with gradient conditions: From 0 to 30 min the composition of B was increased from 0 to 100% and held at 100% B for a further 2 min. The composition of B was decreased from 100% to 0% from 32.0 min to 32.1 min and held at 0% B until 35.0 min. The total duration of the gradient run was 35.0 min. Flow rates used were 1.0 mUmin for analytical, and 20.0 mL/min for preparative HPLC. Detection was at 254 and 280 nm.

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

Exampie 1 (a) (11 S,11aS, 11'3,11a’S)-di-tert-butyi 8,8'-(((5-halo-1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2Hpyran-2-yl)oxy)-2,3,11,11a-tetrahydro~1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)· carboxylate) (2a, 2b, 2c)

<img file="CA2901941C_D0086.tif" />

2b: X= 8r 2c: X= Cl (i) (11S,11aS, 1TS,11a'S)-di-tert-butyl 8,8'-(((5~iodo-1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2Hpyran-2-yl)oxy)-2.3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)carboxylate) (2a).

1,3-bis(bromomethyl)-5-iodobenzene (2.00 g. 5.20 mmol) was added to a stirred solution of Boc/THP-protected PBD capping unit 1 (4.75 g, 10.3 mmol), TBAI (190 mg, 0.52 mmol) and K₂CO₃ (1.42 g, 10.3 mmol) in dry DMF (60 mL). The reaction mixture was heated to 60 °C and stirred under an argon atmosphere for 3 hours at which point analysis by LC/MS revealed substantial product formation at retention time 4.15 min (ES+) m/z 1171 ([M+ Naf ’, -10% relative intensity). The reaction mixture was aiiowed to cool to room temperature and the DMF was removed by evaporation in vacuo. The resulting residue was partitioned between water (50 mL) and EtOAc (50 mL) and the aqueous phase was extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with water (2 x 20 mL), brine (50 mL), dried (MgSCL), filtered and evaporated in vacuo to provide toe crude product. Purification by flash chromatography (gradient elution: 50:50 v/v EtOAc/hexane to 80:20 v/v EtOAc/hexane) gave the bis-ether 2a as a white foam (5.42 g, 91% yield).

(ii) (11S, 11aS, 11'S,11 a'S)-di-tert-butyl 8,8'-(((5-bromo-1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2Hpyran-2~yl)oxy)-2,3,11,11a-tetrahydro~1H-pyrroio[2,1-c][1,4]benzodiazepine-10(5H)~ carboxylate) (2b)

1-bromo-3,5-bis(bromomethyi)benzene (1.54 g, 4.53 mmol) was added to a stirred solution of Boc/THP-protected PBD capping unit 1 (4.20 g, 9.06 mmol), TBAI (167 mg, 0.45 mmol) and KjCOj (1.25 g, 9.06 mmol) in dry DMF (52 mL). The reaction mixture was heated to 60 °C and stirred under an argon atmosphere for 5 hours at which point analysis by LC/MS

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

100 revealed substantial product formation at retention time 4.10 min (ES+) m/z 1101 ([M+ H]⁺, -70% relative intensity). The reaction mixture was allowed to cool to room temperature and the DMF was removed by evaporation in vacuo. The resulting residue was partitioned between water (60 mL) and EtOAc (60 mL) and the aqueous phase was extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with water (30 mL), brine (50 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 50:50 v/v EtOAc/hexane to 100% EtOAc) gave the bis-ether 2b as a white foam (3.37 g, 68% yield).

(Hi) (11S.11aS, 11 'S, 11a'S)-di-tert-butyl 8,8'-(((5-chloro-1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydiO-2Hpyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)carboxylate) (2c)

1,3-bis(bromomethyl)-5-chlorobenzene (1.42 g, 4.80 mmol) was added to a stirred solution of Boc/THP-protected PBD capping unit 1 (4.42 g, 9.60 mmol), TBAI (177 mg, 0.48 mmol) and K₂CO₃ (1.33 g. 9.60 mmol) in dry DMF (55 mL). The reaction mixture was heated to 60 °C and stirred under an argon atmosphere for 1.5 hours at which point analysis by LC/MS revealed substantial product formation at retention time 4.08 min (ES+) m/z 1057 ([M+ HJ*. -30% relative intensity). The reaction mixture was allowed to cool to room temperature and the DMF was removed by evaporation in vacuo. The resulting residue was partitioned between water (60 mL) and EtOAc (60 mL) and the aqueous phase was extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with water (20 mL), brine (40 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 50:50 v/v EtOAc/hexane to 80:20 v/v EtOAc/hexane) gave the bis-ether 2c as a white foam (5.10 g, 99% yield).

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

101 (b) (11S,11aS, 11’S,11a’S)-di-tert-buty! 8,8’-(((5-ethynyl-1,3 phenyiene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4] benzodiazepine-10(5H)carboxylate) (4)

<img file="CA2901941C_D0087.tif" />

<img file="CA2901941C_D0088.tif" />

2c: x= a

<img file="CA2901941C_D0089.tif" />

(i) (11 S,11aS, 11’S,11a'S)-di-tert-butyl 8,8'-(((5-((trimethylsilyl)ethynyl)-1,3 phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H pyran-2-yl)oxy)-2,3,11.11a-tetrahydro-1H-pyrrolo(2,1-c][1,4]benzodiazepine-10(5H)carboxylate) (3)

A catalytic amount of Pd(PPh₃)₄ (15.0 mg, 13.1 pmol) was added to a mixture of the bisether 2a (750 mg, 0.65 mmol), TMS-acetylene (278 pL, 191 mg. 1.96 mmol), Cui (5.0 mg, 26.1 pmol), diethyiamine (1.35 mL, 956 mg, 13.1 mmol) and oven-dried 4 A molecular sieve pellets in dry DMF (5.6 mL) in an oven-dried sealable vessel. The mixture was degased and flushed with argon 3 times then heated in a microwave at 100 ⁿC for 30 minutes at which point analysis by LC/MS revealed complete consumption of starting material and substantial product formation at retention time 4.37 min (ES+) m/z 1142 ([M+ Nap, ~40% relative intensity). Peak at retention time 3.97 min (ES+) m/z 1069 ([M+ Nap. -60% relative intensity) observed which corresponds to TMS-cleavage under LC/MS conditions. The reaction mixture was allowed to cool to room temperature and was then filtered through a sinter to remove the sieves (washed with DMF). The filtrate was evaporated in vacuo and the resulting residue subjected to flash chromatography (gradient elution; 50:50 v/v EtOAc/hexane to 80:20 v/v EtOAc/hexane) to provide the TMS-acetylene 3 as a yellow foam (691 mg, 95% yield).

(ii) (11S,11aS, 11'S,11a'S)-di-tert-butyl 8,8'-(((5-ethynyl-1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2Hpyran-2-yl)oxy)-2.3,1l_:1la-tetrahydro-1H-pyrrolo[2, 1-c][1,4] benzodiazepine-10(5H)carboxylaie) (4)

Solid K₂CO₃ (383 mg, 2.77 mmol) was added to a stirred solution of the TMS-protected compound 3 (1.55 g, 1.39 mmoi) in MeOH (20 mL). After 3 hours stirring at room temperature the reaction was deemed to be complete as judged by LC/MS (desired product

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

102 peak at retention time 4.00 min (ES+) mlz 1047 ([Λ4+ H]⁺·. -30% relative intensity)]. The MeOH was removed by evaporation in vacuo and the resuiting residue was partitioned between water (60 mL) and EtOAc (60 mL). The iayers were separated and the aqueous phase was extracted with EtOAc (3 x 20 mL). The combined organic iayers were washed with water (30 mL), brine (30 mL), dried (MgSOi), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 50:50 v/v EtOAc/hexane to 80:20 v/v EtOAc/hexane) gave the acetylene 4 as an orange foam (1.13 g, 78% yield).

(c) (11S, 11aS, 11'S,11a’S)-di-tert-butyl 8,8'-(((5-(1-(2-(2-(2-(2aminoethoxy)ethoxy)ethoxy)ethy!)-1H-1,2,3-triazol-4-yl)-1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2Hpyran-2-yl)oxy)~2,3,11,11a-telrahydro~1H- pyrrolo[2,1-c][ 1,4]benzodiazepine~10(5H)carboxylate) (5)

<img file="CA2901941C_D0090.tif" />

<img file="CA2901941C_D0091.tif" />

<img file="CA2901941C_D0092.tif" />

Solid CuSO4.5H₂O (13.0 mg, 52.0 pmol) and (+)-sodium L-ascorbate (41.0 mg, 0.21 mmol) were added to a stirred solution of 11-Azido-3,6,9-trioxaundecan-1-amine (227 mg, 207 pL, 1.04 mmol) and the alkyne 4 (1.09 g, 1.04 mmol) in fert-BuOH (6 mL) and H₂O (6 mL) at room temperature. A colour change from yeilow to green was observed as the reaction progressed. After stirring for 16 hours analysis by LC/MS reveaied a substantial of amount of desired product formed corresponding to peak at retention time 3.12 min (ES+) mlz 1265 ([Λ4+ H]⁺ , -100% relative intensity). [NOTE: On some occasions reaction progress stalled, however, toe reaction was driven to completion upon addition of further CuSOî.ôHîO (0.05 equivalents) and (+)-sodium L-ascorbate (0.2 equivalents)]. The reaction mixture was partitioned (without shaking of the separating funnel) between water (50 mL) and EtOAc (50 mL). The aqueous phase was extracted with EtOAc (3 x15 mL) and the combined organic layers were washed with water (30 mL), brine (50 mL), dried (MgSOi), filtered and evaporated in vacuo to provide the crude product 5 as a green foam (1.32 g, 100% crude yield). The crude product was carried through to next step without further purification.

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

103 (d) (11S,11aS, irs,11a’S)-di-tert-butyl 8,8'-(((5-(1-(18~(2,5-dioxo-2.5-dihydro~1H-pyrrol-1-yl)13-oxo-3,6,9-trioxa-12-azaoctadecyi)~1H-1,2,3-triazol-4-yi)-1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2Hpyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)~ carboxylate) (6)

<img file="CA2901941C_D0093.tif" />

Solid 6-maleimidohexanoic acid N-hydroxysuccinimide ester (327 mg. 1.06 mmol) was added to a stirred solution of the primary amine 5 (1.28 g, 1.01 mmol) in dry DCM (30 mL) at room temperature. Progress was monitored by LC/MS and after 3 days stirring the reaction proceeded no further, a substantial amount of desired product was observed at retention time 3.65 min (ES+) m/z 1458 ([M+ Hf. ~100% relative Intensity) accompanied by unreacted starting material at retention time 3.15 min. The reaction mixture was treated with silica gel and the solvent removed by evaporation in vacuo. The resulting residue was subjected to flash chromatography (gradient elution: 100% DCM to 97:3 v/v DCM/MeOH) to give foe maleimide 6 as a foam (658 mg, 45% yield).

(e) N-(2-(2-(2-(2-(4-(3,5-bis((((S)-7-methoxy-2-methy!ene-5-oxo-2,3,5,11a-tetrahydro-1Hpyrrolo[2,1~c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)-1H-1,2,3-triazol-1yl)ethoxy)ethoxy)eihoxy)sthyl)’6-(2,5~dioxo-2,5-dihydro~1H-pyrrol-1-yl)hexanamide (7).

<img file="CA2901941C_D0094.tif" />

A solution of 95:5 v/v TFA/H₂O (5 mL) was added to a sample of the Boc/THP-protected compound 6 (428 mg, 0.29 mmol) at 0 °C (ice/acetone). After stirring at 0 for 1 hour the reaction was deemed complete as judged by LC/MS, desired product peak at retention time 2.72 min (ES+) m/z 1054 ((/W+ Hj⁺ , ~70% relative intensity). The reaction mixture was kept

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

104 cold and added drop wise to a chilled saturated aqueous solution of NaHCO₃ (100 mL). The mixture was extracted with DCM (3 x 30 mL) and the combined organic layers washed with H₂O (20 mL), brine (40 mL), dried (MgSO&lt;), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCI₃ to 96:4 v/v CHCI₃/MeOH) gave 7 as an orange foam (163 mg, 53% yield).

Example 2 (a) (115,11aS, 11 '5,11a'S)-di-tert-butyl 8,8'·(((5~(3·3ηΊΪηορΓορ~1^η·1·^)·1,3phenylene)bis(methyiane))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2Hpyran-2-yl)oxy)-2.3,11,11a-tetrahydro-1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)carboxylate) (8)

<img file="CA2901941C_D0095.tif" />

<img file="CA2901941C_D0096.tif" />

A catalytic amount of Pd(PPh₃)₄ (5.0 mg, 4.2 pmol) was added to a mixture of the bis-ether

2a (242 mg, 0.21 mmol), propargylamine (41 pL, 35 mg, 0.63 mmol), Cui (1.6 mg, 84 pmol), diethylamine (0.42 mL, 309 mg, 4.22 mmol) and oven-dried 4Λ molecular sieve pellets in dry

DMF (1.8 mL) in an oven-dried sealable vessel. The mixture was degased and flushed with argon 3 times then heated in a microwave at 100 °C for 3 minutes at which point analysis by

LC/MS revealed complete consumption of starting material and substantial product formation at retention time 3.18 min (ES+) m/z 1076 ([M+ Hf , -60% relative intensity). The reaction mixture was allowed to cool to room temperature and was then filtered through a sinter to remove the sieves (washed with DMF). The filtrate was evaporated in vacuo to provide the unstable crude product 8 which was used immediately in the next step without purification or analysis.

(b) (11S.11aS. 11'S.11a'S)-di-tert-butyl 8,8'-(((5-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y!)3,19-dioxo-7,10,13,16-tetraoxa-4,20-diazatricos-22-yn-23-yi)-1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-ll-((tetrahydro-2Hpyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrro‘o[2_:1 -c][1,4} benzodiazepine-10(5H)carboxylate) (9)

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

105

<img file="CA2901941C_D0097.tif" />

<img file="CA2901941C_D0098.tif" />

MAL-dPEG®4-acid (88 mg. 0.21 mmol) was added to a stirred solution of EDCI (41 mg, 0.21 mmol) and the crude primary amine 8 in dry DCM (4 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 3 hours at which point analysis by LC/MS showed a substantial amount of desired product at retention time 3.58 min (ES+) m/z 1475 ([Λ4+ H]⁺ , -10% relative intensity), 1498 ([Λ4+ Nap, -5% relative intensity) accompanied by a side product at retention time 3.85 min. The reaction mixture was diluted with DCM (30 mL) and washed with H?O (3x10 mL), brine (20 mL), dried (MgSO-O, filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% DCM to 96:4 v/v DCM/MeOH) gave the maleimide 9 as a foam (67 mg, 22% yield over 2 steps).

