NZ793831A - Steroids and protein-conjugates thereof - Google Patents
Steroids and protein-conjugates thereofInfo
- Publication number
- NZ793831A NZ793831A NZ793831A NZ79383117A NZ793831A NZ 793831 A NZ793831 A NZ 793831A NZ 793831 A NZ793831 A NZ 793831A NZ 79383117 A NZ79383117 A NZ 79383117A NZ 793831 A NZ793831 A NZ 793831A
- Authority
- NZ
- New Zealand
- Prior art keywords
- examples
- alkyl
- formula
- compound
- amino
- Prior art date
Links
- 150000003431 steroids Chemical class 0.000 title abstract description 58
- 150000001875 compounds Chemical class 0.000 abstract description 420
- 108090001123 antibodies Proteins 0.000 abstract description 213
- 102000004965 antibodies Human genes 0.000 abstract description 213
- 239000011230 binding agent Substances 0.000 abstract description 72
- 102000004169 proteins and genes Human genes 0.000 abstract description 13
- 108090000623 proteins and genes Proteins 0.000 abstract description 13
- 239000003862 glucocorticoid Substances 0.000 abstract description 12
- 229940065521 Glucocorticoid inhalants for obstructive airway disease Drugs 0.000 abstract description 7
- 229940037128 Systemic Glucocorticoids Drugs 0.000 abstract description 7
- 125000000217 alkyl group Chemical group 0.000 description 217
- -1 small molecule steroids Chemical class 0.000 description 165
- 125000005647 linker group Chemical group 0.000 description 155
- 239000000203 mixture Substances 0.000 description 145
- 229910003827 NRaRb Inorganic materials 0.000 description 135
- 125000005843 halogen group Chemical group 0.000 description 114
- 239000000243 solution Substances 0.000 description 108
- 125000003118 aryl group Chemical group 0.000 description 90
- XEKOWRVHYACXOJ-UHFFFAOYSA-N acetic acid ethyl ester Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 87
- 125000000592 heterocycloalkyl group Chemical group 0.000 description 87
- 239000007787 solid Substances 0.000 description 87
- 238000005160 1H NMR spectroscopy Methods 0.000 description 83
- 229920000858 Cyclodextrin Polymers 0.000 description 76
- 238000006243 chemical reaction Methods 0.000 description 74
- 125000003710 aryl alkyl group Chemical group 0.000 description 71
- 239000000562 conjugate Substances 0.000 description 71
- 229910052705 radium Inorganic materials 0.000 description 70
- 229910052701 rubidium Inorganic materials 0.000 description 69
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 68
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene dichloride Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 65
- 239000000427 antigen Substances 0.000 description 60
- 108091007172 antigens Proteins 0.000 description 60
- 102000038129 antigens Human genes 0.000 description 60
- 201000010099 disease Diseases 0.000 description 60
- 238000004128 high performance liquid chromatography Methods 0.000 description 51
- 125000004432 carbon atoms Chemical group C* 0.000 description 47
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 46
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 44
- 238000002953 preparative HPLC Methods 0.000 description 43
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 43
- 239000002253 acid Substances 0.000 description 42
- JNWBBCNCSMBKNE-UHFFFAOYSA-N HATU Chemical compound F[P-](F)(F)(F)(F)F.C1=CN=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 JNWBBCNCSMBKNE-UHFFFAOYSA-N 0.000 description 41
- KFZMGEQAYNKOFK-UHFFFAOYSA-N iso-propanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 41
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 40
- KXDHJXZQYSOELW-UHFFFAOYSA-M carbamate Chemical group NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 40
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 40
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 40
- 239000002202 Polyethylene glycol Substances 0.000 description 38
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 38
- 229920001223 polyethylene glycol Polymers 0.000 description 38
- 150000001345 alkine derivatives Chemical class 0.000 description 37
- 230000014759 maintenance of location Effects 0.000 description 37
- 238000000034 method Methods 0.000 description 37
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 37
- 150000001412 amines Chemical class 0.000 description 36
- 239000011541 reaction mixture Substances 0.000 description 36
- 235000001014 amino acid Nutrition 0.000 description 33
- 239000011780 sodium chloride Substances 0.000 description 33
- 125000004404 heteroalkyl group Chemical group 0.000 description 31
- CSCPPACGZOOCGX-UHFFFAOYSA-N acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 30
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 30
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 30
- 239000004472 Lysine Substances 0.000 description 28
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 28
- 125000004429 atoms Chemical group 0.000 description 27
- 210000004027 cells Anatomy 0.000 description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 27
- 150000003839 salts Chemical class 0.000 description 27
- 125000004104 aryloxy group Chemical group 0.000 description 25
- 235000018977 lysine Nutrition 0.000 description 25
- VOVIALXJUBGFJZ-VXKMTNQYSA-N Budesonide Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1C[C@H]3O[C@@H](CCC)O[C@@]3(C(=O)CO)[C@@]1(C)C[C@@H]2O VOVIALXJUBGFJZ-VXKMTNQYSA-N 0.000 description 24
- 150000001413 amino acids Chemical class 0.000 description 24
- 238000003818 flash chromatography Methods 0.000 description 24
- 230000002194 synthesizing Effects 0.000 description 24
- 125000003275 alpha amino acid group Chemical group 0.000 description 23
- 125000001072 heteroaryl group Chemical group 0.000 description 23
- 101700034322 TGAS Proteins 0.000 description 22
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 22
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 22
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 22
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 22
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 21
- 239000012267 brine Substances 0.000 description 21
- 239000004474 valine Substances 0.000 description 21
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 20
- 229910003813 NRa Inorganic materials 0.000 description 20
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 20
- 210000004602 germ cell Anatomy 0.000 description 19
- 125000003827 glycol group Chemical group 0.000 description 19
- 125000004435 hydrogen atoms Chemical group [H]* 0.000 description 19
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 19
- 125000001400 nonyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 19
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 19
- HEDRZPFGACZZDS-MICDWDOJSA-N deuterated chloroform Substances [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 18
- 150000002148 esters Chemical class 0.000 description 18
- 239000000543 intermediate Substances 0.000 description 18
- PMZURENOXWZQFD-UHFFFAOYSA-L na2so4 Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 18
- 238000000746 purification Methods 0.000 description 18
- 229910052938 sodium sulfate Inorganic materials 0.000 description 18
- 235000011152 sodium sulphate Nutrition 0.000 description 18
- 125000001424 substituent group Chemical group 0.000 description 18
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide Chemical compound [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 description 17
- UKJLNMAFNRKWGR-UHFFFAOYSA-N cyclohexatrienamine Chemical group NC1=CC=C=C[CH]1 UKJLNMAFNRKWGR-UHFFFAOYSA-N 0.000 description 17
- 229910052731 fluorine Inorganic materials 0.000 description 17
- 125000003187 heptyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 17
- 239000000047 product Substances 0.000 description 17
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 17
- 239000003039 volatile agent Substances 0.000 description 17
- 229920002574 CR-39 Polymers 0.000 description 16
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 16
- UREBDLICKHMUKA-CXSFZGCWSA-N Dexamethasone Chemical group C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 16
- 239000000706 filtrate Substances 0.000 description 16
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 16
- 108060005723 speA Proteins 0.000 description 16
- 101710017909 24 Proteins 0.000 description 15
- 125000003545 alkoxy group Chemical group 0.000 description 15
- 230000015572 biosynthetic process Effects 0.000 description 15
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 15
- 239000003814 drug Substances 0.000 description 15
- 125000001624 naphthyl group Chemical group 0.000 description 15
- 101700047197 pie-1 Proteins 0.000 description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 15
- ZMANZCXQSJIPKH-UHFFFAOYSA-N N,N-Diethylethanamine Substances CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 14
- 101700007087 PID1 Proteins 0.000 description 14
- 150000001408 amides Chemical class 0.000 description 14
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 14
- 230000021615 conjugation Effects 0.000 description 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 14
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 13
- 238000006736 Huisgen cycloaddition reaction Methods 0.000 description 13
- 230000035492 administration Effects 0.000 description 13
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 13
- 125000005842 heteroatoms Chemical group 0.000 description 13
- AFVFQIVMOAPDHO-UHFFFAOYSA-N methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 13
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- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 12
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- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 12
- 229910052739 hydrogen Inorganic materials 0.000 description 12
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 12
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- 239000002904 solvent Substances 0.000 description 12
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- 229960004436 Budesonide Drugs 0.000 description 11
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 11
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- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 11
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- 125000003342 alkenyl group Chemical group 0.000 description 10
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- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 10
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- 125000000524 functional group Chemical group 0.000 description 9
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- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 8
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 8
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- SAIZFIRMLDAPKH-UHFFFAOYSA-N tert-butyl N-[(2-hydroxyphenyl)methyl]carbamate Chemical compound CC(C)(C)OC(=O)NCC1=CC=CC=C1O SAIZFIRMLDAPKH-UHFFFAOYSA-N 0.000 description 1
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Abstract
Described herein protein steroid conjugates whereby a glucocorticoid compound is conjugated to a binding agent which is preferably an antibody. These are useful, for example, for the target-specific delivery of glucocorticoids (GCs) to cells.