(c) N-(3-(3,5~bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1c][1,4] benzodiazepin-8-yl)oxy)methyl)phenyl)prop-2-yn-1-yl)-1-(3-(2,5-dioxo-2,5-dihydro-1 Hpyrrol-1 -y!)propanamido)-3,6,9.12-tetraoxapentadecan-15-amide (10)

<img file="CA2901941C_D0099.tif" />

A solution of 95:5 v/v TFA/H₂O (1 mL) was added to a sample of the Boc/THP-protected compound 9 (67 mg, 45.5 pmol) at 0 °C (ice/acetone). After stirring at 0 °C for 1.5 hours, the reaction was deemed complete as judged by LC/MS, desired product peak at retention time 2.67 min (ES+) m/z 1070 ([&lt;M+ Hf , ~5% relative intensity). The reaction mixture was kept cold and added drop wise to a chilled saturated aqueous solution of NaHCOs (50 mL). The mixture was extracted with DCM (3x15 mL) and the combined organic layers washed with brine (40 mL), dried (MgSCL), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCls to 96:4 v/v CHCls/MeOH) gave 10 as an orange foam (12 mg, 24% yield).

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

106

Example 3 (a) (11S, 11aS, 11'S,11a'S)-di-tert-butyi 8,8'-(((5-(4-(3-((tertbutoxycarbonyl)amino)propyl)piperazin-1~yl)-1,3-phenylene)bis(methylene))bis(oxy))bis(7~ methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)~2,3,11,11 a-tetrahydro-1H~ pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxylate) ( 11)

OMe Moi

2c

A sample of the bis-ether 2c (250 mg, 0.24 mmol), NaOfeu (57 mg, 0.59 mmol), RuPhos (11 mg, 23.7 pmol) and RuPhosPd (19 mg, 23.7 pmol) were added to an oven-dried sealable tube (which was allowed to cool in a desicator). The mixture was degased and flushed with argon 3 times before the addition of dry THF (5 mL) and then allowed to stir under an inert atmosphere for -10 minutes until the red colour had discharged. A solution of 3-(piperazin-1-yl)propan-1-amine (58 mg, 0.26 mmol) in dry THF (1 mL) was added and the mixture again degased and flushed with argon 3 times. The reaction mixture was heated at 80 °C in a pre-heated oil bath for 2.5 hours at which point analysis by LC/MS revealed a 3component mixture: desired product at retention time 3.35 min (ES+) m/z 1264 ([M+ Hf, ~60% relative intensity), major side product at retention time 3.95 min (de-chlorinated analogue of 2c) and shoulder 4.13 min (trace starting material). After being allowed to cool to room temperature the reaction mixture was partitioned between water (20 mL) and EtOAc (20 mL). The aqueous phase was extracted with EtOAc (3 x15 mL) and the combined organic layers were washed with water (30 mL), brine (50 mL), dried (MgSOi), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution; 100% DCM to 95:5 v/v DCM/MeOH) gave the piperazine 11 as a foam (152 mg, 25% yield).

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

107 (b) ( 11aS, 11a'S)-8,8'-(((5-(4-(3-aminopropyl)piperazin-1-yi)-1,3phenyiene)bis(methylene))bis(oxy))bis(7-methoxy~2~methylene-2,3-dihydro-1H-pyrrolo[2,1c][1,4]benzodiazepin-5( 11aH)-one) ( 12)

<img file="CA2901941C_D0100.tif" />

<img file="CA2901941C_D0101.tif" />

<img file="CA2901941C_D0102.tif" />

A solution of 95:5 v/v TFA/H₂O (2 mL) was added to a sample of the Boc/THP-protected compound 11 (142 mg, 0.11 mmol) at 0 °C (ice/acetone). After stirring at 0 °C for 1 hour, the reaction was deemed complete as judged by LC/MS, desired product peak at retention time 2.23 min (ES+) m/z 778 ([Λ4+ H₂O]*, -5% relative intensity). The reaction mixture was kept cold and added drop-wise to a chilled saturated aqueous solution of NaHCOs (50 mL). The mixture was extracted with DCM (3 x 20 mL) and the combined organic layers washed with brine (15 mL), dried (MgSO^), filtered and evaporated in vacuo to provide the crude product 12 as a waxy solid (22.6 mg). Note that during the NaHCOs neutralisation step the desired product precipitated out of solution as a waxy solid which was only partially soluble in DCM. Additional product was obtained by dissolving DCM-insoluble solids in DMF followed by evaporation in vacuo. The resulting oily residue was triturated with diethylether to provide a solid which was dried in vacuo to provide additional crude 12 (54.4 mg, total amount = 77 mg, 85% yield) which was carried through to the next step without further purification or anaiysis.

(c) N-(3-(4-(3,5-bis((((S)-7-methoxy-2-methyiene-5-oxo-2,3,5,11a-tetrahydro- 1H-pyirolo[2,1cJ[1,4]benzodiazepin-8-yl)oxy)methy!)pheny!)piperazin-1 -yl)propyl)-1 -(3-(2.5-dioxo-2,5dihydro-1H-pymol-1-yi)propanamido)-3,6,9.12-tetraoxapentadecan-15-amide (13).

<img file="CA2901941C_D0103.tif" />

<img file="CA2901941C_D0104.tif" />

<img file="CA2901941C_D0105.tif" />

<img file="CA2901941C_D0106.tif" />

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

108

MAL-dPEG®4-acid (42 mg, 0.10 mmol) was added to a stirred solution of EDCI (20 mg, 0.10 mmol) and the crude primary amine 12 (77 mg, 0.10 mmol) in dry DCM (4 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 3 hours at which point analysis by LC/MS showed complete consumption of starting material, a substantial amount of desired product al retention time 2.42 min (ES+) miz 1176 ([M+ H₂O]*·, -5% relative intensity) and excess MAL-dPEG®4-acid at retention time 2.05 min (weak signa! on diode array but detectable on ES+/ES-). The reaction mixture was diluted with DCM (30 mL) and washed with H₂O (15 mL), brine (20 mL), dried (MgSCh). filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCI₃ to 93:7 v/v CHCh/MeOH) gave the maleimide 13 as a foam (46 mg, 55%). Note that trace amounts of excess MAL-dPEG®4-acid could not be removed using flash chromatography.

Example 4 (a) (11$, 11a$, 11'$,1 la'S)-di-tert-butyl 8,8'-(((5-(4-(tert-butoxycaibonyl)piperazin-1-yl)-1,3~ phenylene)bis(methyiene))bis(oxy))bis(7-methoxy-2‘methy!ene-5-oxo-11-((tetrahydro-2Hpyran-2-yi)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1 -c][1,4}benzodiazepine-10(5H)carboxylate) (14)

<img file="CA2901941C_D0107.tif" />

ÇOC

<img file="CA2901941C_D0108.tif" />

2-Dicyclohexylphosphino-2’,6’-diisopropoxybiphenyl (18 mg, 38 pmol, 0.2 eq), chloro(2dicyclohexylphosphino-2',6 -diisopropoxy-1,1 -biphenyi)[2-(2 -amino-1,1 biphenyl)]pa!ladium(ll) (18 mg, 22 pmol, 0.12 eq), caesium carbonate (0.36 g,1,1 mmol, 5.0 eq) and iodo derivative (2a) (0.307 g, 0.27 mmol, 1.0 eq) were placed in a microwave vial which was evacuated and flushed with Argon (x 3). Anhydrous THF (5 mL) was added followed by tert-butyl piperazine-1-carboxylate (70 mg, 0.37 mmol, 1.1 eq) and the resultant mixture was heated at 85°C for 4h then overnight at room temperature. The reaction mixture was diluted with saturated sodium hydrogen carbonate and extracted with ethylacetate (3 x 100 mL). The combined ethylacetate extracts were washed with brine (100 mL), dried (MgSO,&lt;) and evaporated under reduced pressure. The product 14 was purified by flash column chromatography [CHCk/MeOH 0% to 1.5% in 0.5% increments] (0.111 g, 51%)

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

109

Analytical Data: RT 4.12 min; MS (ES*) m/z (relative intensity) 1207 ([M + 1]', 30).

(b) ( 11aS,11a'S)-8,8'-(((5-(piperazin-1-y')-1,3-phenylene)bis(methylene))bis(oxy))bis(7methoxy-2-methylene-2,3-dihydro-1H-pyrrolo[2,1-c][1,4]benzodiazepin-5(11aH)-one) (15)

<img file="CA2901941C_D0109.tif" />

<img file="CA2901941C_D0110.tif" />

A cold (ice bath) solution of 95% trifluoroacetic acid (4 mL) was added to compound (14) (0.2 g. 0.165 mmol, 1 eq.) which had been cooled in an ice bath. The solution was stirred at 0^eC for 30 min when reaction was shown to be complete by LCMS. The reaction mixture was added drop-wise to a mixture of ice and saturated sodium bicarbonate solution to neutralise the trifluoroacetic acid. The mixture was extracted with DCM (4 x 75 mL) and the combined extracts were washed with water (100 mL) saturated brine (100 mL), dried (MgSO₄) and evaporated under reduced pressure to give the product 15 as a yellow solid which was used without further purification (0.116 g, 100%)

Analytical Data: RT 2.33 min; MS (ES*) m/z (relative intensity) 703 ([M + 1p, 100).

(c) N-( 15-(4-(3,5-bis(((($)-7-methaxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1Hpyrrolo[2_! 1 -c][1,4]benzod!azepin-8-yl)Qxy)methyl)phenyl)piperazin·· 1 -yl)-15-oxo-3,6,9,12tetraoxapentadecyl)-3-(2,5-dioxo-2.5-dihydro-1H-pyrrof-1-yl)propananiide (17)

<img file="CA2901941C_D0111.tif" />

N-(3-Dimethylaminopropyf)-A/’-ethylcarbodiimide hydrochloride (35 mg, 0.18 mmol, 1.1 eq) was added to a solution of compound (15) (116 mg, 0.165 mmol, 1.0 eq) and 1-(2,5-dioxo2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16-tetraoxa-4-azanonadecan-19-oic acid (16) (69 mg, 0.165 mmol 1.0 eq) in anhydrous DCM (5 mL) under Argon. The resultant solution was stirred at room temperature for 2h. The reaction mixture was diluted with DCM (50 mL), washed with water (100 mL), saturated sodium hydrogen carbonate solution (100 mL). water (100 mL), brine (100 mL), dried (MgSO₄) and evaporated under reduced pressure.

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

110

Purification by flash column chromatography [CHCiyMeOH 0% to 5% in 1% increments] gave the product 17 as a yellow glass (0.058 g, 32%)

Analytical Data: [o]¹⁸d = [+628’] (c = 0.25. CHCI3); RT 2.65 min; MS (ES⁴) m/z (relative intensity) 1101 {[M + 1]*, 40)

Example 5 (a) tert-butyl (42-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1~yl)-37-oxo3,6,9,12,15,18,21,24,27,30,33-undecaoxa-36-azadotetracontyl)oxycarbamate (19) nh₂

BOC'^N'_h

<img file="CA2901941C_D0112.tif" />

6-Maleimidohexanoic acid (64 mg, 0.30 mmol) was added to a stirred solution of EDCi (64 mg, 0.33 mmol) and the primary amine 18 (200 mg, 0.30 mmol) in dry DCM (6 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 16 hours at which point analysis by LC/MS showed a substantial amount oi desired product at retention time 1.38 min {(ES*) m/z 854 ([M+ H]*„ -30% relative intensity). 877 ([M+ Na]*., -100% relative intensity)} accompanied by unreacted 18 at retention time 1.07 min, note that both starting material and product had weak UV absorption (214 and 254 nm) and were best detected on ES* TIC. Additional 6-maieimidohexanoio acid (32 mg, 0.15 mmol) and EDCI (32 mg, 0.17 mmol) were added to the stirred mixture until starting material was completely consumed (as judged by LC/MS). The reaction mixture was diluted with DCM (100 mL) and washed with H₂O (3 x 30 mL), brine (40 mL), dried (MgSCU), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution in 1% increments: 100% DCM to 96:4 v/v DCM/MeOH) gave the amide 19 as an oil (214 mg, 83% yield).

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

111 (b) N-(35-(aminooxy)-3.6,9,12,15,18,21,24,27_l30_!33-undecaQXdpentatriacontyl)-6-(2,5-dioxo2,5-dihydro-1H-pyrrol-1-yl)hexanamide (20)

<img file="CA2901941C_D0113.tif" />

<img file="CA2901941C_D0114.tif" />

A solution of 95:5 v/v TFA/H₂O (2 mL) was added to a sample of the Boc/THP-protected compound 19 (214 mg, 0.25 mmol) at 0 °C (ice/acetone). After stirring at 0 'C for 1 hour the reaction was deemed complete as judged by LC/MS, desired product peak at retention time 1.06 min {(ES¹) m/z 754 ([Λ4+ Hf., -100% relative intensity)}, note that both starting material and product had weak UV absorption (214 and 254 nm) and were best detected on ES⁺ TIC. The reaction mixture was kept cold and added drop wise to a chilled saturated aqueous solution of NaHCOj (100 mL). The mixture was extracted with DCM (3 x 30 mL) and the combined organic layers washed with brine (50 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide the oxyamine 20 as an oil (161 mg, 85% yield) which was carried through to toe next step without further purification.

(c) (118,11aS, 11'8,11a'S)-di-tert-butyl 8,8'-(((5-foimyl~1,3phenylene)bis(methylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2Hpyran-2-yl)oxy)-2,3,11,11a-tetrahydrO‘1H‘pyrrolo[2,1 -c][1,4]benzodiazep!ne-10(5H)carboxylate) (21)

<img file="CA2901941C_D0115.tif" />

<img file="CA2901941C_D0116.tif" />

3,5-bis(bromomethyl)benzaldehyde (260 mg, 0.90 mmol) [Enrique Di'ez-Barra et al J. Org. Chem. 2001, 66, 5664-5670] was added to a stirred solution of Boc/THP-protected PBD capping unit 7 (826 mg, 1.79 mmol), TBAI (33 mg, 89.7 pmol) and K₂CO₃ (247 mg,

1.79 mmol) in dry DMF (12 mL). The reaction mixture was heated to 60 °C and stirred under an argon atmosphere for 2.5 hours at which point analysis by LC/MS revealed substantial product formation at retention time 1.92 min {(ES) m/z 1051 ([/W+ H]⁺., -65% relative

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

112 intensity) 1073 ([Λί+ Na]⁺., -25% relative intensity)}. The reaction mixture was allowed to cool to room temperature and the DMF was removed by evaporation in vacuo. The resulting residue was partitioned between water (50 mL) and EtOAc (50 mL) and the aqueous phase was extracted with EtOAc (3x15 mL). The combined organic layers were washed with water (2 x 20 mL), brine (30 mL), dried (MgSÛ4), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution in 10% increments: 50:50 v/v EtOAc/hexane to 80:20 v/v EtOAc/hexane) gave the bis-ether 21 as a white foam (717 mg, 76% yield). Note that 21 was isolated as a mixture of diasteroisomers arising from THP protecting group.

(d) (11S, 11aS,irS,11a’S)-di-tert-butyl8,8'-(((5-((syn/anti)-45-(2,5-dioxo-2,5-dihydro-1Hpyrrol-1 -yi) -40-oxo-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxa-2,39-diazapentatetracont1-en-1-yl)-1,3-phenylene)bis(meihylene))bis(oxy))bis(7-methoxy-2-methylene-5-oxo-11((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrroio[2,1-c][1,4]benzodiazepine10(5H)-carboxytate) (22)

<img file="CA2901941C_D0117.tif" />

<img file="CA2901941C_D0118.tif" />

PTSA (4.1 mg, 21.4 pmol) was added to a stirred solution of the aldehyde 21 (224 mg, 0.21 mmol) and the oxy-amine 20 (161 mg, 0.21 mmol) in dry DCM (3 mL) at 0 ^eC (ice/acetone). The reaction mixture was allowed to stir at 0 °C under an argon atmosphere and stirring for 3 hours analysis by LC/MS revealed complete consumption of oxyamine 20 (retention time 1.06 min), presence of desired product {retention time 1.85 min (ES⁺) m/z 1787 ([Λ4+ H]*., -25% relative intensity) 1810 ([Λ4+ Na]*., -90% relative intensity)} and unreacted aldehyde 21 (retention time 1.91 min). In order to avoid unwanted THP cleavage (observed in earlier test reactions), the reaction was quenched at this point although aldehyde had not been completely consumed: The mixture was diluted with DCM (50 mL) and washed with NaHCO₃(3 x 15 ml), brine (30 ml), dried (MgSO₄), filtered and evaporated in vacuo to provide the

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

113 crude product. Purification by flash chromatography (gradient elution in 1% increments:

100% DCM to 96:4 v/v DCM/MeOH) gave the syn/anti oximes 22 as a white foam (215 mg,

56% yield). Unreacted aldehyde 21 (83 mg) was recovered during flash chromatography.

Note that 22 was isolated as a mixture of diasteroisomers arising from THP protecting group.

(e) N-((syn/anti)-1-(3,5-bis((((S)-7-methoxy-2-mathytene-5-oxo-2,3,5,1 la-tetrahydro-IHpyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)-3_l6,9,12,15,18,21,24,27,30,33,36dodecaoxa-2~azaoctatriacont-1 -en-38~yi)-6-(2,5-dioxo~2,5-dihydro-1 H-pyrrol· 1 yl)hexanamide (23)

<img file="CA2901941C_D0119.tif" />

<img file="CA2901941C_D0120.tif" />

A solution of 95:5 v/v TFA/H₂O (1 mL) was added to a sample of the Boc/THP-protected compound 22 (204 mg, 0.11 mmol) at 0 °C (ice/acetone). After stirring at 0 °C for 30 minutes, the reaction was deemed complete as judged by LC/MS, desired product peak at retention time 1.42 min {(ES⁺) m/z 1383 Hf., &lt;5% relative intensity)}. The reaction mixture was kept coid and added drop wise to a chilled saturated aqueous solution of NaHCCh (50 mL). The mixture was extracted with DCM (3x15 mL) and the combined organic layers washed with brine (30 mL), dried (MgSOi), filtered and evaporated in vacuo to provide the crude product Purification by flash chromatography (gradient elution in 1% increments: 100% CHCh to 96:4 v/v CHClVMeOH) gave the deprotected syn/anti oximes 23 as a yellow thin film (85 mg, 54% yield). Analysis by reverse-phase ultra-high-performance liquid chromatography (see General Information section for conditions) revealed predominantly two peaks at 16.15 min (syn isomer, minor component) and 16.42 min (anti isomer, major component).

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564 (f) A/-((anti)-?-(3.5-b/s((((S}-7-metooxy-2-met/iy/ene-5-oxo-2,3,5,11a-tetrahydro-1H~ pyrrolo[2,1-cji1,4]benzodiazepin-8-y!)oxy)methyi)phenyi)-3_:6,9,12,15,18,21,24,27,30,33,36dode caoxa-2-azaoctatriacont-1 -en-38-yi)-6-(2,5-dioxo-2,5-dihydro-1 H-pyrro!-1 yl)hexanamide (24)

114

<img file="CA2901941C_D0121.tif" />

Compound 23 was subjected to purification by preparative HPLC (see genera! information section for conditions). The peak eluting at a retention time of 16.42 min was isolated and lyophilised to provide the anf/-oxime 24 (9.9 mg): ^H NMR (400 MHz, CDCI3) δ 8.12 (s, 1Η), 7.66 (d, 2Η, J = 4.4 Hz), 7.62-7.59 (m, 2H), 7.53-7.51 (m, 3H), 6.82 (s, 2Η), 6.68 (s, 2H), 6.16 (br s. 2H), 5.25-5.14 (m, 8Η), 4.33 (t, 2Η, J = 4.8 Hz), 4.28 (br $. 4Η), 3.96 (s. 6H), 3.90-3.84 (m, 2Η), 3.82-3.78 (m, 2H), 3.67-3.49 (m, 43Η), 3.46-3.42 (m, 2Η), 3.15-3.08 (m, 2Η), 2.98-2.90 (m, 2H), 2.16 (t, 2H, J = 7.6 Hz), 1.70-1.54 (m, 4Η), 1.36-1.24 (m, 2H).

(g) N-((syn)-1-(3,5~bis((((S)-7-methoxy-2-methylene-5oxo-2,3,5,11a-tetrahydro~lHpyrrolo[2,1-c][1,4]benzod!azepin-8-yl)oxy)methyl)phenyl)-3,6,9,12,15,18,21,24,27,30,33,36 dodecaoxa-2-dzaoctatriacont-l'en-38-ylF6~(2,5-d!Oxo-2,5'dihydro-1H-pyrrol-i yl)hexanamide (25)

<img file="CA2901941C_D0122.tif" />

0

Compound 23 was subjected to purification by preparative HPLC (see general information section for conditions). The peak eluting at a retention time of 16.15 min was isolated and lyophilised to provide the syn-oxime 25 (5.2 mg): ¹H NMR (400 MHz, CDCI3) δ 7.93-7.91 (m, 2Η), 7.66 (d, 2Η, J = 4.4 Hz), 7.57-7.55 (m, 1Η), 7.52 (s, 2Η), 7.32 (s, 1Η), 6.82 (s, 2H), 6.68 (s, 2H), 6.18 (br s, 2H), 5.26-5.14 (m, 8H), 4.35 (t, 2Η, J = 5.1 Hz), 4.28 (br s, 4H), 3.96 (s, 6H). 3.90-3.84 (m, 2Η), 3.81 (t, 2H, J = 5.1 Hz), 3.68-3.48 (m, 43H). 3.46-3.42 (m, 2Η), 3.15-3.07 (m, 2Η), 2.97-2.90 (m, 2H), 2.16 (t, 2Η, J = 7.6 Hz), 1.70-1.54 (m, 4H), 1.36-1.24 (m, 2Η).

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

115

Example 6 (a) Di-tert-butyl 8,8-(((5-( 1-amino-15-oxo-3,6,9,12-tetraoxa-16-azanonadec-18-yn-19-y!)-1,3phenylene)bis(methylene))bis(oxy))( 11S,11aS,11'S,11a'S)-bis(7-methoxy-2-methylene-5oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-lH-pyrrolo[2,1~c][1,4]benzo diazepine- 10(5H)~carboxylate) (29)

<img file="CA2901941C_D0123.tif" />

H

<img file="CA2901941C_D0124.tif" />

0

2S

<img file="CA2901941C_D0125.tif" />

0

2S (i) tert-Butyl (15-oxo-3_s6,9,12-tetraoxa-16-azanonadec-18-yn-1-yl)carbamate (27)

EDCI (263 mg, 1.37 mmol) was added to a stirred solution of i-boc-A/-amido-dPEG®₄-acid (26) (500 mg, 1.37 mmol, Stratech Scientific Limited) and propargylamine (88 pL, 76 mg, 1.37 mmol) in dry DCM (10 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 16 hours at which point analysis by LC/MS showed a substantial amount of desired product at retention time 1.26 minutes (ES+) miz 403 ([M+ H]⁺. ~50% relative intensity), 425 ([Λ4+ Naf, ~100% relative intensity), note that both starting material and product had weak UV absorption (214 and 254 nm) and were best detected on ES+ TIC. The reaction mixture was diluted with DCM (100 mL) and washed with H₂O (30 mL), brine (40 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution in 1% increments: 100% DCM to 98:2 v/v DCM/MeOH) gave the amide 27 as an oil (392 mg, 71% yield).

(ii) Di-tert-butyl 8,8'-(((5-(2,2-dimethyl-4,20-dioxo-3,8,11,14,17-pentaoxa-5,21-diazatetracos23-yn-24-yl)-1_!3-phenylene)bis(methylene))bis(oxy))(11S,11aS,11'S,11a’S)-bis(7-methoxy-2CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

116 methylene-5-oxo-11-((tetrahydro-2H-pyran-2-y/)oxy)-2,3,11,11a-1eirahydro-1H~pyrrolo[2,1c][1,4]benzodiazepine-10(5H)-carboxylate) (28)

A catalytic amount ot Pd(PPh₃)₄ (23.0 mg, 19.5 pmol) was added to a mixture of the iodoaryi compound 2a (1.02 g, 0.89 mmol), Boc-acetylene 27 (393 mg, 0.98 mmol), Cui (7.4 mg, 39.1 pmol), diethylamine (2.02 mL, 1.43 g, 19.5 mmol) and oven-dried 4A molecular sieve pellets in dry DMF (9 mL) in an oven-dried sealable vessel. The mixture was degased and flushed with argon 3 times then heated in a microwave at 100 °C for 26 minutes at which point analysis by LC/MS revealed substantial product formation at retention time 1.89 minutes (ES+) m/z 1446 ([IW+ Na]⁺, -100% relative intensity, 1424 ([M+ Hf , -15% relative intensity). The reaction mixture was allowed to cool to room temperature and was then filtered through a sinter to remove the sieves (washed with DMF). The filtrate was evaporated in vacuo and the resulting residue dissolved in DCM (100 mL) and washed with H₂O (20 mL), brine (30 mL), dried (MgSO₄), filtered and evaporated in vacuo to give the crude product. Purification by flash chromatography (gradient elution in 1% increments: 100% DCM to 97:3 v/v DCM/MeOH) provided the alkyne 28 as a yellow foam (882 mg, 70% yield).

(iii) Di-tert-butyl 8,8 '-(((5-( 1 -amino-15-oxo-3,6,9,12-tetraoxa-16-azanonadec-18-yn-19-yl)1,3-phenyiene)bis(meihylene))bis(oxy))(11S,11aS, 11 'S, 11 a’S)~bis(7-methoxy-2-methyiene5-oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3.17,71a-tetrahydro- 7 H-pyrrolo[2,1-c][1,4]benzo diazepine-10(5H)-carboxylate) (29)

TBDMSOTf (1.42 mL, 1.64 g, 6.2 mmol) was added to a stirred solution of the tri-Boc protected compound 28 (882 mg, 0.62 mmol) and 2.6-lutidine (0.96 mL. 883 mg, 8.25 mmol) in dry DCM (15 mL) at room temperature. The reaction mixture was allowed to stir under an argon atmosphere for 16 hours during which time analysis by LC/MS revealed formation of the TBS carbamate at retention time 2.09 minutes (ES+) m/z 1504 (ÎM+ Nap, -100% relative intensity). The reaction mixture was diluted with DCM (60 mL) and washed with saturated NH₄C1 (2 x 20 mL), H₂O (20 mL), brine (30 mL), dried (MgSO₄), filtered and evaporated in vacuo to give the crude T3S carbamate. The product was re-dissolved in THF (15 mL) and treated with a solution of TBAF (744 pL of a 1.0M solution in THF, 0.744 mmol) at room temperature. The reaction mixture was allowed to stir for 1 hour at room temperature at which point analysis by LC/MS revealed substantial product formation at retention time 1.45 minutes (ES+) m/z 1324 ([A4+ H]*, -60% relative intensity) along with product corresponding to 1 N10Boc/1 THP cleaved at retention time 1.29 minutes (ES+) m/z 1121 ([Λ4+ Hf , -10% relative intensity), 1138 ([Λ4+ H₂O]⁺ , -20% relative intensity) and product corresponding to 2 N10 Boc/2 THP cleaved at retention time 1.12 minutes (ES+)

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

117 m/z 919 ([M+ H]⁺, -2.5% relative intensity), 937 ({M+ H20]⁺’, -3% relative intensity), 955 ([/W+ 2H2O]\ -5% relative intensity). The THF was removed by evaporation in vacuo and the resulting residue re-dissolved in DCM (60 mL) and washed with saturated NH₄CI (2 x 20 mL), H₂O (20 mL), brine (30 mL), dried (MgSO₄), filtered and evaporated in vacuo to give the key amine 29 as a pinkish foam.

(b) (R)-2‘(pyridin-2-yldisuifanyl)pfopyl ( 19-(3_!5-bis((((S)-7-methoxy‘2-methyierte-5-oxo2,3,5,11 a-tetrahydro· 1 H~pyrmlo[2,1 -c][1.4]benzodiazepin-8-yl)oxy)methyl)phenyi)· 15-oxo3,6,9,12-tetraoxa-16-azanonadec-18-yn-1-y!)carbamate (33)

<img file="CA2901941C_D0126.tif" />

<img file="CA2901941C_D0127.tif" />

?