Description
Described herein protein steroid conjugates whereby a glucocorticoid compound is conjugated
to a binding agent which is preferably an dy. These are useful, for example, for the targetspecific
delivery of glucocorticoids (GCs) to cells.
NZ 793831
STEROIDS AND PROTEIN-CONJUGATES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of New Zealand patent application ,
which is the national phase entry in New Zealand of PCT international ation
(published as
application claims priority to and the benefit of U.S. Provisional Patent Application No.
62/508,317 filed on May 18, 2017, and also U.S. Provisional Patent Application No.
62/419,365, filed on November 8, 2016, the entire contents of each of which are herein
incorporated in their entirety for all purposes.
FIELD
Provided herein are novel steroids, protein conjugates thereof, and methods for
treating diseases, disorders, and conditions comprising administering the steroids and
ates.
dy-drug conjugates (ADCs) are dies that are covalently linked to
biologically active small molecule drugs, thus combining the targeting specificity of antibodies
with the mode-of-action and potency of small molecule drugs. The therapeutic utility of
ADC(s) has been validated in cancer treatment and is a major ongoing focus of study.
ADCETRIS® (bentruximab vedotin) and KADCYLA® (ado-trastuzumab emtansine) are ADCs
ed for the treatment of n cancer types, and at least forty ADCs are currently in
clinical development.
Glucocorticoids (GCs) are small molecule steroids that bind to glucocorticoid
receptors (GRs) and are utilized in anti-inflammatory and immunosuppressive therapies.
However, due to the ubiquitous expression of glucocorticoid receptors in many cell types,
glucocorticoid treatments are compromised by toxicities to most organ systems. Thus, there is
need for both novel glucocorticoids as well as novel therapies that minimize the side effects
arising from glucocorticoid stration, particularly those arising from activating
orticoid receptors in non-target cells. The instant disclosure provides solutions to the
entioned needs as well as other unmet needs in the field to which the instant disclosure
pertains. Included in the instant disclosure are antibody-drug conjugates comprising
orticoid payloads.
SUMMARY
Provided herein are compounds and methods useful for the treatment of various
diseases, disorders, or conditions. In certain aspects, the compounds have the structure of
Formula (A):
or a pharmaceutically able salt, solvate, stereoisomer, or derivative thereof,
wherein:
R1 and R2 are, independently, –H, alkyl, alkyl-C(O)-O–, –OH, or halo; or R1 and
R2 together form ,
wherein R4 is alkyl, aryl, arylalkyl, or an N-containing heterocycloalkyl,
wherein the alkyl, aryl, arylalkyl, and aining heterocycloalkyl are,
independently in each instance, optionally substituted with -NRaRb;
R3 is –OH, RZ-C(O)-X–, heteroalkyl, piperidinyl, –NRaRb, yl–NRaRb
or P)t;
R5 is, independently in each instance, –OH, halo, alkyl, or arylalkyl;
RZ is alkyl;
X is O or NRa;
Z is S, S(O), S(O)2, SO2NRa, O, C(O)NRa, C(O), or NRa;
A is aryl, arylalkyl, or heteroaryl;
RP is, ndently in each instance, halo, optionally substituted alkyl, –OH,
or -NRaRb;
Ra and Rb are, independently in each instance, –H, optionally substituted alkyl, or
optionally subtitued aryl;
n is an integer from 0-19; and
t is an r from 1-3;
with the o that:
(1) R3 is not –OH (a) when R1 is –OH or (b) when R1 and R2 er form ,
wherein R4 is C1-9alkyl or and
(2) R3 is not .
In n aspects, the compounds are protein-drug conjugates, e.g., antibodydrug
conjugates, comprising an antigen-binding protein, e.g., antibody and a nd of
Formula (A).
In certain aspects, the compounds are protein-drug conjugates, e.g., antibodydrug
conjugates, comprising an antigen-binding protein, e.g., antibody, a compound of
Formula (A), and a cyclodextrin moiety.
BRIEF DESCRIPTIONS OF THE DRAWINGS
shows a sequence for synthesizing the certain steroids described .
shows a sequence for modifying the primary alcohol position of
nide.
shows a sequence for modifying the primary alcohol position of
Flumethasone.
shows a sequence for modifying the primary alcohol position of
dexamethasone.
shows a two–dimensional nuclear Overhauser effect (NOE) magnetic
resonance spectrum (hereinafter “2D–NOESY”) for compound 7-1R in Table 1.
shows a SY for compound 7-1S in Table 1.
shows a 2D–NOESY spectrum for 11-5R in Table 1.
shows a 2D–NOESY spectrum for nd 11-5S in Table 1.
shows a general approaches for synthesizing certain Linker-Payloads.
shows a sequence for sizing DIBAC–Suc–NHS (Compound
(V)).
shows a sequence for synthesizing DIBAC–Suc–PEG4–acid/NHS
(Compound (VI)).
shows a sequence for synthesizing BCN–PEG4–Acid und
(VII)).
shows a sequence for synthesizing DIBAC–Suc–PEG4–VC–pAB–PNP
(Compound (VIII)).
shows a sequence for synthesizing Linker–Payload 1 (LP1).
shows a sequence for synthesizing Linker–Payload 2 (LP2) and
Linker–Payload 3 (LP3).
shows a sequence for synthesizing Linker–Payloads 4-11 (LP4-LP11).
shows a sequence for sizing Linker–Payload 12 (LP12).
shows a synthesis sequence for making Linker–Payload 12 (LP13) and
Linker–Payload 14 (LP14).
shows a general synthetic process for an ADC conjugation via a [2+3]
click reaction with LP4.
shows a Coomassie-stained SDS–PAGE Gel of an anti–PRLR
antibody, azido–functionalized anti–PRLR antibody, and anti–PRLR antibody–LP4 conjugate
as described in Example 59
shows size ion chromatography (SEC) of an anti–PRLR
antibody, azido–functionalized antibody, and 4DAR anti–PRLR–LP4 Conjugate as described
in Example 59.
shows an ESI-MS of anti–PRLR dy, azido–functionalized anti–
PRLR antibody and anti–PRLR antibody–LP4 ate as described in Example 59.
shows selective GR activation in 293/PRLR/GRE–Luc cells (A)
and 293/MMTV–Luc cells (B) by steroid ADCs and budesonide control as described in
Example 64.
shows the linker–payload contribution to GR activation by steroid ADC
and budesonide control as tested in 293/PRLR/GRE–Luc cells as described in Example 65.
shows activation of glucocorticoid receptor in a HEK293/MMTV–
luc/IL–2Rγ/IL7R cell line by Budesonide, 11-5 in Table 1, and anti–IL2Rγ ncADC at twentyfour
(24), forty-eight (48), or seventy-two (72) hours as described in Example 66.
shows a sequence for synthesizing Linker–Payload (LP7).
shows a synthetic process for preparing compound (27b).
shows a sequence for synthesizing –Payloads (LP15 and LP16).
shows a general synthetic process for an ADC ation via a [2+3]
click reaction with Cyclodextrin-Linker-Payloads.
shows bioactivity of steroid ADCs with and without cyclodextrin
linkers in a plot of relative light units (RLU) vs. Log10 [M].
shows a sequence for synthesizing certain steroids (payloads 1-6)
described herein.
shows a sequence for sizing certain linker-steroids (LP101 to
shows a general tic process for an ADC ation via [2+3]
click reaction.
shows ESI–MS of anti-PRLR Ab, anti–PRLR Ab–N3, and anti-PRLRLPs.
shows ESI–MS of anti-Fel d1 Ab, anti– Fel d1 Ab–PEG3–N3, and anti-
Fel d1 Ab-LPs.
shows bioactivity of steroid ADCs in antigen positive cells
RLR_PBind GR/UAS-Luc cells, A) vs in n negative cells (293_PBind
GR/UAS-Luc cells, B) in a plot of relative light units (RLU) vs. Log10 [M].