<img file="CA2901941C_D0128.tif" />

0 (i) (R)-2-(pyridin-2-y!disutfanyl)propyl carbonochloridate (31)

Triphosgene (9.36 mg, 31.5 pmol) was added to a stirred solution of (R)-2-(pyridin-2yldisulfanyl)propan-1-ol (30) (18 mg, 0.09 mmol) and pyridine (6.7 pL, 6.6 mg, 0.08 mmol) in dry DCM (1 mL). The reaction mixture was allowed to stir under an argon atmosphere for 30 minutes after which time the solvent was removed by evaporation in vacuo to provide the crude chioroformate 31 as a white foam. Note: The product was carried through to the next step without purification or analysts.

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

118 (ii) Di-tert-butyi 8,8'-(((5-((R)-5,21-dioxo-2~(pyridin-2~yldisulfanyl)-4.9,12,15,18-pentaoxa-

6,22-diazapentacos-24-yn-25-yi)-1,3phenylene)bis(methylene))bis(oxy))(11S, 11aS,irS.11a'S)-bis(7-methoxy-2-methylene~5oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1-c}[1,4] benzodiazepine-10(5H)-carboxylate) (32)

A solution of 31 (-23 mg, -0.09 mmol) in dry DCM (1 mL) was added drop-wise to a stirred solution of amine 29 (-116 mg, -0.09 mmol) and pyridine (7.8 pL, 7.7 mg, 0.1 mmol) in dry DCM (1 mL) at room temperature. The reaction mixture was allowed to stir under an argon atmosphere for 3 hours at which point analysis by LC/MS (Kinetex® column) revealed substantial product formation at retention time 2.02 minutes (ES+) miz 1550 ([.M+ Hf , -20% relative intensity) along with persistence of unreacted starting material 29 at retention time

1.51 minutes. The solvent was removed by evaporation in vacuo to provide the crude carbamate 32 which was carried through to the next step without further purification or analysis.

(iii) (R)-2-(pyridin-2-yldisulfanyl)propyl (19-(3,5-bis((((S)-7-methoxy-2-methylene-5-oxo2.3.5,11a-tetrahydro- 1H-pyrrolo[2.1-c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)-15-oxo3,6,9,12-tetraoxa-16-azanonadec~18-yn-1-yi)carbamate (33)

A solution of 95:5 v/v TFA/H₂O (1 mL) was added to a crude sample of the Boc/THPprotected compound 32 (-136 mg, 88 pmol) at 0 °C (ice/acetone). After stirring at 0 °C for 1 hour the reaction was deemed complete as judged by LC/MS (Kinetex® column), desired product peak at retention time 1.42 minutes (ES+) miz 1146 ([M+ H]*, -90% relative intensity). The reaction mixture was kept cold and added drop-wise to a chilled saturated aqueous solution of NaHCOs (50 mL). The mixture was extracted with DCM (3x15 mL) and the combined organic layers washed with brine (20 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCI3 to 95:5 v/v CHClVMeOH) gave 33 as a film (10 mg, 7% yield): LC/MS (15-minute run), retention time 5.79 minutes (ES+) miz 1146 ([Λ/+ H]⁺, -8% relative intensity); ’H NMR (400 MHz, CDCI3) δ 8.44 (d, 1H, J- 4.8 Hz), 7.75-7.58 (m, 2H), 7.66 (d, 2H, J = 4.4 Hz), 7.52 (s, 2H), 7.45-7.40 (m, 3H), 7.08-7.05 (m, 1H), 6.95-6.85 (m, 1H), 6.79 (s. 2H), 5.43-5.41 (m, 1H). 5.23-5.09 (m, 8H), 4.30-4.23 (m, 6H), 4.19-4.10 (m, 4H), 3.973.94 (m, 2H), 3.96 (s, 6H), 3.91-3.85 (m, 2H), 3.75 (t, 2H, J = 5.8 Hz), 3.66-3.58 (m, 8H).

3.52 (t, 2H, J = 5.1 Hz), 3.34 -3.30 (m, 2H), 3.23-3.08 (m, 3H), 2.96-2.90 (m, 2H), 2.52 (t, 2H, J = 5.7 Hz), 1.31 (d, 3H, J = 7.0 Hz).

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

119

Example 7

<img file="CA2901941C_D0129.tif" />

<img file="CA2901941C_D0130.tif" />

<img file="CA2901941C_D0131.tif" />

<img file="CA2901941C_D0132.tif" />

(a) (±)-4-(pyridin-2-yldisulfanyl)pentanoic acid (35)

Aldrithiol^TIW-2 (176 mg, 0.86 mmol) was added to a stirred solution of (±)-4mercaptopentanoic acid 34 (107 mg, 0.80 mmol, Aurora Fine Chemicals LLC) in EtOH (2 mL) at room temperature. The reaction mixture was allowed to stir for 16 hours under an argon atmosphere at which point analysis by LC/MS revealed substantial product formation at retention time 1.32 minutes (ES+) m/z 244 ([M+ Hp, -95% relative intensity). The solvent was removed by evaporation in vacuo and the resulting residue purified by flash chromatography (gradient elution: 90:10 v/v hexane/EtOAc to 80:20 v/v hexane/EtOAc) to give 35 as a white solid (92 mg, 47% yield).

(b) (±)-Di-tert-butyi 8,8^(((5-(5,21~dioxo-24-(pyridin-2-yldisuifanyl)-8,11,14,17-tetraoxa~4,20diazapentacos-1-yn-1-yl)-1,3-phenytene)bis(methy!ene))bis(oxy))(11S, 11aS, 11’S, 11a’S)bis(7-methoxy-2-maihylene-5-oxo-11-((tetrahydrc:-2H-pyran-2-yl)oxy)-2.3,11,11a-tetrahyd!O1H-pyrrolo[2,1-c][1,4]benzodiazepine-10(5H)-carboxy!ate) (36)

EDCi (22 mg, 0.12 mmol) was added to a stirred solution of (±)-4-(pyridin-2yldisulfanyl)pentanoic acid (35) (26 mg, 0,10 mmol) and amine 29 (-139 mg, 0.1 mmol) in

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

120 dry DCM (2 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 20 hours at which point anaiysis by LC/MS showed a substantia! amount of desired product (split peak) at retention time 1.88 minutes (ES+) miz 1548 ([M+ Hf; -40% relative intensity) along with product corresponding to 1 N10Boc/1 THP cleaved at retention time 1.67 minutes (ES+) m/z 1346 ([A4+ Hf, -20% relative intensity), 1138 ([M+ H₂0f·, -20% relative intensity) and product corresponding to 2 N10 Boc/2 THP cleaved at retention time 1.43 minutes (ES+ Λ4+ not observed). The reaction mixture was diluted with DCM (30 mL) and washed with H₂O (15 mL), brine (20 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide the crude product 36 as a foam.

(c) (+)-N-(3-(3,5-bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1c][1, 4]benzodiazepin-8-yl)oxy)methyl)phenyl)prop-2-yn -1 -yl) -1 -(4-(pyridin-2yldisulfanyl)pentanamido)-3,6,9.12-tetraoxapeniadecan-15-amide (37)

A solution of 95:5 v/v TFA/H₂O (1 mL) was added to a crude sample of the Boc/THPprotected compound 36 (-163 mg, 0.10 mmol) at 0 ^CC (ice/acetone). After stirring at 0 °C for 1 hour the reaction was deemed complete as judged by LC/MS, desired product peak at retention time 1.44 minutes (ES+) miz 1144 ([M+ H]*, -3% relative intensity). The reaction mixture was kept cold and added drop-wise to a chilled saturated aqueous solution of NaHCCh (60 mL). The mixture was extracted with DCM (3 x 30 mL) and the combined organic layers washed with brine (30 mL), dried (MgSO₄). filtered and evaporated in vacuo to provide the crude product Purification by flash chromatography (gradient elution: 100% CHCI3 to 96:4 v/v CHCls/MeOH) gave 37 as an orange foam (69 mg, 57% yield): LC/MS (15minute run), retention time 5.72 minutes (ES+) miz 1144 ([M+ Hf, -3% relative intensity); ’H NMR (400 MHz, CDCI₃) δ 8.42 (d, 1 H, J = 4.3 Hz), 7.72-7.59 (m, 2H), 7.66 (d, 2H, J =

4.3 Hz), 7.52 (s, 2H), 7.45-7.40 (m, 3H). 7.09-7.04 (m, 1H), 6.98-6.94 (m, 1H). 6.80 (s, 2H). 6.40-6.35 (m, 1H), 5.23-5.09 (m, 8H), 4.30-4.23 (m, 6H), 4.19-4.10 (m, 4H), 3.96 (s, 6H), 3.91-3.85 (m, 2H), 3.75 (t, 2H, J = 5.7 Hz), 3.66-3.58 (m, 8H), 3.51 (t, 2H, J = 5.0 Hz), 3.423.39 (m, 2H), 3.23-3.08 (m, 2H), 2.96-2.90 (m, 3H), 2.52 (t, 2H, J = 5.6 Hz), 2.34 (t, 2H, J =

7.4 Hz), 1.94 (q, 2H, J = 7.4 Hz), 1.30 (d, 3H, J = 6.7 Hz).

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

121

Example 8

<img file="CA2901941C_D0133.tif" />

<img file="CA2901941C_D0134.tif" />

<img file="CA2901941C_D0135.tif" />

<img file="CA2901941C_D0136.tif" />

(a) 3-(((prop-2-yn-1-y!oxy)carbonyl)amino)propanoic acid (39)

A solution of propargyl chloroformate (315 pL, 383 mg, 3.23 mmol) in toluene (2 mL) was added drop-wise to a stimed mixture of β-aianine (38) (250 mg, 2.81 mmol) and NaHCOs (678 mg, 8.1 mmol) in H₂O (7 mL) at room temperature. The reaction mixture was allowed to stir vigorously for 16 hours after which time it was partitioned. The aqueous layer was diluted with H₂O (20 mL), washed with Et₂O (4x10 mL), chilled to 0-5 ^aC (ice/acetone) and acidified to pH 2 with concentrated HCi. The acidic solution was extracted with EtOAc (3 x 20 mL) and the combined organic layers washed with H_ZO (10 mL), brine (20 mL), dried (MgSOn), filtered and evaporated in vacuo to provide the crude product 39 as an oil, which was carried through to the next step without further purification.

(b) Di-tert-butyl 8,8'-(((5-(5,9,25-trioxo-4,13,16,19,22-pentaoxa-6_;10,26-triazanonacosa-1,28diyn-29-yl)-1_!3-phenylene)bis(methyiene))bis(oxy))( 11S,11aS, 1 TS,11a'S)-bis(7-methoxy-2rrtethylene-5-oxo-11-((tetrahydfO-2H-pyi'an-2-yl)oxy)-2,3.11,11a-tetrahydro-1H-pyrrolo[2,1c][1,4/benzodiazepine- 10(5H)-carboxylate) (40)

CA 02901941 2015-08-19

WO 2014/159981 PCT/US2014/025564

122

EDCI (24 mg, 0.13 mmol) was added to a stirred solution of 3-(((prop-2-yn-1yloxy)carbonyl)amino)propanoic acid (39) (18 mg, 0.10 mmol) and amine 29 (-139 mg, 0.10 mmol) in dry DCM (3 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 1.5 hours at which point analysis by LC/MS showed a substantial amount of desired product at retention time 1.84 minutes (ES+) m/z 1477 ([JU+ Hf, -20% relative intensity), 1499 ([M+ Naf, -22% relative intensity) along with product corresponding to 1 N10Boc/1 THP cleaved at retention time 1.60 minutes (ES*) m/z 1274 ([M+ Hf, -10% relative intensity) and unreacted 5 at retention time 1.47 minutes (ES+) m/z 1324 ([M+ Hf, -5% relative intensity). The reaction mixture was diluted with DCM (20 mL) and washed with H₂O (2 x 10 mL), brine (20 mL), dried (MgSO₄). filtered and evaporated in vacuo to provide the crude product 40 as a foam.

(c) Prop-2-yn-1-yl (23-(3,5-bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,5,11a-ietrahydro-1Hpyrrolo[2,1-c][1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)-3,19-dioxo-7,10,13,16-tetraoxa4,20-diazatricos-22-yn-1 -ytycarbamate (41)

A solution of 95:5 v/v TFA/H₂O (2 mL) was added to a crude sample of the Boc/THPprotected compound 40 (-155 mg, 0.10 mmol) at 0 ^eC (ice/acetone). After stirring at 0 ^eC for

1.5 hours the reaction was deemed complete as judged by LC/MS, desired product peak at retention time 1.41 minutes (ES+) m/z 1073 ([Λ4+ Hf, -30% relative intensity). The reaction mixture was kept coid and added drop-wise to a chilled saturated aqueous solution of NaHCOs (60 mL). The mixture was extracted with DCM (3 x 20 mL) and the combined organic layers washed with brine (25 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCI3 to 95:5 v/v CHCIÿ/MeOH) gave 41 as a yellow foam (51 mg, 45% yield): LC/MS (15· minute run), retention time 5.71 minutes (ES+) m/z 1073 ((M+ H]~, -30% relative intensity); ¹H NMR (400 MHz, CDCI₃) δ 7.67 (d, 2H, J * 4.5 Hz), 7.52 (s, 2H), 7.46-7.43 (m, 3H), 7.077.02 (m ,1H), 6.80 (s, 2H), 6.64-6.57 (m ,1H), 5.78-5.72 (m ,1H), 5.21-5.09 (m, 8H), 4.64 (d, 2H, J = 2.2 Hz), 4.29-4.25 (m ,6H), 3.96 (s, 6H), 3.90-3.85 (m, 2H), 3.76 (t, 2H, J = 5.9 Hz), 3.65-3.35 (m ,18H), 3.16-3.07 (m, 2H), 2.93 (d, 2H, J = 16 Hz), 2.52 (t, 2H, J - 5.9 Hz), 2.48-2.45 (m, 1H), 2.40 (t, 2H, J - 5.9 Hz).