A shows mean blood concentration-time provides for compounds 4b
and 6-I.
B shows TNF-α level in blood samples of payloads 4b and 6-I as
described in Examples 120-121.
DETAILED DESCRIPTION
A. DEFINITIONS
As used herein, “alkyl” refers to a monovalent and saturated hydrocarbon
radical moiety. Alkyl is optionally tuted and can be linear, branched, or cyclic, i.e.,
cycloalkyl. Alkyl includes, but is not limited to, those having 1–20 carbon atoms, i.e., C1–20
alkyl; 1–12 carbon atoms, i.e., C1–12 alkyl; 1–8 carbon atoms, i.e., C1–8 alkyl; 1–6 carbon
atoms, i.e., C1–6 alkyl; and 1–3 carbon atoms, i.e., C1–3 alkyl. es of alkyl moieties
include, but are not d to , ethyl, n–propyl, i–propyl, l, s–butyl, t–butyl, i–
butyl, a pentyl moiety, a hexyl moiety, cyclopropyl, cyclobutyl, cyclopentyl, and exyl.
“Alkylene” is divalent alkyl.
As used herein, “haloalkyl” refers to alkyl, as defined above, wherein the alkyl
includes at least one substituent selected from a halogen, e.g., F, Cl, Br, or I.
As used herein, “alkenyl” refers to a monovalent hydrocarbon radical moiety
containing at least two carbon atoms and one or more non–aromatic carbon–carbon double
bonds. Alkenyl is optionally substituted and can be linear, ed, or cyclic. Alkenyl
includes, but is not limited to, those having 2–20 carbon atoms, i.e., C2–20 alkenyl; 2–12 carbon
atoms, i.e., C2–12 alkenyl; 2–8 carbon atoms, i.e., C2–8 l; 2–6 carbon atoms, i.e., C2–6
alkenyl; and 2–4 carbon atoms, i.e., C2–4 alkenyl. es of alkenyl moieties include, but
are not limited to vinyl, yl, butenyl, and cyclohexenyl. “Alkenylene” is divalent alkenyl.
As used herein, “alkynyl” refers to a lent hydrocarbon radical moiety
containing at least two carbon atoms and one or more carbon–carbon triple bonds. Alkynyl is
optionally substituted and can be linear, branched, or cyclic. Alkynyl includes, but is not
limited to, those having 2–20 carbon atoms, i.e., C2–20 alkynyl; 2–12 carbon atoms, i.e., C2–12
alkynyl; 2–8 carbon atoms, i.e., C2–8 alkynyl; 2–6 carbon atoms, i.e., C2–6 alkynyl; and 2–4
carbon atoms, i.e., C2–4 alkynyl. Examples of alkynyl moieties include, but are not limited to
ethynyl, propynyl, and butynyl. “Alkynylene” is divalent alkynyl.
As used herein, “alkoxy” refers to a lent and saturated hydrocarbon
radical moiety wherein the hydrocarbon includes a single bond to an oxygen atom and wherein
the radical is localized on the oxygen atom,e.g, CH3CH2–O· for ethoxy. Alkoxy substituents
bond to the nd which they substitute through this oxygen atom of the alkoxy
substituent. Alkoxy is ally substituted and can be linear, branched, or cyclic, i.e.,
cycloalkoxy. Alkoxy includes, but is not limited to, those having 1–20 carbon atoms, i.e., C1–
alkoxy; 1–12 carbon atoms, i.e., C1–12 alkoxy; 1–8 carbon atoms, i.e., C1–8 alkoxy; 1–6
carbon atoms, i.e., C1–6 alkoxy; and 1–3 carbon atoms, i.e., C1–3 alkoxy. Examples of alkoxy
moieties include, but are not limited to methoxy, ethoxy, n–propoxy, i–propoxy, n–butoxy, s–
butoxy, t–butoxy, i–butoxy, a pentoxy moiety, a hexoxy moiety, cyclopropoxy, cyclobutoxy,
cyclopentoxy, and cyclohexoxy.
As used herein, “haloalkoxy” refers to alkoxy, as defined above, wherein the
alkoxy includes at least one substituent ed from a halogen, e.g., F, Cl, Br, or I.
As used herein, “aryl” refers to a monovalent moiety that is a radical of an
aromatic compound wherein the ring atoms are carbon atoms. Aryl is optionally substituted
and can be monocyclic or polycyclic, e.g., bicyclic or tricyclic. Examples of aryl moieties
include, but are not limited to those having 6 to 20 ring carbon atoms, i.e., C6–20 aryl; 6 to 15
ring carbon atoms, i.e., C6–15 aryl, and 6 to 10 ring carbon atoms, i.e., C6–10 aryl. Examples of
aryl moieties include, but are limited to phenyl, naphthyl, fluorenyl, yl, anthryl,
phenanthryl, and pyrenyl.
As used herein, lkyl” refers to an lent moiety that is a radical of
an alkyl compound, wherein the alkyl compound is substituted with an aromatic substituent,
i.e., the aromatic compound includes a single bond to an alkyl group and n the l is
localized on the alkyl group. An arylalkyl group bonds to the rated chemical structure via
the alkyl group. An arylalkyl can be represented by the structure, e.g.,
wherein B is an aromatic moiety, e.g., phenyl. Arylalkyl is ally substituted, i.e., the aryl
group and/or the alkyl group, can be substituted as disclosed . Examples of arylalkyl
include, but are not limited to, benzyl.
As used herein, “aryloxy” refers to a monovalent moiety that is a l of an
aromatic compound wherein the ring atoms are carbon atoms and wherein the ring is
substituted with an oxygen radical, i.e., the aromatic compound includes a single bond to an
oxygen atom and wherein the radical is zed on the oxygen atom¸ e.g.¸ for
y. Aryloxy substituents bond to the compound which they substitute through this
oxygen atom. Aryloxy is optionally substituted. y includes, but is not limited to those
having 6 to 20 ring carbon atoms, i.e., C6–20 aryloxy; 6 to 15 ring carbon atoms, i.e., C6–15
aryloxy, and 6 to 10 ring carbon atoms, i.e., C6–10 aryloxy. Examples of aryloxy es
include, but are not limited to phenoxy, naphthoxy, and anthroxy.
As used herein, “RaRbN–aryloxy” refers to a monovalent moiety that is a radical
of an aromatic compound wherein the ring atoms are carbon atoms and n the ring is
tuted with an RaRbN– substituent and an oxygen radical, i.e., the aromatic compound
includes a single bond to an RaRbN– substituent and a single bond to an oxygen atom and
wherein the radical is localized on the oxygen atom¸ e.g.¸ . RaRbN–aryloxy
substituents bond to the compound which they substitute through this oxygen atom. RaRbN–
aryloxy is ally substituted. RaRbN–aryloxy includes, but is not limited to those having
6 to 20 ring carbon atoms, 6 to 15 ring carbon atoms; and 6 to 10 ring carbon atoms. An
example of an RaRbN–aryloxy moiety includes, but is not limited to 4–(dimethyl–amino)–
phenoxy, .
As used herein, “arylene” refers to a divalent moiety of an aromatic compound
wherein the ring atoms are only carbon atoms. Arylene is optionally substituted and can be
monocyclic or polycyclic, e.g., bicyclic or tricyclic. Examples of arylene moieties include, but
are not limited to those having 6 to 20 ring carbon atoms, i.e., C6–20 arylene; 6 to 15 ring
carbon atoms, i.e., C6–15 arylene, and 6 to 10 ring carbon atoms, i.e., C6–10 arylene.
As used herein, “heteroalkyl” refers to an alkyl in which one or more carbon
atoms are replaced by heteroatoms. As used herein, “heteroalkenyl” refers to an alkenyl in
which one or more carbon atoms are replaced by heteroatoms. As used herein, oalkynyl”
refers to an alkynyl in which one or more carbon atoms are replaced by heteroatoms. Suitable
heteroatoms include, but are not limited to, nitrogen, oxygen, and sulfur atoms. Heteroalkyl is
ally substituted. Examples of heteroalkyl es include, but are not limited to,
aminoalkyl, sulfonylalkyl, sulfinylalkyl. Examples of heteroalkyl moieties also include, but
are not limited to, methylamino, methylsulfonyl, and methylsulfinyl.
As used herein, “heteroaryl” refers to a monovalent moiety that is a radical of
an aromatic nd wherein the ring atoms contain carbon atoms and at least one oxygen,
sulfur, nitrogen, or phosphorus atom. es of heteroaryl moieties e, but are not
limited to those having 5 to 20 ring atoms; 5 to 15 ring atoms; and 5 to 10 ring atoms.
Heteroaryl is ally substituted.
As used herein, “heteroarylene” refers to an arylene in which one or more ring
atoms of the aromatic ring are replaced with an oxygen, sulfur, nitrogen, or phosphorus atom.
Heteroarylene is optionally substituted.
As used , “heterocycloalkyl” refers to a cycloalkyl in which one or more
carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited
to, nitrogen, oxygen, and sulfur atoms. Heterocycloalkyl is optionally tuted. es
of heterocycloalkyl moieties include, but are not limited to, morpholinyl, piperidinyl,
tetrahydropyranyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, dioxolanyl,
lanyl, oxanyl, or thianyl.
As used herein, “N–containing heterocycloalkyl,” refers to a cycloalkyl in
which one or more carbon atoms are replaced by atoms and wherein at least one
heteroatom is a nitrogen atom. Suitable heteroatoms in addition to nitrogen, include, but are
not limited to oxygen and sulfur atoms. N–containing heterocycloalkyl is optionally
substituted. Examples of N–containing heterocycloalkyl moieties include, but are not limited
to, morpholinyl, piperidinyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, or lidinyl.
As used herein, “optionally substituted,” when used to describe a l
moiety, e.g., optionally substituted alkyl, means that such moiety is ally bonded to one
or more substituents. es of such substituents include, but are not limited to halo,
cyano, nitro, haloalkyl, azido, epoxy, optionally substituted heteroaryl, optionally substituted
heterocycloalkyl, , , , , , ,
, , , , , , ,
or , wherein RA, RB, and RC are, independently at each occurrence, a hydrogen atom, alkyl,
alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heteroaryl, or heterocycloalkyl, or RA
and RB, together with the atoms to which they are bonded, form a saturated or rated
carbocyclic ring, wherein the ring is optionally substituted and wherein one or more ring atoms
is optionally replaced with a heteroatom. In certain embodiments, when a radical moiety is
optionally substituted with an optionally substituted heteroaryl, optionally substituted
heterocycloalkyl, or optionally substituted saturated or unsaturated carbocyclic ring, the
substituents on the optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or
optionally substituted saturated or unsaturated carbocyclic ring, if they are substituted, are not
substituted with substituents which are further optionally substituted with additional
tuents. In some embodiments, when a group described herein is optionally substituted,
the substituent bonded to the group is tituted unless otherwise ied.
As used herein, ng agent” refers to any molecule capable of binding with
specificity to a given binding partner. In some embodiments, the binding agent is an antibody,
or an antigen g fragment thereof.
As used herein, “linker” refers to a nt moiety that covalently links the
binding agent to the steroid described herein.
As used herein, “amide synthesis conditions” refers to reaction conditions
le facilitate the formation of an amide, e.g., by the reaction of a carboxylic acid, activated
carboxylic acid, or acyl halide with an amine. In some examples, "amide synthesis conditions"
refers to reaction conditions suitable to facilitate the formation of an amide bond between a
carboxylic acid and an amine. In some of these examples, the carboxylic acid is first converted
to an activated carboxylic acid before the activated ylic acid reacts with an amine to
form an amide. Suitable conditions to effect the formation of an amide e, but are not
limited to, those utilizing reagents to effect the reaction n a carboxylic acid an amine,
including, but not d to, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide
(DIC), (benzotriazol–1–yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP),
(benzotriazol–1–yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7–
azabenzotriazol–1–yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP),
ripyrrolidinophosphonium hexafluorophosphate (PyBrOP), O–(benzotriazol–1–yl)–
N,N,N’,N’–tetramethyluronium hexafluorophosphate (HBTU), O–(benzotriazol–1–yl)–
N,N,N’,N’–tetramethyluronium tetrafluoroborate (TBTU), 1–
[Bis(dimethylamino)methylene]1H–1,2,3–triazolo[4,5–b]pyridinium 3–oxid
hexafluorophosphate (HATU), xy–1–ethoxycarbonyl–1,2–dihydroquinoline (EEDQ),
1–Ethyl–3–(3–dimethylaminopropyl)carbodiimide (EDC), 2–Chloro–1,3–
dimethylimidazolidinium hexafluorophosphate (CIP), 2–chloro–4,6–dimethoxy–1,3,5–triazine
(CDMT), and carbonyldiimidazole (CDI). In some examples, a carboxylic acid is first
converted to an activated carboxylic ester before ng with an amine to form an amide
bond. In certain embodiments, the carboxylic acid is reacted with a reagent. The t
activates the carboxylic acid by deprotonating the carboxylic acid and then forming a product
complex with the deprotonated carboxylic acid as a result of nucleophilic attack by the
deprotonated carboxylic acid onto the protonated t. For certain carboxylic acids, this
ted ester is more susceptible subsequently to nucleophilic attack by an amine than the
carboxylic acid is before it is converted. This results in amide bond formation. As such, the
carboxylic acid is described as activated. ary reagents include DCC and DIC.
As used herein, peutically effective amount” refers to an amount (of a
compound) that is sufficient to provide a therapeutic benefit to a patient in the treatment or
management of a disease or disorder, or to delay or minimize one or more symptoms
associated with the disease or disorder.
As used herein, “pharmaceutically able derivative” refers to any form,
e.g., ester or prodrug of a compound, which provides said compound upon administration to a
patient.
As used herein, “pharmaceutically acceptable salt” refers to any salt suitable for
administration to a patient. Suitable salts include, but are not limited to, those disclosed in.
Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977, 66:1, incorporated herein by
reference. Examples of salts include, but are not limited to, acid–derived, base–derived,
organic, inorganic, amine, and alkali or alkaline earth metal salts, including but not limited to
calcium salts, ium salts, potassium salts, sodium salts, salts of hydrochloric acid,
hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid,
glycolic acid, pyruvic acid, oxalic acid, maleic acid, c acid, succinic acid, fumaric acid,
ic acid, citric acid, c acid, cinnamic acid, mandelic acid, methanesulfonic acid,
ethanesulfonic acid, enesulfonic acid, and salicylic acid, and the like.
Certain groups, moieties, substituents, and atoms are depicted with a wiggly
line that intersects or caps a bond or bonds to indicate the atom through which the groups,
moieties, tuents, atoms are bonded. For example, a phenyl group that is substituted with
a propyl group depicted as:
has the following structure:
. As used herein, illustrations showing substituents bonded to a cyclic group
(e.g., aromatic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or
heterocycloalkyl) through a bond n ring atoms are meant to indicate, unless ied
otherwise, that the cyclic group may be substituted with that substituent at any ring position in
the cyclic group or on any ring in the fused ring group, according to techniques set forth herein
or which are known in the field to which the instant disclosure pertains. For example, the
group, , wherein subscript q is an integer from 0 to 4 and in which the positions of
substituent R1 are described generically, i.e., not directly ed to any vertex of the bond
line structure, i.e., specific ring carbon atom, includes the following, non–limiting examples of,
groups in which the tuent R1 is bonded to a specific ring carbon atom: ,
, , , , , ,
, , , , , , ,
, , , , , ,
, , , , , , ,
, , and . Also, for example, the group,
, wherein subscript n is an integer from 0 to 19 and in which the positions
of substituent R5 are described generically, i.e., depicted as not ly attached to any vertex
of the bond line structure, includes the following, non–limiting examples of, groups in which
the substituent R5 is bonded to a specific ring carbon atom: ,
, , , ,
, , , ,
, , , ,
, , , ,
R5 5
R5 R R5
, , , ,
, or .