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

123

Example 9

<img file="CA2901941C_D0137.tif" />

0 Oa^NH₂

<img file="CA2901941C_D0138.tif" />

<img file="CA2901941C_D0139.tif" />

(a) Di-tert-butyl 8,8-(((5-( 1-(4-((S)~2-((S)-2-(6-(2,5-dioxo-2,5~dihydro-1H-pyrrol-1yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)phenyl)-3,19-dioxo2,7,10,13.16-pentaoxa-4,20-diazatricos-22~yn-23-yl)-1,3phenylene)bis(methylene))bis(oxy))( 11S,11aS, 11'S,11a 'S)-bis(7-methoxy-2-methylene-5oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1c][1,4]benzodiazepine-10(5H)-carboxylate) (43)

DIPEA (44 pL, 32 mg, 0.25 mmol) was added to a stirred solution of key amine 29 (-155 mg, 0.11 mmol) and the nitrophenyi carbonate 42 (84 mg, 0.11 mmol) in dry DMF (3 mL) at room temperature. The reaction mixture was allowed to stir under an argon atmosphere for 3 days after which time analysis by LC/MS revealed desired product observed at retention time 1.80 minutes (ES+) mlz 1922 ([M+ H]⁺ , -40% relative intensity), 1944 ([M+ Na]⁴, -20% relative intensity) along with product corresponding to 1 N10Boc/1 THP cleaved at retention time 1.61 minutes (ES+) m/z 1720 ([M+ H]⁴, -20% relative intensity). The DMF was removed by

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

124 evaporation in vacuo and the resuiting product 43 carried through to the next step 'without further purification or analysis.

(b) 4-((S)-2-((S)-2-(0-(2_l5-d!Oxo~2_l5-dihydro-1H-pyrroi-1-yl)hexanamido)-3methy!butanamido)-5~ureidopentanamido)benzyl (19-(3,5-bis((((S)-7-methoxy-2-methylene5-oxo-2,3,5,11a-tetrahydro-1H-pyrro!o[2,1-c]{1,4]benzodiazepin-8-yl)oxy)methyi)pheny!)-15oxo-3,6,9,12-tetraoxa-16-azanonadec-1d-yn-1-yi)carbamate (44)

A solution of 95:5 v/v TFA/H2O (3 mL) was added to a crude sample of the Boc/THPprotected compound 43 (-173 mg, 0.11 mmol) at 0 °C (ice/acetone). After stirring at 0 °C for

1.5 hours the reaction was deemed complete as judged by LC/MS, desired product peak at retention time 1.42 minutes (ES+) m/z 1518 ([M+ H]* , -40% relative intensity). The reaction mixture was kept cold and added drop-wise to a chilled saturated aqueous solution of NaHCOs (100 mL). The mixture was extracted with DCM (3 x 30 mL) and the combined organic layers washed with brine (20 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCI3 to 80:20 v/v CHCls/MeOH) gave crude product as a yellow foam (72 mg, 42% crude yield). The material was further purified by preparative HPLC to provide pure 44 as a thin film (4.5 mg, 3% yield): LC/MS (15-minute run), retention time 5.44 minutes (ES+) m/z 1518 ([Af+ Hj⁺, -30% relative intensity).

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

125 (a) 1-(3-aminopropanamido)~N-(3-(3,5-bis((((S)-7-methoxy-2~methylene-5-oxo-2,3,5,11atetrahydro-1H-pyrrolol2,1-cft1,4]benzodiazepin-8-yl)oxy)methyl)phenyl)prop-2-yn-1-yl)3,6,9,12-tetraoxapentadecan-15-amide (47)

Example 10

<img file="CA2901941C_D0140.tif" />

<img file="CA2901941C_D0141.tif" />

<img file="CA2901941C_D0142.tif" />

<img file="CA2901941C_D0143.tif" />

<img file="CA2901941C_D0144.tif" />

(i) Di-tert-butyl 8,8'-(((5-(1 -(9H-fluoren-9-yl)-3,7.23-trioxo-2,11,14.17,20-pentaoxa-4,8,24 triazaheptacos-26-yn-27-yl)-1,3-phenylene)bis(methylene))bis(oxy))(11S, 11aS.11'S, 11a'S) bis(7-methoxy-2-methylene-5-oxo-11-((tetrahydro-2H-pyran-2-yi)oxy)-2,3,11,11 a-tetrahydro

H-pyrrolo[2,1 -eft 1,4]benzodiazepine-1 û(ôH)-carboxylate) (45)

EDCi (49 mg, 0.25 mmol) was added to a stirred solution of Fmoc-p-alanine (66 mg. 0.21 mmol) and amine 29 (-279 mg, 0.21 mmol) in dry DCM (5 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 3 hours at which point analysis by LC/MS showed a substantial amount of desired product at retention time 1.76 minutes (ES+) mlz 1617 ([M+ Hf , -10% relative intensity), 1639 ([A4+ Na]’, -80% relative intensity) along with product corresponding to 1 N10Boc/1 THP cleaved at retention time 1.56 minutes (ES+) mlz 1415 ([Λ4+ H]⁺ , -10% relative intensity). The reaction mixture was diluted with DCM (30 mL) and washed with H₂O (20 mL). brine (20 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide the crude product 45 as a foam.

(ii) (9H-fluoren-9-yl)methyl (23-(3,5-bis((((S)-7-methoxy-2-methylene-5-oxo-2,3,6,11atetrahydro-1H-pyrrolo[2_;1-cft1_;4jbenzodiazepin-8-yi)oxy)methyl)phenyl)-3,19-dioxo7,10,13,16-tetraoxa-4,20-diazatricos-22-yn-1-yl)carbamate (46)

A solution of 95:5 v/v TFA/H₂O (4 mL) was added to a crude sample of the Boc/THPprotected compound 45 (-341 mg, 0.21 mmol) at 0 °C (ice/acetone). After stirring at 0 °C for hour the reaction was deemed complete as judged by LC/MS, desired product peak at

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

126 retention time 1.44 minutes (ES+) m/z 1212 {[M+ H]‘ , -30% relative intensity). The reaction mixture was kept cold and added drop-wise to a chilled saturated aqueous solution of NaHCO₃ (80 mL). The mixture was extracted with DCM (3 x 20 mL) and the combined organic layers washed with NaHCO₃ (2 x 20 mL), brine (20 mL), dried (MgSCXf), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCI₃ to 95:5 v/v CHCL/MeOH) gave pure product 46 as a yellow foam (179 mg, 70% yield).

(Hi) 1-(3-aminopropanamido)-N-(3-(3_!5-bis((((S)-7-mathoxy-2-methylene-5-oxo-2,3,5,11atetrahydro-1H-pyrrolo[2,1-c][1..4]benzodiazepin-8-yl)oxy)methyl)phenyl)prop-2-yn-1-yl)3,6,9,12-tetraoxapentadecan-15-amide (47)

Dimethylamine (735 pL of a 2.0M solution in THF, 1.47 mmol) was added to a stirred solution of the Fmoc protected compound 46 (89 mg, 73.5 pmol) in THF (3 mL) at room temperature. After stirring for 3 hours at room temperature, analysis by LC/MS revealed reaction completion with desired product at retention time 1.14 minutes (ES+) m/z 990 ((M+ H]⁺ , -8% relative intensity), 1008 ([Λ4+ H₂Of, -10% relative intensity), 1026 ((M+ 2H₂O]⁺ , -15% relative intensity) along with Fmoc cleavage by-product at retention time 1.88 minutes. The mixture was evaporated in vacuo and crude 47 was carried through to next step without further purification or analysis.

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

127 (b) N-(3f3₎5-bis((((S)'7-methoxy-2-methy!ône-5-oxo-2_l3_;5_r11adetrahydro-1H-pyrro!0[2.1c][1.4]benzodiazepin-8-y!)oxy)methyl)phenyl)prop~2-yn-1 -yi)-1 -(3-(2bromoacetamido)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (48)

<img file="CA2901941C_D0145.tif" />

<img file="CA2901941C_D0146.tif" />

Bromoacetic anhydride (23 mg, 88.2 pmol) was added to a stirred solution of the crude amine 47 (-73 mg, 73.5 pmol) in DCM (3 mL). The reaction mixture was allowed to stir under an argon atmosphere at room temperature for 3 hours at which point analysis by LC/MS revealed completion of reaction with desired product observed at retention time 1.34 minutes (ES+) m/z 1112 ([M+ H]⁺, -30% relative intensity). The solvent was removed by evaporation in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCi₃ to 93:7 v/v CHClj/MeOH) gave the product as a yellow foam (38 mg, 46% crude yield). The material was further purified by preparative HPLC to provide pure 48 as a thin film (5 mg, 6% yield): LC/MS (15-minute run), retention time 4.96 minutes (ES+) m/z 1112 ([M+ Hf, -10% relative intensity); ¹H NMR (400 MHz, CDCI₃) 5 7.67 (d, 2H,

J = 4.4 Hz), 7.52 (s, 2H), 7.46-7.43 (m, 4H), 7.10-7.07 (m ,1H), 6.80 (s, 2H). 5.21-5.09 (m,

8H), 4.29-4.25 (m ,6H), 3.96 (s, 6H), 3.90-3.85 (m, 2H), 3.82 (s. 2H), 3.77 (t, 2H, J = 5.9

Hz), 3.65-3.40 (m ,18H), 3.16-3.07 (m, 2H), 2.94 (d, 2H, J ~ 16 Hz), 2.53 (t, 2H, J - 5.9 Hz),

2.43 (t, 2H, J=5.9 Hz).

CA 02901941 2015-09-19

WO 2014/159981

PCT7US2014/025564

128

Example 11 - Alternate synthesis of 10

<img file="CA2901941C_D0147.tif" />

<img file="CA2901941C_D0148.tif" />

(a) di-tert-Buiyl 8,8'-(((5-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,l9-dioxo~7,10,13,16tetraoxa-4,20-diazatricos-22-yn-23-yl)~ 1,3phenylene)bis(rriethylerie))bis(oxy))( 11S, 11 aS, 11'S,11a ‘S)-bis(7-rnethoxy-2-melhylerie-5oxo-11-((tetrahydro-2H-pyran-2-yl)oxy)-2,3,11,11a-tetrahydro-1H-pyrrolo[2,1c][1,4Jbenzodiazepine-10(5H)-carboxylate (9)

EDCI (61 mg, 0.32 mmol) was added to a stirred solution of N-maleoyl-3-alanine (53 mg, 0.32 mmol) and amine 29 (-418 mg, 0.32 mmol) in dry DCM (6 mL) at room temperature. The reaction mixture was stirred under an argon atmosphere for 3 hours at which point analysis by LC/MS showed a substantial amount of desired product at retention time 1.80 minutes (ES+) m/z 1474 ([A4+ Hf, -15% relative intensity), 1497 ([iW+ Na]’, -100% relative intensity), along with product corresponding to 1 N10Boc/1 THP cleaved at retention time 1.56 minutes 1272 ([M+ H]* , -80% relative intensity), 1295 ([M+ Na]⁺ , -45% relative intensity) and product corresponding to 2 N10 Boc/2 THP cleaved at retention time 1.31 minutes (ES+ M+ not observed). The reaction mixture was diluted with DCM (30 mL) and washed with H₂O (15 mL), brine (20 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide the crude product 9 as a foam.

(b) N-(3-(3,5-bis((((S)-7-methoxy-2-methylene-5~oxo-2,3,5,11a-tetrahydro-1H-pyrrolo[2,1c][1,4]benzodiazepin~8-y!)oxy)methyl)phenyl)prop-2-yn-1-y!)-1-(3-(2,5-dioxo-2,5-dihydro-1Hpyrrol- 1-yl)propanamido)-3,6,9.12-tetraoxapentadecan-15-amide (10)

A solution of 95:5 v/v TFA/H₂O (5 mL) was added to a crude sample of the Boc/THPprotected compound 9 (-466 mg, 0.32 mmol) at 0 °C (ice/acetone). After stirring at 0 °C for 1

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

129 hour the reaction was deemed complete as judged by LC/MS. desired product peak at retention time 1.32 minutes (ES+) m/z 1070 ([M+ Hp, -100% relative intensity). The reaction mixture was kept cold and added drop-wise to a chilled saturated aqueous solution of NaHCOa (120 mL). The mixture was extracted with DCM (3 x 40 mL) and the combined organic layers washed with brine (50 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide the crude product. Purification by flash chromatography (gradient elution: 100% CHCI₃ to 96:4 v/v CHCls/MeOH) gave 10 as an orange foam (202 mg, 60% yield): [a]²¹Cl = +351° (c = 0.47, CHCI3): LC/MS (15-minute run), retention time 4.88 minutes (ES+) mlz 1070 (ÎA4+ Hp, -100% relative intensity); ¹H NMR (400 MHz, CDCI3) δ 7.66 (d, 2H, J = 4.4 Hz), 7.52 (s, 2H), 7.45-7.40 (m, 3H), 6.98-6.94 (m, 1H), 6.80 (s, 2H), 6.66 (s, 2H), 6.556.50 (m, 1H), 5.22-5.07 (m. 8H), 4.30-4.22 (m, 6H), 3.96 (s, 6H). 3.91-3.85 (m, 2H), 3.82 (t, 2H, J = 7.2 Hz), 3.76 (t, 2H, J = 5.8 Hz), 3.65-3.43 (m, 16H), 3.16-3.08 (m, 2H), 2.94 (d, 2H, J = 15.7 Hz), 2.54-2.44 (m, 4H).

Reduction/Oxidation of ThioMabs for Conjugation

Full length, cysteine engineered monoclonal antibodies (ThioMabs - Junutula, et al.. 2008b Nature Biotech., 26(8):925-932; Doman et al (2009) Blood 114(13):2721-2729; US 7521541; US 7723485; W02009/052249, Shen etai (2012) Nature Biotech., 30(2):184-191: Junutula et al (2008) Jour of Immun. Methods 332:41-52) expressed in CHO cells were reduced with about a 20-40 fold excess of TCEP (tris(2-carboxyethyl)phosphine hydrochloride or DTT (dithiothreitol) in 50 mM Tris pH 7.5 with 2 mM EDTA for 3 hrs at 37°C or overnight at room temperature.(Getz et al (1999) Anal. Blochem. Vol 273:73-80; Soltec Ventures, Beverly, MA). The reduced ThioMab was diluted and loaded onto a HiTrap S column in 10 mM sodium acetate, pH 5, and eluted with PBS containing 0.3M sodium chloride. Alternatively, the antibody was acidified by addition of 1/20⁸¹ volume of 10% acetic acid, diluted with 10 mM succinate pH 5, loaded onto the column and then washed with 10 column volumes of succinate buffer. The column was eluted with 50 mM Tris pH7.5, 2 mM EDTA.