As used herein, the phrase “reactive ,” or the iation “RL” refers to a
monovalent group that comprises a reactive group and linking group, ed as ,
wherein RG is the reactive group and L is the linking group. The linking group is any divalent
moiety that bridges the reactive group to a d. The reactive linkers (RL), together with
the payloads to which they are bonded, comprise intermediates (“linker–payloads”) useful as
synthetic precursors for the preparation of the antibody steroid conjugates described herein.
The reactive linker contains a ve group (“RG”), which is a functional group or moiety
that reacts with a reactive portion of an dy, modified antibody, or antigen binding
nt thereof. The moiety resulting from the reaction of the ve group with the
antibody, modified antibody, or antigen binding fragment f, together with the linking
group, comprise the “binding agent linker” (“BL”) portion of the conjugate, described herein.
In certain embodiments, the “reactive group” is a functional group or moiety (e.g., maleimide
or NHS ester) that reacts with a cysteine or lysine residue of an antibody or antigen–binding
fragment thereof. In certain embodiments, the “reactive group” is a functional group or moiety
that is capable of undergoing a click chemistry reaction. In some embodiments of said click
chemistry reaction, the reactive group is an alkyne that is capable of undergoing a 1,3
cycloaddition reaction with an azide. Such suitable reactive groups include, but are not limited
to, strained alkynes, e.g., those suitable for strain–promoted alkyne–azide cycloadditions
(SPAAC), cycloalkynes, e.g., cyclooctynes, benzannulated alkynes, and alkynes capable of
undergoing 1,3 cycloaddition reactions with azides in the absence of copper catalysts. le
alkynes also include, but are not limited to, DIBAC, DIBO, BARAC, DIFO, substituted, e.g.,
fluorinated s, aza–cycloalkynes, BCN, and derivatives thereof. Linker–payloads
comprising such reactive groups are useful for conjugating antibodies that have been
functionalized with azido groups. Such functionalized antibodies include antibodies
functionalized with azido–polyethylene glycol groups. In certain ments, such
onalized antibody is derived by ng an antibody comprising at least one glutamine
residue, e.g., heavy chain Q295 (EU numbering), with a compound according to the formula
H2N–LL–N3, wherein LL is a divalent polyethylene glycol group, in the presence of the
enzyme transglutaminase.
In some examples, the reactive group is an alkyne, e.g., , which can
react via click chemistry with an azide, e.g., , to form a click chemistry product,
e.g., , its regioisomer, or mixture thereof. In some examples, the reactive group
is an , e.g., or , which can react via click chemistry with an
azide, e.g., to form a click chemistry product, e.g., . In some
examples, the reactive group is an alkyne, e.g., , which can react via click chemistry
with an azide, e.g., , to form a click chemistry product, e.g., , its
regioisomer, or mixture thereof. In some examples, the reactive group is a functional group,
e.g., ,which reacts with a cysteine e on an antibody or antigen–binding fragment
thereof, to form a bond thereto, e.g., , wherein Ab refers to an antibody or antigen–
binding fragment f and S refers to the S atom on a cysteine residue h which the
functional group bonds to the Ab. In some es, the reactive group is a functional group,
e.g., ,which reacts with a lysine residue on an antibody or antigen–binding
fragment thereof, to form a bond thereto, e.g., , wherein Ab refers to an antibody or
antigen–binding fragment thereof and N refers to the N atom on a lysine residue through which
the functional group bonds to the Ab.
As used herein, the phrase “binding agent linker,” or “BL” refers to any divalent
group or moiety that links, connects, or bonds a binding agent (e.g., an antibody or an antigen–
binding fragment f) with a payload compound set forth herein (e.g., steroid). lly,
suitable binding agent s for the antibody conjugates described herein are those that are
sufficiently stable to exploit the circulating half–life of the antibody and, at the same time,
capable of releasing its payload after antigen–mediated internalization of the conjugate.
Linkers can be cleavable or non–cleavable. Cleavable linkers are linkers that are cleaved by
intracellular metabolism following internalization, e.g., cleavage via hydrolysis, ion, or
enzymatic on. Non–cleavable linkers are linkers that e an attached payload via
lysosomal ation of the antibody following internalization. Suitable linkers include, but
are not limited to, acid–labile linkers, hydrolysis–labile linkers, enzymatically cleavable
linkers, reduction labile linkers, self–immolative linkers, and non–cleavable linkers. Suitable
linkers also include, but are not limited to, those that are or comprise glucuronides,
succinimide–thioethers, polyethylene glycol (PEG) units, hydrazones, proyl units,
disulfide units (e.g., –S–S–, –S–C(R1R2) –, wherein R1 and R2 are independently hydrogen or
hydrocarbyl), carbamate units, para-amino-benzyl units (PAB), phosphate units, e.g., mono-,
bis-, or tris- ate units, and peptide units, e.g., peptide units containing two, three four,
five, six, seven, eight, or more amino acids, including but not limited to valine–citrulline and
units. In some embodiments, the binding agent linker (BL) comprises a moiety that is formed
by the reaction of the reactive group (RG) of a reactive linker (RL) and reactive portion of the
g agent, e.g., antibody, modified antibody, or antigen binding fragment thereof.
In some examples, the BL comprises the ing moiety: ,
its somer, or mixture thereof, wherein is the bond to the binding agent. In some
examples, the BL comprises the following moiety: , its somer, or
mixture thereof, wherein is the bond to the binding agent. In some examples, the BL
ses the ing moiety: 1 , its regioisomer, or mixture thereof,
wherein is the bond to the binding agent. In some examples, the BL comprises the following
moiety: , its regioisomer, or mixture thereof, wherein is the bond to the binding
agent. In some examples, the BL ses the following moiety: , wherein is the
bond to the cysteine of the antibody or antigen–binding fragment thereof. In some examples,
the BL comprises the following moiety: , wherein is the bond to the lysine of the
antibody or antigen–binding fragment f. In these examples, the bond to the binding
agent is direct or via a linker. In particular embodiments, the binding agent is modified with an
azide to facilitate linkage to BL. Examples are described below.
B. STEROIDS
Provided herein are compounds having the structure of Formula (A):
or a pharmaceutically acceptable salt, solvate, stereoisomer, or derivative thereof,
R1 and R2 are, independently, –H, alkyl, alkylene-C(O)-O–, –OH, or halo; or R1 and
R2 together form ,
wherein R4 is alkyl, aryl, arylalkyl, or an N-containing heterocycloalkyl,
wherein the alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl are,
independently in each instance, optionally substituted with
–NRaRb;
R3 is –OH, RZ-C(O)-X–, heteroalkyl, piperidinyl, –NRaRb, –oxyaryl-NRaRb,
or P)t;
R5 is, ndently in each instance, –OH, halo, alkyl, or arylalkyl;
RZ is alkyl;
X is O or NRa;
Z is S, S(O), S(O)2, SO2NRa, O, C(O)NRa, C(O), or NRa;
A is aryl or heteroaryl;
RP is, independently in each instance, halo, ally substituted alkyl, –OH,
or -NRaRb;
Ra and Rb are, ndently in each instance, –H or optionally substituted alkyl;
n is an integer from 0-19; and
t is an integer from 1-3;
with the proviso that (1) R3 is not –OH (a) when R1 is –OH or (b) when R1 and R2
together form , wherein R4 is C1-9alkyl or and (2) R3 is not
In some embodiments, the compound of Formula (A) has the structure of
Formula (A1):
wherein R1-R3 are as defined above and R5A and R5B are each, independently, halo or a
hydrogen atom.
In some ments of the compound of Formula (A1), R5A and R5B are
hydrogen atoms. In some embodiments of the compound of Formula (A1), R5A and R5B are
fluoro. In some embodiments of the compound of Formula (A1), R5A is a hydrogen atom and
R5B is fluoro.
In some embodiments of the compound of Formula (A1), R1 is alkylene-C(O)-
O– or –OH and R2 is alkyl.
In some embodiments of the compound of Formula (A1), R1 and R2 together
form , wherein R4 is aryl, arylalkyl, or alkyl, wherein the aryl, arylalkyl, and alkyl are
optionally substituted with –NRaRb. In some embodiments, R4 is –aryl- NRaRb. In some
embodiments, R4 is –phenyl- NRaRb.
In some embodiments of the compound of Formula (A1), R1 and R2 together
form , wherein R4 is , , , or
In some ments of the compound of Formula (A1), R3 is –OH, ,
)-X–, or , wherein RP is halo, t is an integer from 0 to 2, Ra is H,
Rb is H or alkyl, X is O or NH, and RZ is alkyl.
In some embodiments of the compound of Formula (A1), R3 is –OH, –NH2,
–NHCH3, )2, , , ,
, , , ,
, , , or .
In some embodiments of the compound of Formula (A1), R1 and R2 together
form , wherein R4 is aryl, arylalkyl, or alkyl, wherein the aryl, arylalkyl, and alkyl are
optionally tuted with –NRaRb; R3 is –OH, –NRaRb, RZ-C(O)-X–, or
, n RP is halo, t is an integer from 0 to 2, Ra is H, Rb is H or alkyl,
X is O or NH, and RZ is alkyl; and R5, independently at each occurrence, is fluoro or a
hydrogen atom.
Set forth are also compounds of Formula (A2):
wherein n is an integer from 0 to 4 and R3 is –OH or RZ-C(O)-O–; wherein RZ is alkyl. In
certain embodiments, n is 0 or 1.
Set forth are also compounds of Formula (A3):
wherein n is an r from 1-4 and R3 is –OH or RZ-C(O)-O–; wherein RZ is alkyl. In certain
embodiments, n is 2.
Set forth are also compounds of Formula (A4):
wherein R3 is –NRaRb and R4 is alkyl, wherein Ra and Rb are each, independently, a hydrogen
atom or alkyl, or Ra and Rb, taken together form a 3-7 membered ring. In certain
embodiments, R4 is C1-4 alkyl. In some embodiments, R4 is propyl. In certain embodiments,
R3 is –NH2,
–NHCH3, or –N(CH3)2.
Set forth are also compounds of Formula (A5):
wherein R4 is alkyl, RP1 is halo or a hydrogen atom, and RP2 is –NRaRb or –OH, wherein Ra
and Rb are each, independently, a hydrogen atom or alkyl. In some embodiments, R4 is C1-4
alkyl and RP2 is –NH2.
Set forth are also compounds of Formula (A6):
wherein R3 is , RZC(O)X–, , or NRaRb, wherein
X is O or NRa, is aryl or heteroaryl, RP is halo, t is an integer from 0-2, Ra and Rb are each,
independently, a hydrogen atom or alkyl, RZ is alkyl, and RQ is alkoxy, and R4 is alkyl. In
some embodiments, R3 is .
Set forth herein are also compounds of Formula (A7)
wherein R3 is in X is O or NRa, is aryl or aryl,
RP is halo, t is an integer from 0-2, Ra and Rb are each, independently, a hydrogen atom or
alkyl, R5A is a hydrogen atom or fluoro, and R5B is . In some ments, R3 is
In some examples, set forth herein is a compound having the structure of
Formula (I):
or a pharmaceutically acceptable salt, solvate, stereoisomer, or derivative thereof,
wherein:
R1 and R2 are, independently, –H, alkyl, alkyl–C(O)–O–, –OH, or halo; or R1 and R2
together form ,
n R4 is alkyl, aryl, arylalkyl, or an N–containing heterocycloalkyl,
wherein the alkyl, aryl, arylalkyl, and N–containing cycloalkyl
are, independently in each instance, optionally substituted with -NRaRb;
R3 is –OH, alkyl–C(O)–O–, heteroalkyl, –NRaRb, -NRaRb–aryloxy, or
RaRbN–aryloxy–, wherein the alkyl–C(O)–O– , heteroalkyl, –NRaRb, and
RaRbN–aryloxy– are optionally substituted with halo;
R5 is, independently in each instance, –OH, halo, alkyl, or arylalkyl;
Ra and Rb are, independently in each instance, H or alkyl; and
n is an integer from 0–19;
with the proviso that R3 is not –OH when either (a) or (b): (a) R1 is –OH or (b) R1 and
R2 together form and R4 is a kyl or .
In some of these examples, R1 and R2 are, independently, selected from –H,
alkyl, alkyl–C(O)–O–, –OH, and halo. In some other examples, R1 and R2 together form
. In n examples, R1 is –H. In certain other examples, R1 is alkyl. In some
examples, R1 is alkyl–C(O)–O–. In some other examples, R1 is –OH. In certain examples, R1
is halo. In certain other examples, R1 is –F. In some examples, R1 is –Cl. In some other
examples, R1 is –Br. In certain examples, R1 is –I. In certain other examples, R2 is –OH. In
some examples, R2 is halo. In some other examples, R2 is –F. In certain examples, R2 is –Cl.
In certain other examples, R2 is –Br. In some examples, R2 is –I.
In some es, in Formula (I), R5 is –OH. In some examples, R5 is halo
such as but not limited to –F, –Cl, –Br, or –I. In some es, R5 is –F. In some examples,
R5 is –Cl. In some examples, R5 is –Br. In some examples, R5 is –I. In some examples, R5 is
alkyl such as, but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, or
nonyl. In some es, R5 is benzyl.
In some examples, in Formula (I), R3 is selected from –OH, alkyl–C(O)–O–,
and RaRbN–aryloxy. In some of these examples, alkyl–C(O)–O– or RaRbN–aryloxy is
optionally substituted with halo. In some examples, R3 is –OH. In some examples, R3 is
alkyl–C(O)–O–. In some es, R3 is RaRbN–aryloxy. In some es, R3 is
. In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is . In some examples, R3
is .
In some examples of Formula (I), R3 is –OH, alkyl–C(O)–O–, heteroalkyl,
–NRaRb, or RaRbN–aryloxy, wherein alkyl–C(O)–O– , heteroalkyl, –NRaRb, or RaRbN–
aryloxy is optionally tuted with halo. Ra and Rb are, independently in each instance,
–H or alkyl.
In some examples, R3 is RaRbN–aryloxy, wherein Ra and Rb are, independently
in each instance, –H or alkyl.
In some examples, R3 is . In some es, R3 is
. In some examples, R3 is . In some
examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is .
In some examples, R3 is . In some es, R3 is
In some es, R3 is RaRbN–aryloxy, wherein Ra and Rb are, independently
in each instance, –H or alkyl.
In some examples, in Formula (I), R4 is selected from the group consisting of
alkyl, aryl, arylalkyl, and an N–containing heterocycloalkyl. In some of these examples, alkyl,
aryl, arylalkyl, or N–containing heterocycloalkyl are optionally substituted with
–NRaRb. In some examples, R4 is alkyl such as, but not limited to, methyl, ethyl, propyl, butyl,
pentyl, hexyl, heptyl, octyl, or nonyl. In some examples, R4 is methyl. In some es, R4
is ethyl. In some examples, R4 is n–propyl. In some examples, R4 is i–propyl. In some
examples, R4 is n–butyl. In some examples, R4 is i–butyl. In some examples, R4 is t–butyl. In
some examples, R4 is sec–butyl. In some examples, R4 is pentyl. In some examples, R4 is
hexyl. In some examples, R4 is . In some examples, R4 is octyl, or nonyl. In some
examples, R4 is aryl such as but not d to phenyl or naphthyl. In some examples, R4 is
phenyl. In some examples, R4 is naphthyl. In some examples, R4 is arylalkyl–such as but not
limited to . In some examples, R4 is N–containing heterocycloalkyl such as but not
limited to piperidinyl. In some examples, R4 is 4–amino–phenyl. In some examples, R4 is 4–
aminophenyl optionally substituted with halo.
In some examples, R4 is , wherein Ra and Rb are, independently
in each instance, H or alkyl.
In some examples, R4 is .
RbRaN
In some examples, R4 is .
In some examples, R4 is .