The eluted reduced ThioMab was treated with 15 fold molar excess of DHAA (dehydroascorbic acid) or 200 nM aqueous copper sulfate (CuSO,·.). Oxidation of the interchain disulfide bonds was complete in about three hours or more. Ambient air oxidation was also effective. The re-oxidized antibody was dialyzed into 20 mM sodium succinate pH 5, 150 mM NaCl, 2 mM EDTA and stored frozen at -20°C.

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

130

Conjugation of Thlo-Mabs with Compounds to prepare antibody-drug conjugates The deblocked, reoxidized, thio-antibodies (ThioMab) were reacted with 6-8 fold molar excess of compounds 7,10,13,17, 24,25, 33,37, 44, 48 (from a DMSO stock at a concentration of 20 mM) in 50 mM Tris, pH 8, until the reaction was complete (16-24 hours) as determined by LC-MS analysis of the reaction mixture.

The crude antibody-drug conjugates (ADC) were then applied to a cation exchange column after dilution with 20 mM sodium succinate, pH 5. The column was washed with at least 10 column volumes of 20 mM sodium succinate, pH 5, and the antibody was eluted with PBS. The antibody drug conjugates were formulated into 20 mM His/acetate, pH 5, with 240 mM sucrose using gel filtration columns. The antibody-drug conjugates were characterized by UV spectroscopy to determine protein concentration, analytical SEC (size-exclusion chromatography) for aggregation analysis and LC-MS before and after treatment with Lysine C endopeptidase.

Size exclusion chromatography was performed using a Shodex KW802.5 column in 0.2M potassium phosphate pH 6.2 with 0.25 mM potassium chloride and 15% ÎPA at a flow rate of 0.75 ml/min. Aggregation state of the conjugate was determined by integration of eluted peak area absorbance at 280 nm.

LC-MS analysis was performed using an Agilent QTOF 6520 ESI instrument. As an example, an antibody-drug conjugate generated using this chemistry was treated with 1:500 w/w Endoproteinase Lys C (Promega) in Tris. pH 7.5. for 30 min at 37^eC. The resulting cleavage fragments were loaded onto a 1000A, 8 um PLRP-S column heated to 80°C and eluted with a gradient of 30% B to 40% B in 5 minutes. Mobile phase A was H₂O with 0.05% TFA and mobile phase B was acetonitrile with 0.04% TFA. The flow rate was 0.5ml/min. Protein elution was monitored by UV absorbance detection at 280 nm prior to electrospray ionization and MS analysis. Chromatographic resolution of the unconjugated Fc fragment, residual unconjugated Fab and drugged Fab was usually achieved. The obtained m/z spectra were deconvolved using Mass Hunter™ software (Agilent Technologies) to calculate the mass of the antibody fragments.

ADC Thio-ooniugates with 7

<td> Ab</td><td> ADC</td><td> I DAR (drug to antibody ratio)</td><td> ί LCMS results</td>

<td> Thio Hu Anti-</td><td> 110</td><td> I 1.8 1</td><td> | 23439.82 LC</td>

<td> Her2 4D5-8</td><td></td><td></td><td> | 51681.86 HC J</td>

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

131

<td> HC A118C</td><td colspan="3"> i i i</td>

<td> Thio Hu Anti- Her2 4D5-8 HC A118C</td><td> 111</td><td colspan="2"> i¹⁹ | ! i ! i ! i I I</td>

<td> Thio hu anti- CD22 10F4v3 HC A118C</td><td> 115</td><td> i 1.8 ! 1 t t</td><td> 1 24034.96 LC | 51727.97 HC I i i I i</td>

ADC Thio-coniugates with 10

<td> Ab</td><td> ADC</td><td> DAR (drug to antibody ratio)</td><td> LCMS results</td>

<td rowspan="2"> Thio Hu Anti· Her2 4D5-8 HC A118C</td><td> 120</td><td> 1.9</td><td rowspan="2"> 23440.18 LC 51697.51 HC</td>

<td colspan="2"> t 1 t</td>

<td> Thio Hu Anti- Her2 4D5-8 HC A118C</td><td> 121</td><td> 1.9</td><td></td>

<td> Thio hu anti- CD22 10F4v3 HC A118C</td><td> 125</td><td> 1.7</td><td> 24035.14 LC 51744.95 HC</td>

<td> Thio Hu Anti- Her2 4D5-8 HC A118C</td><td> 201</td><td> 1.8</td><td> 23440.16 LC 51697.07 HC</td>

<td> Thio Hu Anti- Her2 4D5-8 HC A118C</td><td> 202</td><td> 1.7</td><td> 51698.55 HC</td>

<td> Thio Hu Anti- CD33 GM 15.33 HC A118C</td><td> 203</td><td> 1.6</td><td> 23932.47 LC 51116.61 HC</td>

<td> Thio Hu AntiLGR5 8E11.V2HC A118C</td><td> 204</td><td> 1.6</td><td> 23956.85 LC 51432.43 HC</td>

<td> Thio Hu Anti- Napi3b 10H1.11.4B HC A118C</td><td> 205</td><td> 1.4</td><td></td>

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

132

<td> Thio hu anti- CD22 10F4v3 HC A118C</td><td> 206</td><td> I 1.9 t | t !</td><td> i i i I i i i I</td>

<td> Thio Hu Anti- Hera 4D5-8 HC A118C</td><td> 207</td><td> | 2.0 ! I I t</td><td> I 48502 Fab i I I i i</td>

ADC Thio-coniugates with 13

<td> Ab</td><td> ADC</td><td> DAR (drug to antibody ratio)</td><td> LCMS results</td>

<td> Thio Hu Anti- Her2 4D5-8 HC A118C</td><td> 211</td><td> 1.7</td><td></td>

<td> Thio Hu Anti- CD22 10F4v3 HC A118C</td><td> 212</td><td> 1.7</td><td></td>

<td> Thio Hu Anti- CD33 GM 15.33 HC A118C</td><td> 213</td><td> 1.5</td><td> 23934.64 LC 51210.40 HC</td>

<td> Thio Hu Anti- MUC16 3A5 HC A118C</td><td> 214</td><td> 1.8</td><td> 146970 ADC</td>

<td> Thio Hu Anti- CD33 GM 15.33 HC A118C</td><td> 215</td><td> 1.6</td><td> 147407 ADC</td>

ADC Thio-coniugates with 17

<td> Ab</td><td> ADC</td><td> i DAR (drug to antibody ratio)</td><td> LCMS results</td>

<td> Thio Hu Anti-</td><td> 130</td><td> | 1.8</td><td> 23440.15 LC</td>

<td> Her2 4D5-8</td><td></td><td> t !</td><td> 51728.25 HC</td>

<td> HC A118C</td><td></td><td></td><td></td>

<td> Thio Hu Anti-</td><td> 131</td><td> I 1.9</td><td> 51727.48 HC</td>

<td> Her2 4D5-8</td><td></td><td> È</td><td></td>

<td> HCA118C</td><td></td><td> :</td><td></td>

<td> Thio Hu Anti-</td><td> 135</td><td> I 1.8 t</td><td> 23440.15 LC</td>

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

133

<td> CD22 10F4v3 HO A118C</td><td></td><td></td><td> 51728.25 HC</td>

<td> Thio Hu Anti- CD22 10F4v3 HC A118C</td><td> 221</td><td> 1.9</td><td> 24035.11 LC 51774.92 HC</td>

<td> Thio Hu Anti- CD33 GM 15.33 HC A118C</td><td> 222</td><td> 1.7</td><td> 23932.93 LC 51148.14 HC</td>

<td> Thio Hu Anti- Her2 4D5-8 HC A118C</td><td> 223</td><td> 2.0</td><td></td>

<td> Thio Hu Anti- Her2 4D5-8 HC A118C</td><td> 224</td><td> 0.9</td><td></td>

ADC Thio-coniuqates with 24

<td> Ab</td><td> ADC i DAR (drug to antibody ratio) | LCMS resuits</td>

<td> Thio Hu Anti- CD33 GM 15.33 HC A118C</td><td> I 1.3 j 23932.14 LC | I 51428.37 HC i ! : I I I i ! Î I</td>

<td> Thio Hu Anti- MUC16 3A5 HC A118C</td><td> I 0.8 I 23479.80 LC | | 51645.56 HC I i I I</td>

ADC Thio-coniuqates with 25

<td> Ab</td><td> ADC I DAR (drug to antibody ratio)</td><td> LCMS results</td>

<td> Thio Hu Anti- CD33 GM 15.33 HC A118C</td><td> I 17 t t ! t</td><td> 23933 LC 51431 HC</td>

<td> Thio Hu Anti- MUC16 3A5 HC A118C</td><td> I 1.7 t I t</td><td></td>

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

134

ADC Thio-coniuqates with 33

<td> Ab</td><td> ADC I DAR (drug to antibody ratio)</td><td> LCMS results</td>

<td> Thio Hu Anti- CD33 GM 15.33 HC A118C</td><td> | 1.5 ! t !</td><td></td>

<td> Thio Hu Anti- Napi3b 10H1.11.4B HC A118C</td><td> 1¹⁴ I t t Î</td><td></td>

ADC Thio-coniuqates with 37

<td> Ab</td><td> ADC</td><td> DAR (drug to antibody ratio) | LCMS results I</td>

<td rowspan="2"> Thio Hu Anti- CD33 GM15.33 HC A118C Thio Hu Anti· Napi3b 10H1.11.4B HC A118C</td><td rowspan="2"></td><td> 1.4 i i I I i i I I</td>

<td> Ï.6 [ I I I I I I i</td>

ADC Thio-coniuqates with 44

<td> Ab</td><td> ADC Î DAR (drug to antibody ratio) I LCMS results</td>

<td> Thio Hu anti- CD22 10F4v3 HC A118C</td><td> | 1.6 ! ! i I I : i ! i ! i I I</td>

<td> Thio Hu anti- CD33 15G15.3 HC A118C</td><td> i 1.6 ! : I I I i ! ! I : I I I i !</td>

ADC Thio-coniuqates with 48

<td> Ab</td><td> ADC</td><td> I DAR (drug to antibody ratio)</td><td> LCMS results</td>

<td> Thio Hu Anti- Her2 4D5-8 HC A118C</td><td></td><td> | 1.9 t :</td><td> 147329 ADC</td>

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

135

<td> Thio Hu Anti- CD22 10F4v3 HC A118C</td><td> i 1.7 I î I : I I I I i ! i ! i I I</td>

The following in vitro and in vivo assays are also described in Phillips et ai (2008) Cancer Res. 68(22):9280-9290.

In vitro ceil proliferation assay

Efficacy of ADC were measured by a cell proliferation assay employing the following protocol (CellTIter Gio Luminescent Cell Viability Assay, Promega Corp. Technical Bulletin TB288; Mendoza et al (2002) Cancer Res. 62:5485-5488). All cell lines were obtained from American Type Culture Collection;

1. An aliquot of 100 gl of cell culture containing about 10⁴ cells (for example, KPL-4, a human breast cancer cell line, Kurebayashi et al (1999) Brit. Jour. Cancer 79(5-6):707-717), or SKBR-3) in medium was deposited in each well of a 96-weil, opaque-wailed plate.

2. Control wells were prepared containing medium and without cells.

3. ADC was added to the experimental wells and incubated for 3-5 days,

4. The plates were equilibrated to room temperature for approximately 30 minutes.

5. A volume of CeliTiter-Gio Reagent equal to the volume of cel! culture medium present in each well was added.

6. The contents were mixed for 2 minutes on an orbital shaker to induce cell lysis.

7. The plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal.

8. Luminescence was recorded and reported in graphs as RLU = relative luminescence units.

Certain cells are seeded at 1000-2000/well or 2000-3000/well in a 96-well plate, 50 uL/well. After one or two days, ADC are added in 50 gL volumes to final concentration of 9000, 3000, 1000, 333,111, 37, 12.4, 4.1, or 1.4 ng/mL, with no ADC control weils receiving medium alone. Conditions are in duplicate or triplicate After 3-5 days, 100 gL/well Ceil TiterGlo II is added (luciferase-based assay; proliferation measured by ATP levels) and cell counts are determined using a iuminometer. Data are plotted as the mean of luminescence for each set of replicates, with standard deviation error bars. The protocol is a modification of the CeilTiter Gio Luminescent Ceil Viability Assay (Promega):

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

136

1. Plate 1000 cells/ well in 50 pL/well of FBS/glutamine media. Allow cells to attach overnight.

2. ADC is serially diluted 1:3 in media beginning at at working concentration 18 pg/ml (this results in a final concentration of 9 pg/ml). 50 μΙ_ of diluted ADC is added to the 50 pL of cells and media already in the well.

3. Incubate 72-96 hrs (the standard is 72 hours, but watch the 0 ug/mL concentration to stop assay when the cells are 85-95% confluent).

4. Add 100 pL/well of Promega Cell Titer Gio reagent, shake 3 min. and read on luminometer

Results

Antibody-drug conjugates, trastuzumab-7 (110) trastuzumab-10 (120) and trastuzumab-17 (130) were tested against SK-BR-3, KPL-4, and MCF-7 (Levenson et ai (1997) Cancer Res. 57(15):3071-3078) cells to measure in vitro cell viability in five day studies. The iCso value (ng/mL) for 110 against SK-BR-3 was 22.90. The IC50 value for 120 against SK-BR-3 was 11.14. The iC₅₀ value for 130 against SK-BR-3 was 16.8. SK-BR-3 cells are HER2+ expressing, trastuzumab sensitive. 110.120 and 130 were effectively inactive against MCF-7, which is a HER2 non-expressing human breast adenocarcinoma cell line. Thus, conjugates 110,120 and 130 demonstrate targetted cell killing potency.

Tumor growth inhibition, in vivo efficacy in high expressing HER2 transgenic explant mice

Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Taconic (Germantown, N.Y.). Many strains are suitable, but FVB female mice are preferred because of their higher susceptibility to tumor formation. FVB males were used for mating and vasectomized CD.1 studs were used to stimulate pseudopregnancy. Vasectomized mice can be obtained from any commercial supplier. Founders were bred with either FVB mice or with 129/BL6 x FVB p53 heterozygous mice. The mice with heterozygosity at p53 allele were used to potentially increase tumor formation. However, this has proven unnecessary. Therefore, some F1 tumors are of mixed strain. Founder tumors are FVB only. Six founders were obtained with some developing tumors without having litters.

Animals having tumors (allograft propagated from Fo5 mmtv transgenic mice) were treated with a single or multiple dose by IV injection of ADC. Tumor volume was assessed at various time points after injection.

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

137

Tumors arise readily in transgenic mice that express a mutationaiiy activated form of neu, the rat homolog of HER2. but the HER2 that is overexpressed in human breast cancers is not mutated and tumor formation is much less robust in transgenic mice that overexpress nonmutated HER2 (Webster ei al (1994) Semin. Cancer Biol. 5:69-76).

To improve tumor formation with nonmutated HER2, transgenic mice were produced using a HER2 cDNA plasmid in which an upstream ATG was deleted in order to prevent initiation of translation at such upstream ATG codons, which would otherwise reduce the frequency of translation initiation from the downstream authentic initiation codon of HER2 (for example, see Child et al (1999) J. Biol. Chem. 274: 24335-24341). Additionally, a chimeric intron was added to the 5’ end, which should also enhance the level of expression as reported earlier (Neuberger and Williams (1988) Nucleic Acids Res. 16:6713; Buchman and Berg (1988) Mol. Cell. Biol. 8:4395; Brinster et al (1988) Proc. Natl. Acad. Sci. USA 85:836). The chimeric intron was derived from a Promega vector, Pci-neo mammalian expression vector (bp 890-1022). The cDNA 3’-end is flanked by human growth hormone exons 4 and 5, and polyadenylation sequences. Moreover, FVB mice were used because this strain is more susceptible to tumor development. The promoter from MMTV-LTR was used to ensure tissue-specific HER2 expression in the mammary gland. Animals were fed the AIN 76A diet in order to increase susceptibility to tumor formation (Rao et ai (1997) Breast Cancer Res. and Treatment 45:149-158).

Fo5 murine mammary tumor model

The Fo5 model is a transgenic mouse model in which the human HER2 gene, under transcriptional regulation of the murine mammary tumor virus promoter (MMTV-HER2), is overexpressed in mammary epithelium. The overexpression causes spontaneous development of mammary tumors that overexpress the human HER2 receptor. The mammary tumor of one of the founder animals (founder #5 [Fo5]) has been propagated in subsequent generations of FVB mice by serial transplantation of tumor fragments. Before being used for an in vivo efficacy study, the MMTV-HER2 Fo5 transgenic mammary tumor was surgically transplanted into the No. 2/3 mammary fat pad of nu/nu mice (from Charles River Laboratories) in fragments that measured approximately 2x2 mm. When tumors reached desired volumes, the tumor-bearing mice were randomized and given a single dose by IV injection of the ADC.

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

138

Results

Figure 1 shows a plot of the in vivo mean tumor volume change over time in breast cancermodel MMTV-HER2 Fo5 mammary allograft tumors inoculated into CRL nu/nu mice after single iv dosing on day 0 with: (1) Vehicle 20mM Histidine acetate. pH 5.5, 240mM sucrose.

(2) xCD22-7 (115) at 6 mg/kg, (3) trasturumab-7 (110) at 1 mg/kg, (4) trastuzumab-7 (110) at 3 mg/kg, and (5) trastuzumab-7 (110) at 6 mg/kg. The lines in the figure are indicated with the following symbols:

χ Vehicle —θ— ADC110 Tmab-7 HC A118C, 1 mg/kg

A ADC110 Tmab-7 HC A118C, 3 mg/kg □ ADC110 Tmab-7 HC A118C, 6 mg/kg --ψ—ADC 115 CD22-7 HC A118C, 6 mg/kg

Figure 2 shows a plot of the in vivo mean tumor volume change over time in breast cancer-model MMTV-HER2 Fo5 mammary aliograft tumors inoculated into CRL nu/nu mice after single IV dosing on day 0 with: (1) Vehicle 20 mM Histidine acetate, pH 5.5, 240 mM sucrose, (2) XCD22-10 (125) at 3 mg/kg, (3) trastuzumab-10 (120) at 0.3 mg/kg, (4) trastuzumab-10 (120) at 1 mg/kg, and (5) trastuzumab-10 (120) at 3 mg/kg. The lines in the figure are indicated with the following symbols:

-X— Vehicle

-θ—ADC120 Tmab-10 HC A118C, 0.3 mg/kg

-φ— ADC120 Tmab-10 HC A118C_S 1 mg/kg

4g—ADC120 Tmab-10 HC A118C, 3 mg/kg -V—ADC 125 CD22 HC A118C, 3 mg/kg

Figure 3 shows a plot of the in vivo mean tumor volume change over time in breast cancermodel MMTV-HER2 Fo5 mammary allograft tumors inoculated into CRL nu/nu mice after single iv dosing on day 0 with: (1) Vehicle 20 mM Histidine acetate, pH 5.5, 240 mM sucrose, (2) XCD22-17 (135) at 3 mg/kg, (3) trastuzumab-17 (130) at 0.3 mg/kg, (4) trastuzumab-17 (130) at 1 mg/kg, and (5) trastuzumab-17 (130) at 3 mg/kg. The lines in the figure are indicated with the following symbols:

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

139 )( Vehicle —ta-ADC130 Tmab-17 HC A118C, 03 mg/kg —O— ADC130 Tmab-17 HC A118C, 1 mg/kg —Θ— ADC130 Tmab-17 HC Al 18C, 3 mg/kg —ADC135 CD22 HC Al 18C, 3 mg/kg

Abbreviations

<td> Ac</td><td> acetyl</td>

<td> Acm</td><td> acetamidomethyl</td>

<td> Alioc</td><td> ailyloxycarbonyi</td>

<td> Boc</td><td> di-fert-butyl dicarbonate</td>

<td> t-Bu</td><td> tert-butyl</td>

<td> Bzl</td><td> benzyl, where Bzl-OMe is methoxybenzyl and Bzl-Me is methylbenzene</td>

<td> Cbz or Z</td><td> benzyloxy-carbonyl, where Z-CI and Z-Br are chloro- and bromobenzyloxy carbonyl respectively</td>

<td> DMF</td><td> A/,N-dimethylformamide</td>

<td> Dnp DTT</td><td> dinitrophenyl dithiothreitol</td>

<td> Fmoc</td><td> 9H-fluoren-9-ylmethoxycarbonyl</td>

<td> imp MC-OSu</td><td> Λ/-10 imine protecting group: 3-(2-methoxyethoxy)propanoate-Val-Ala-PAB maleimidocaproyl-O-N-succinimide</td>

<td> Moc</td><td> methoxycarbonyl</td>

<td> MP</td><td> maleimidopropanamide</td>

<td> Mtr</td><td> 4-methoxy-2,3,6-trimethtylbenzenesulfonyl</td>

<td> PAB</td><td> para-aminobenzyloxycarbonyl</td>

<td> PEG</td><td> ethyleneoxy</td>

<td> PNZ</td><td> p-nitrobenzyl carbamate</td>

<td> Psec</td><td> 2-(phenylsulfonyl)ethoxycarbonyi</td>

<td> TBDMS</td><td> tert-butyldi methylsi lyl</td>

<td> TBDPS</td><td> tert-butyldiphenylsilyl</td>

<td> Teoc</td><td> 2-(tri methylsi lyl)ethoxyca rbony I</td>

<td> Tos</td><td> tosyl</td>

<td> Troc</td><td> 2,2,2-trichlorethoxycarbonyl chloride</td>

<td> Trt</td><td> trityl</td>

<td> Xan</td><td> xanthy!</td>

140

References

EP 0522868 EP 0875569 EP 1295944 EP 1347046 EP 1394274 EP 1394274 EP 1439393 JP 05003790 JP 2004113151 JP 58180487 US 2001/055751 US 2002/034749 US 2002/042366 US 2002/150573 US 2002/193567 US 2003/0228319 US 2003/060612 US 2003/064397 US 2003/065143 US 2003/091580 US 2003/096961 US 2003/105292 US 2003/109676 US 2003/118592 US 2003/119121 US 2003/119122 US 2003/119125 US 2003/119126 US 2003/119128 US 2003/119129 US 2003/119130 US 2003/119131 US 2003/124140 US 2003/124579 US 2003/129192 US 2003/134790-A1 US 2003/143557 US 2003/157089 US 2003/165504 US 2003/185830 US 2003/186372 US 2003/186373 US 2003/194704 US 2003/206918 US 2003/219806 US 2003/224411 US 2003/224454 US 2003/232056 US 2003/232350 US 20030096743

CA 2901941 2019-03-08

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

141

US 20030130189

US 2003096743

US 2003130189

US 2004/0001827

US 2004/005320

US 2004/005538

US 2004/005563

US 2004/005598

US 2004/0101899

US 2004/018553

US 2004/022727

US 2004/044179

US 2004/044180

US 2004/101874

US 2004/197325

US 2004/249130

US 20040018194

US 20040052793

US 20040052793

US 20040121940

US 2005/271615

US 2006/116422

US 4816567

US 5362852

US 5440021

US 5583024

US 5621002

US 5644033

US 5674713

US 5700670

US 5773223

US 5792616

US 5854399

US 5869445

US 5976551

US 6011146

US 6153408

US 6214345

US 6218519

US 6268488

US 6518404

US 6534482

US 6555339

US 6602677

US 6677435

US 6759509

US 6835807

US 7223837

US 7375078

US 7521541

US 7723485

WO 00/012508

WO 00/12507

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

142

WO 00/12508

WO 01/16318

WO 01/45746

WO 02/088172

WO 03/026577

WO 03/043583

WO 04/032828

WO 2000/12130

WO 2000/14228

WO 2000/20579

WO 2000/22129

WO 2000/32752

WO 2000/36107

WO 2000/40614

WO 2000/44899

WO 2000/55351

WO 2000/75655

WO 200053216

WO 2001/00244

WO 2001/38490

WO 2001/40269

WO 2001/40309

WO 2001/41787

WO 2001/46232

WO 2001/46261

WO 2001/48204

WO 2001/53463

WO 2001/57188

WO 2001/62794

WO 2001/66689

WO 2001/72830

WO 2001/72962

WO 2001/75177

WO 2001/77172

WO 2001/88133

WO 2001/90304

WO 2001/94641

WO 2001/98351

WO 2002/02587

WO 2002/02624

WO 2002/06317

WO 2002/06339

WO 2002/101075

WO 2002/10187

WO 2002/102235

WO 2002/10382

WO 2002/12341

WO 2002/13847

WO 2002/14503

WO 2002/16413

WO 2002/16429

WO 2002/22153

WO 2002/22636

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

143

WO 2002/22660

WO 2002/22808

WO 2002/24909

WO 2002/26822

WO 2002/30268

WO 2002/38766

WO 2002/54940

WO 2002/59377

WO 2002/60317

WO 2002/61087;

WO 2002/64798

WO 2002/71928

WO 2002/72596

WO 2002/78524

WO 2002/81646

WO 2002/83866

WO 2002/86443

WO 2002/88170

WO 2002/89747

WO 2002/92836

WO 2002/94852

WO 2002/98358

WO 2002/99074

WO 2002/99122

WO 2003/000842

WO 2003/002717

WO 2003/003906

WO 2003/003984

WO 2003/004989

WO 2003/008537

WO 2003/009814

WO 2003/014294

WO 2003/016475

WO 2003/016494

WO 2003/018621

WO 2003/022995

WO 2003/023013

WO 2003/024392

WO 2003/025138

WO 2003/025148

WO 2003/025228

WO 2003/026493

WO 2003/029262

WO 2003/029277

WO 2003/029421

WO 2003/034984

WO 2003/035846

WO 2003/042661

WO 2003/045422

WO 2003/048202

WO 2003/054152

WO 2003/055439

WO 2003/055443

CA 02901941 2015-09-19

WO 2014/159981

PCT/US2014/025564

144

WO 2003/062401

WO 2003/062401

WO 2003/072035

WO 2003/072036

WO 2003/077836

WO 2003/081210

WO 2003/083041

WO 2003/083047

WO 2003/083074

WO 2003/087306

WO 2003/087768

WO 2003/088808

WO 2003/089624

WO 2003/089904

WO 2003/093444

WO 2003/097803

WO 2003/101283

WO 2003/101400

WO 2003/104270

WO 2003/104275

WO 2003/105758

WO 2003004529

WO 2003042661

WO 2003104399

WO 2004/000997

WO 2004/001004

WO 2004/009622

WO 2004/011611

WO 2004/015426

WO 2004/016225

WO 2004/020595

WO 2004/022709

WO 2004/022778

WO 2004/027049

WO 2004/031238

WO 2004/032828

WO 2004/032842

WO 2004/040000

WO 2004/043361

WO 2004/043963

WO 2004/044178

WO 2004/045516

WO 2004/045520

WO 2004/045553

WO 2004/046342

WO 2004/047749

WO 2004/048938

WO 2004/053079

WO 2004/063355

WO 2004/063362

WO 2004/063709

WO 2004/065577

WO 2004/074320

CA 02901941 2015-08-19

WO 2014/159981

145

PCT/US2014/025564

WO 2004000221

WO 2004020583

WO 2004042346

WO 2004065576

WO 2005/023814

WO 2005/082023

WO 2005/085251

WO 2006/111759

WO 2007/044515

WO 2007/085930

WO 2009/052249

WO 2010/091150

WO 91/02536

WO 92/07574

WO 92/17497

WO 94/10312

WO 94/28931

WO 9630514

WO 97/07198

WO 97/44452

WO 98/13059

WO 98/37193

WO 98/40403

WO 98/51805

WO 98/51824

WO 99/28468

WO 99/46284

WO 99/58658

Am. J. Hum. Genet. 49 (3):555-565 (1991)

Amie! J., et al Hum. Mol. Genet. 5, 355-357,1996 Amir et al (2003) Angew. Chem. Int. Ed. 42:4494-4499 Amsberry, et al (1990) J. Org. Chem. 55:5867

Angew Chem. Inti. Ed. Engl. (1994) 33:183-186 Annu. Rev. Neurosci. 21:309-345 (1998)

Arai H., et al J. Biol. Chem. 268, 3463-3470,1993

Arai H., et al Jpn. Cire. J. 56, 1303-1307,1992 Arima, et al., J. Antibiotics, 25, 437-444 (1972)