In some examples, R4 is . In some es, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some es, R4 is
F . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples,
R4 is . In some examples, R4 is . In some
examples, R4 is . In some examples, R4 is . In
some examples, R4 is . In some examples, R4 is .
In some es, R4 is . In some examples, R4 is
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some
examples, R4 is . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4
is . In some examples, R4 is . In some
examples, R4 is .
In some examples, R4 is alkyl substituted with amino such as, but not d to,
methyl–amino, ethyl–amino, propyl–amino, butyl–amino, pentyl–amino, hexyl–amino, heptyl–
amino, octyl–amino, or amino. In some examples, R4 is methyl–amino. In some
examples, R4 is ethyl–amino. In some es, R4 is n–propyl–amino. In some examples, R4
is i–propyl–amino. In some examples, R4 is n–butyl–amino. In some examples, R4 is i–butyl–
amino. In some examples, R4 is t–butyl–amino. In some examples, R4 is pentyl–amino. In
some examples, R4 is hexyl–amino. In some examples, R4 is heptyl–amino. In some
examples, R4 is octyl–amino. In some es, R4 is nonyl–amino.
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is .
In some examples, , Ra and Rb are, independently in each instance,
selected from H and alkyl. In some examples, both Ra and Rb are H. In some examples, both
Ra and Rb are methyl. In some examples, both Ra and Rb are ethyl. In some examples, both
Ra and Rb are propyl. In some examples, one of Ra or Rb is –H and the other is alkyl. In some
examples, one of Ra or Rb is –H and the other is methyl. In some examples, one of Ra or Rb is
–H and the other is ethyl. In some examples, one of Ra or Rb is –H and the other is propyl.
In some es, n is an r from 0–19. In some examples, n is 0. In
some other examples, n is 1. In certain examples, n is 2. In some other examples, n is 3. In
n examples, n is 4. In some examples, n is 5. In some other examples, n is 6. In certain
examples, n is 7. In some other examples, n is 8. In certain examples, n is 9. In some
es, n is 10. In some other examples, n is 11. In n examples, n is 12. In some
other examples, n is 13. In certain examples, n is 14. In some examples, n is 15. In some
other examples, n is 16. In certain examples, n is 17. In some other examples, n is 18. In
certain es, n is 19.
In some examples, in a (I), R3 is not –OH when R1 is –OH.
In some examples, in Formula (I), R3 is not –OH when R1 and R2 together form
wherein R4 is a C1–9alkyl or 4–(dimethyl–amino)–phenyl.
In some examples, set forth herein is a compound of Formula (I), wherein R1
and R2 together form . In some of these examples, R4 is alkyl, aryl, arylalkyl, or a N–
containing heterocycloalkyl. In certain examples, alkyl, aryl, heteroaryl, arylalkyl, or N–
containing heterocycloalkyl are optionally substituted with –NRaRb. In some of these
examples, R4 is alkyl. In some of these examples, R4 is aryl. In some of these examples, R4 is
kyl. In some of these examples, R4 is N–containing heterocycloalkyl. In some
examples, R4 is alkyl such as, but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl,
heptyl, octyl, or nonyl. In some examples, R4 is methyl. In some examples, R4 is ethyl. In
some examples, R4 is n–propyl. In some examples, R4 is i–propyl. In some examples, R4 is n–
butyl. In some examples, R4 is i–butyl. In some examples, R4 is t–butyl. In some examples,
R4 is tyl. In some examples, R4 is pentyl. In some examples, R4 is hexyl. In some
examples, R4 is heptyl. In some examples, R4 is octyl, or nonyl. In some examples, R4 is aryl
such as but not limited to phenyl or naphthyl. In some examples, R4 is phenyl. In some
examples, R4 is naphthyl. In some examples, R4 is heteroaryl–such as but not limited to
ene or phenol. In some examples, R4 is arylalkyl–such as but not limited to benzyl. In
some examples, R4 is N–containing heterocycloalkyl such as but not d to dinyl. In
some examples, R4 is 4–amino–phenyl. In some examples, R4 is 4–aminophenyl optionally
substituted with halo.
In some examples, set forth herein is a compound of Formula (I), n R1
and R2 together form wherein R4 is selected from the group consisting of alkyl, aryl,
arylalkyl, and a aining heterocycloalkyl; and n alkyl, aryl, arylalkyl, or N–
containing heterocycloalkyl are optionally substituted with –NRaRb; and n the
chemistry of the carbon indicated by * is the R configuration.
In some examples, set forth herein is a compound of Formula (I), wherein R1
and R2 together form wherein R4 is selected from the group consisting of alkyl, aryl,
arylalkyl, and a N–containing heterocycloalkyl; and wherein alkyl, aryl, arylalkyl, or N–
containing heterocycloalkyl are optionally substituted with –NRaRb; and wherein the
stereochemistry of the carbon indicated by * is the S configuration.
In some examples, set forth herein is a compound of Formula (I), wherein the
compound has the structure of Formula (PIa):
(PIa).
In some of these examples, R1 and R2 are, independently, selected from –H, alkyl, alkyl–C(O)–
O–, –OH, and halo. In some other examples, R1 and R2 together form . In certain
examples, R1 is –H. In certain other es, R1 is alkyl. In some examples, R1 is alkyl–
C(O)–O–. In some other examples, R1 is –OH. In certain examples, R1 is halo. In certain
other es, R1 is –F. In some examples, R1 is –Cl. In some other es, R1 is –Br. In
certain examples, R1 is –I. In n other examples, R2 is –OH. In some examples, R2 is
halo. In some other examples, R2 is –F. In certain examples, R2 is –Cl. In certain other
examples, R2 is –Br. In some examples, R2 is– I.
In some examples in Formula (PIa), R5 is –OH. In some examples, R5 is halo
such as but not limited to –F, –Cl, –Br, or –I. In some examples, R5 is –F. In some examples,
R5 is –Cl. In some examples, R5 is –Br. In some examples, R5 is –I. In some examples, R5 is
alkyl such as, but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, or
nonyl.
In some examples, in Formula (PIa), R3 is selected from –OH, alkyl–C(O)–O–,
and RaRbN–aryloxy. In some of these examples, C(O)–O– or RaRbN–aryloxy is
optionally substituted with halo. In some examples, R3 is –OH. In some es, R3 is alkyl–
C(O)–O–. In some examples, R3 is RaRbN–aryloxy–. In some examples, R3 is
. In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is . In some examples, R3
is . In some examples, R3 is RaRbN–aryloxy–. In some examples,
R3 is -NRaRb–aryloxy.
In some es, in Formula (PIa), R4 is selected from the group consisting of
alkyl, aryl, kyl, and an N–containing heterocycloalkyl. In some of these examples, alkyl,
aryl, arylalkyl, or N–containing heterocycloalkyl are optionally substituted with
–NRaRb. In some examples, R4 is alkyl such as, but not limited to, methyl, ethyl, propyl, butyl,
pentyl, hexyl, heptyl, octyl, or nonyl. In some es, R4 is methyl. In some examples, R4
is ethyl. In some examples, R4 is n–propyl. In some examples, R4 is i–propyl. In some
examples, R4 is n–butyl. In some examples, R4 is i–butyl. In some examples, R4 is t–butyl. In
some examples, R4 is . In some examples, R4 is hexyl. In some examples, R4 is heptyl.
In some examples, R4 is octyl, or nonyl. In some examples, R4 is aryl such as but not limited
to phenyl or naphthyl. In some examples, R4 is phenyl. In some examples, R4 is naphthyl. In
some examples, R4 is arylalkyl–such as but not limited to benzyl. In some examples, R4 is N–
ning heterocycloalkyl such as but not limited to piperidinyl. In some examples, R4 is 4–
amino–phenyl. In some examples, R4 is 4–aminophenyl optionally substituted with halo.
In some examples, R4 is . In some es, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some es, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
F . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples,
R4 is . In some examples, R4 is . In some
examples, R4 is . In some es, R4 is . In
some examples, R4 is . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is . In
some examples, R4 is . In some examples, R4 is . In
some examples, R4 is . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some
examples, R4 is . In some es, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is .