Attie T„ et al, Hum. Mol. Genet. 4, 2407-2409,1995 Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996

Barel M., et al Moi. Immunol. 35,1025-1031, 1998 Barella et al (1995) Biochem. J. 309:773-779 Barnett T., et al Genomics 3, 59-66,1988

Beck et al (1992) J. Mol. Biol. 228:433-441

Beck et a! (1996) J. Mol. Bioi. 255:1-13

Berge, et al„ J. Pharm. Sci., 66, 1-19 (1977)

Biochem. Biophys. Res. Commun. (2000) 275(3):783-788 Biochem. Biophys. Res. Commun. 255 (2). 283-288 (1999) Blood (2002) 100 (9):3068-3076

Blood 99 (8):2662-2669 (2002)

Blumberg H., et al Ceil 104, 9-19, 2001

Bose, et al., Tetrahedron, 48, 751-758 (1992)

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

146

Bourgeois C., etai J. Ciin. Endocrinol Metab. 82, 3116-3123,1997

Brinster et ai (1988) Proc. Nat!. Acad. Sci. USA 85:836

Buchman and Berg (1988) Moi. Ceil. Biol. 8:4395

Cancer Res. 61 (15), 5857-5860 (2001)

Carl et al (1981) J. Med. Chem. 24:479-480

Calisson et al (1978) Biochem. J. 173:723-737

Carter, P. (2006) Nature Reviews Immunology 6:343-357

Cell 109 (3):397-407 (2002)

CeilTiter Gio Luminescent Cell Viability Assay, Promega Corp. Technical Bulletin

TB288

Chakravarty et al (1983) J. Med. Chem. 26:638-644

Chan.J. and Watt, V.M., Oncogene 6 (6), 1057-1061 (1991)

Child et al (1999) J. Biol. Chem. 274: 24335-24341

Cho H.-S., et al Nature 421, 756-760, 2003

Ciccodicola, A., et al EMBO J. 8(7):1987-1991 (1989)

Clackson eta! (1991) Nature, 352:624-628

Clark H.F.. et al Genome Res. 13, 2265-2270,2003

Corey E, Quinn JE, Buhler KR, et al. LuCap35: a new model of prostate cancer progression to androgen independence. The Prostate 2003;55:239-46

Coussens L., et al Science (1985) 230(4730):1132-1139

Cree et al (1995) AntiCancer Drugs 6:398-404

Crouch et al (1993) J. Immunol. Meth. 160:81-88

Davis et al (2001 ) Proc. Natl. Acad. Sci USA 98(17):9772-9777 de Groot et al (2001 ) J. Org. Chem. 66:8815-8830 de Groot et al (2003) Angew. Chem. Int. Ed. 42:4490-4494

Dennis et al. (2002) Albumin Binding As A General Strategy For improving The Pharmacokinetics Of Proteins J Biol Chem. 277:35035-35043

Dobner et al (1992) Eur. J. Immunol. 22:2795-2799

Doman et a! (2009) Blood 114(13):2721-2729

Doronina et a! (2006) Bioconj. Chem. 17:114-124

Dubowchik et al. Bioconjugate Chemislry, 2002, 13,855-869

Dubowchik, et al. (1997) Tetrahedron Letters, 38:5257-60

Dumoutier L., et al J. Immunol. 167, 3545-3549,2001

E. Schroder and K. Lübke, The Peptides, volume 1, pp 76 -136 (1965) Academic Press

Ehsani A., et al (1993) Genomics 15, 426-429

Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds, John Wiley &amp; Sons,

Inc., New York, 1994

Elshourbagy N.A., et al J. Biol. Chem. 268, 3873-3879,1993

Erickson et al (2006) Cancer Res. 66(8):1-8

Feiid, J.A., et al (1999) Biochem. Biophys. Res. Commun. 258 (3):578-582

Fields, G. and Noble, R. (1990) “Solid phase peptide synthesis utilizing 9fluoroenylmethoxycarbonyl amino acids, Int. J. Peptide Protein Res. 35:161-214

Fuchs S., et al Mol. Med. 7,115-124, 2001

Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125)

Gary S.C.. et al Gene 256,139-147. 2000

Gaugitsch, H.W., et al (1992) J. Biol. Chem. 267 (16):11267-11273)

Geiser et al Automation of solid-phase peptide synthesis* in Macromolecular

Sequencing and Synthesis, Alan R. Liss, Inc., 1988, pp. 199-218

Genome Res. 13 (10):2265-2270 (2003)

Genomics 62 (2):281-284 (1999)

Geoghegan &amp; Stroh, (1992) Bioconjugate Chem. 3:138-146

Getz et al (1999) Anal. Biochem. Vol 273:73-80

Glynne-Jones et al (2001) Int J Cancer. Oct 15; 94(2):178-84

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

147

Gregson et al., Chem. Commun. 1999, 797-798

Gregson et al.. J. Med. Chem. 2001, 44,1161-1174

Gu Z., et al Oncogene 19,1288-1296, 2000

Ha et al (1992) J. Immunol. 148(5):1526-1531

Haendîer B., et al J. Cardiovasc. Pharmacol. 20, s1-S4, 1992

Hamann P. (2005) Expert Opin. Ther. Patents 15(9):1087-1103

Hamblettetal (2004) Clin. Cancer Res. 10:7063-7070

Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, New York, USA)

Handbook of Pharmaceutical Excipients, 2nd edition, 1994

Hara, et al., J. Antibiotics, 41, 702-704 (1988)

Hashimoto et al (1994) Immunogenetics 40(4):287-295

Hay et al. (1999) Bloorg. Med. Chem. Lett. 9:2237

Herdwijn, P. et al., Canadian Journal of Chemistry. 1982, 60, 2903-7

Hermanson. G.T. (1996) Bioconjugate Techniques; Academic Press: New York, p 234242

Hochiowski, et al.. J. Antibiotics, 40,145-148 (1987)

Hofstra R.M.W., et al Eur. J. Hum. Genet. 5. 180-185,1997

Hofstra R.M.W., et al Nat. Genet. 12, 445-447,1996

Horie et a! (2000) Genomics 67:146-152

Hubert, R.S., et al (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (25):14523-14528)

Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986) Immunogenetics 54 (2):87-95 (2002)

Int. Rev. Cytol. 196:177-244 (2000)

Itoh. et al.. J. Antibiotics, 41,1281-1284 (1988)

J. Biol. Chem. 270 (37):21984-21990 (1995)

J. Biol. Chem. 276 (29):27371-27375 (2001)

J. Biol. Chem. 277 (22):19665-19672 (2002)

J. Biol. Chem. 278 (33):30813-30820 (2003)

Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York

Jeffrey et al (2005) J. Med. Chem. 48:1344-1358

Jonsson et al (1989) Immunogenetics 29(6):411-413

Junutuia, et al„ 2008b Nature Biotech., 26(8):925-932

Kang, G-D„ et al., Chem. Commun., 2003,1680-1689

Kasahara et al (1989) Immunogenetics 30(1):66-68

King et al (2002) Tetrahedron Letters 43:1987-1990

Kingsbury et al (1984) J. Med. Chem. 27:1447

Kohler et al (1975) Nature 256:495

Kohn, in Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975).

Konishi, et ai.. J. Antibiotics, 37. 200-206 (1984)

Kovtun et ai (2006) Cancer Res. 66(6):3214-3121

Kuhns J.J., et al J. Biol. Chem. 274, 36422-36427,1999

Kuminoto. et al., J. Antibiotics. 33, 665-667 (1980)

Kurebayashi et al (1999) Brit. Jour. Cancer 79(5-6):707-717

Lab. Invest. 82 (11):1573-1582 (2002)

Lambert J. (2005) Current Opin. in Pharmacol. 5:543-549

Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)

Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119

Law el al /2006) Cancer Res. 66/4):2328-2337

Le et al (1997) FEBS Lett 418(1-2):195-199

Leber, et al.. J. Am. Chem. Soc., 110, 2992-2993 (1988)

Lelmgruber, et at. J. Am. Chem. Soc., 87, 5791-5793 (1965)

CA 02901941 2015-09-19

WO 2014/159981 PCT/US2014/025564

148

Leîmgruber, et al. J. Am. Chem. Soc.. 87, 5793-5795 (1965)

Levenson et al (1997) Cancer Res. 57(15):3071-3078

Liang et al (2000) Cancer Res. 60:4907-12

Manfré, F. et ai., J. Org. Chem. 1992, 57,2060-2065

Marks et al (1991) J. Mol. Biol., 222:581-597

McDonagh (2006) Protein Eng. Design &amp; Sel., 19(7): 299-307

Mendoza et al (2002) Cancer Res. 62:5485-5488

Miller et al (2003) Jour, of Immunology 170:4854-4861

Miura et al (1996) Genomics 38(3):299-304

Miura et al (1998) Blood 92:2815-2822

Moore M., et al Proc. Natl. Acad. Sci. U.S.A. 84, 9194-9198,1987

Morrison et al (1984) Proc. Natl. Acad. Sci. USA. 81:6851-6855

Muller et al (1992) Eur. J. Immunol. 22 (6):1621-1625

Mungall A.J., et al Nature 425, 805-811, 2003

Nagase T., et al (2000) DNA Res. 7 (2):143-150)

Nakamuta NI., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991

Nakayama et al (2000) Biochem. Biophys. Res. Commun. 277(1):124-127

Naruse et al (2002) Tissue Antigens 59:512-519

Nature 395 (6699):288-291 (1998)

Neuberger and Williams (1988) Nucleic Acids Res. 16:6713

Novabiochem Catalog 2006/2007

Ogawa Y., et al Biochem. Biophys. Res. Commun. 178. 248-255,1991

Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997

Oncogene 10 (5):897-905 (1995)

Oncogene 14(11):1377-1382 (1997))

Parrish-Novak J., et al J. Biol. Chem. 277. 47517-47523. 2002

Payne, G. (2003) Cancer Cell 3:207-212

Phillips et al (2008) Cancer Res. 68(22):9280-9290

Pingault V., étal (2002) Hum. Genet. 111, 198-206

Pletnev S., et al (2003) Biochemistry 42:12617-12624

Preud'homme et al (1992) Clin. Exp. Immunol. 90(1):141-146

Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126-4131

Proc. Natl. Acad. Sci. U.S.A. 93 (1):136-140 (1996)

Proc. Natl. Acad. Sci. U.S.A. 98 (17):9772-9777 (2001)

Proc. Natl. Acad. Sci. U.S.A. 99 (26):16899-16903 (2002)

Proc.Natl. Acad. Sci. U.S.A. 96 (20):11531-11536 (1999)

Protective Groups in Organic Synthesis, Greene and Wuts, 3^rd Edition, 1999, John

Wiley &amp; Sons Inc.

Puffenberger EG., et al Cell 79. 1257-1266, 1994

Rao et al (1997) Breast Cancer Res. and Treatment 45:149-158

Reiter R.E., et al Proc. Natl. Acad. Sci. U.S.A. 95,1735-1740, 1998

Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams &amp;

Wilkins, 2000

Rodrigues et al (1995) Chemistry Biology 2:223

Ross et al (2002) Cancer Res. 62:2546-2553

S. P. Parker, Ed., McGraw-Hil! Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York

Sakaguchi et al (1988) EMBO J. 7(11):3457-3464

Sakamoto A., Yanagisawa M., et ai Biochem. Biophys. Res. Commun. 178, 656-663. 1991

Sanderson et a! (2005) Clin. Cancer Res. 11:843-852

Semba K., et al Proc. Natl. Acad. Sci. U.S.A. 82, 6497-6501, 1985

Servenius et al (1987) J. Biol. Chem. 262:8759-8766

CA 02901941 2015-08-19

WO 2014/159981

PCT/US2014/025564

149

Shamis et al (2004) J. Am. Chem. Soc. 126:1726-1731

Sheikh F., et al (2004) J. Immunol. 172, 2006-2010

Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982)

Sinha S.K., et al (1993) J. Immunol. 150, 5311-5320

Storm et al (1972) J. Amer. Chem. Soc. 94:5815

Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99:16899-16903

Sun et a! (2002) Bioorganic &amp; Medicinal Chemistry Letters 12:2213-2215

Sun et ai (2003) Bioorganic &amp; Medicinal Chemistry 11:1761-1768

Svensson P.J.. et al Hum. Genet. 103,145-148,1998

Swiercz J.M., et a! J. Cell Biol. 165, 869-880, 2004

Syrigos and Epenetos (1999) Anticancer Research 19:605-614

Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)

Tawaragi Y., et al Biochem. Biophys. Res. Commun. 150, 89-96,1988 ten Dijke,P., et al Science 264 (5155):101-104 (1994)

Thompson, J.S., et al Science 293 (5537), 2108-2111 (2001 ) WO 2004/058309

Thurston, et al., Chem. Brit., 26, 767-772 (1990)

Thurston, et al.. Chem. Rev. 1994, 433-465 (1994)

Toki et al (2002) J. Org. Chem. 67:1866-1872

Tonnelle et al (1985) EMBO J. 4(11):2839-2847

Touchman et al (2000) Genome Res. 10:165-173

Trail et al (2003) Cancer Immunol. Immunother. 52:328-337

Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)

Tsutsumi M., et al Gene 228, 43-49, 1999

Uchida et al (1999) Biochem. Biophys. Res. Commun. 266:593-602

Verheij J.B., et al Âm. J. Med. Genet. 108, 223-225,2002

Von Hoegen et al (1990) J. Immunol. 144(12):4870-4877

Webster et al (1994) Semin. Cancer Biol. 5:69-76

Weis J.J., et al J. Exp. Med. 167, 1047-1066,1988

Weis J.J., et al Proc. Natl. Acad. Soi. U.S.A. 83, 5639-5643,1986

Wilson et al (1991) J. Exp. Med, 173:137-146

Wu et al (2005) Nature Biotech. 23(9):1137-1145

Xie et al (2006) Expert. Opin. Biol. Ther. 6(3):281-291

Xu. M.J.. et al (2001 ) Biochem. Biophys. Res. Commun. 280 (3):768-775 WO 2004/016225

Xu, X.Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697 (2001) Yamaguchi, N„ et al Biol. Chem. 269 (2), 805-808 (1994)

Yamamoto T„ et al Nature 319, 230-234. 1986

Yu et al (1992) J. Immunol. 148(2) 633-637