In some examples, R4 is alkyl substituted with amino such as, but not limited to,
methyl–amino, ethyl–amino, propyl–amino, butyl–amino, pentyl–amino, hexyl–amino, heptyl–
amino, octyl–amino, or nonyl–amino. In some examples, R4 is methyl–amino. In some
examples, R4 is ethyl–amino. In some examples, R4 is n–propyl–amino. In some es, R4
is i–propyl–amino. In some examples, R4 is n–butyl–amino. In some examples, R4 is l–
amino. In some examples, R4 is t–butyl–amino. In some es, R4 is pentyl–amino. In
some examples, R4 is hexyl–amino. In some examples, R4 is heptyl–amino. In some
examples, R4 is octyl–amino. In some examples, R4 is nonyl–amino.
In some examples, R4 is . In some es, R4 is
. In some examples, R4 is .
In some examples, herein, Ra and Rb are, independently in each instance,
selected from –H or alkyl. In some examples, both Ra and Rb are –H. In some examples, both
Ra and Rb are methyl. In some examples, both Ra and Rb are ethyl. In some examples, both
Ra and Rb are propyl. In some es, one of Ra or Rb is –H and the other is alkyl. In some
es, one of Ra or Rb is –H and the other is methyl. In some examples, one of Ra or Rb is
–H and the other is ethyl. In some examples, one of Ra or Rb is –H and the other is propyl.
In some examples, in Formula (PIa), n is an integer from 0–19. In some
examples, n is 0. In some other examples, n is 1. In certain examples, n is 2. In some other
examples, n is 3. In certain examples, n is 4. In some examples, n is 5. In some other
examples, n is 6. In certain examples, n is 7. In some other examples, n is 8. In certain
examples, n is 9. In some examples, n is 10. In some other examples, n is 11. In certain
examples, n is 12. In some other examples, n is 13. In certain examples, n is 14. In some
es, n is 15. In some other examples, n is 16. In certain examples, n is 17. In some
other examples, n is 18. In certain examples, n is 19.
In some examples, in Formula (PIa), R3 is not –OH when R1 is –OH.
In some examples, in Formula (PIa), R3 is not –OH when R1 and R2 together
form wherein R4 is a C1–9alkyl or 4–(dimethyl–amino)–phenyl. In some examples, R4
is . In some examples, R4 is .
In some es, set forth herein is a compound of Formula (PIa), n the
compound has the structure of Formula (PIb–1) or (PIb–2):
or .
(PIb–1) ( P1b–2)
In some examples, set forth herein is a compound of Formula (PIa), wherein the
compound has the ure of Formula (PIc–1) or (PIc–2):
or .
(PIc–1) ( PIc–2)
In some examples, set forth herein is a compound of Formula (PIa), wherein the
compound has the structure of Formula (PId–1) or (PId–2):
or .
(PId–1) ( PId–2)
In some examples, n is 0. In some examples, n is 1. In some examples, n is 2.
In some examples, set forth herein is a compound of Formula (I), n the
compound has the structure of Formula (PIe–1) or (PIe–2):
or .
(PIe–1) ( PIe–2)
In some examples, set forth herein is a compound of Formula (PIa), (PIb–1),
(PIb–2), (PIc–1), (PIc–2), (PId–1), (PId–2), (PIe–1), or (PIe–2) wherein R3 is ed from
alkyl–C(O)–O– or RaRbN–aryloxy–; wherein alkyl–C(O)–O–, or RaRbN–aryloxy– are
optionally substituted with halo.
In some examples, set forth herein is a compound of Formula (PIa), (PIb–1),
(PIb–2), (PIc–1), (PIc–2), (PId–1), (PId–2), (PIe–1), or (PIe–2) , wherein R3 is alkyl–C(O)–
O– optionally tuted with halo.
In some examples, set forth herein is a compound of Formula (PIa), (PIb–1),
(PIb–2), (PIc–1), (PIc–2), (PId–1), (PId–2), (PIe–1), or (PIe–2) , wherein R3 is .
In some examples, set forth herein is a compound of Formula (PIa), (PIb–1),
(PIb–2), (PIc–1), (PIc–2), (PId–1), (PId–2), (PIe–1), or (PIe–2) , wherein R3 is RaRbN–
aryloxy– optionally tuted with halo.
In some examples, set forth herein is a compound of Formula (PIa), (PIb–1),
(PIb–2), ), (PIc–2), ), (PId–2), (PIe–1), or ), wherein R3 is
In some examples, set forth herein is a compound of a (PIa), ),
(PIb–2), (PIc–1), (PIc–2), (PId–1), (PId–2), (PIe–1), or (PIe–2), wherein R3 is
In some examples, set forth herein is a compound of Formula (PIa), (PIb–1),
(PIb–2), (PIc–1), (PIc–2), (PId–1), (PId–2), (PIe–1), or (PIe–2), wherein R3 is selected from
–OH, alkyl–C(O)–O–, and RaRbN–aryloxy–. In some of these examples, alkyl–C(O)–O– or
RaRbN–aryloxy– is optionally substituted with halo. In some examples, R3 is –OH. In some
examples, R3 is alkyl–C(O)–O–. In some examples, R3 is RaRbN–aryloxy–. In some
es, R3 is . In some examples, R3 is . In some
examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is RaRbN–aryloxy–.
In some examples, R3 is -NRaRb–aryloxy.
In some examples, set forth herein is a compound of Formula (PIa), (PIb–1),
(PIb–2), (PIc–1), (PIc–2), (PId–1), (PId–2), (PIe–1), or ), wherein R3 is RaRbN–
aryloxy–, wherein Ra and Rb are, independently in each instance, H or alkyl.
In some examples, set forth herein is a compound of a (PIa), (PIb–1),
(PIb–2), (PIc–1), (PIc–2), (PId–1), (PId–2), (PIe–1), or (PIe–2), wherein R4 is selected from
the group consisting of alkyl, aryl, arylalkyl, and an N–containing heterocycloalkyl. In some
of these examples, alkyl, aryl, arylalkyl, or N–containing heterocycloalkyl are optionally
substituted with –NRaRb. In some examples, R4 is alkyl such as, but not limited to, methyl,
ethyl, propyl, butyl, pentyl, hexyl, , octyl, or nonyl. In some examples, R4 is methyl. In
some examples, R4 is ethyl. In some examples, R4 is n–propyl. In some examples, R4 is i–
propyl. In some examples, R4 is n–butyl. In some es, R4 is i–butyl. In some examples,
R4 is t–butyl. In some examples, R4 is pentyl. In some es, R4 is hexyl. In some
examples, R4 is heptyl. In some examples, R4 is octyl, or nonyl. In some es, R4 is aryl
such as but not d to phenyl or naphthyl. In some examples, R4 is phenyl. In some
examples, R4 is naphthyl. In some examples, R4 is arylalkyl–such as but not limited to benzyl.
In some examples, R4 is N–containing heterocycloalkyl such as but not limited to piperidinyl.
In some examples, R4 is 4–amino–phenyl. In some examples, R4 is 4–aminophenyl optionally
substituted with halo.
In some examples, R4 is . In some es, R4 is
. In some es, R4 is . In some es, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
F . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples,
R4 is . In some examples, R4 is . In some
examples, R4 is . In some examples, R4 is . In
some es, R4 is . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some
es, R4 is . In some examples, R4 is . In
some examples, R4 is . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples,
R4 is .
In some examples, set forth herein is a compound of a (PIa), (PIb–1),
(PIb–2), ), (PIc–2), (PId–1), (PId–2), ), or (PIe–2), n R4 is alkyl
tuted with amino such as, but not limited to, methyl–amino, ethyl–amino, propyl–amino,
butyl–amino, pentyl–amino, hexyl–amino, heptyl–amino, octyl–amino, or nonyl–amino. In
some examples, R4 is methyl–amino. In some examples, R4 is ethyl–amino. In some
examples, R4 is n–propyl–amino. In some examples, R4 is i–propyl–amino. In some examples,
R4 is n–butyl–amino. In some examples, R4 is i–butyl–amino. In some examples, R4 is t–
butyl–amino. In some examples, R4 is pentyl–amino. In some examples, R4 is hexyl–amino.
In some examples, R4 is heptyl–amino. In some examples, R4 is octyl–amino. In some
examples, R4 is amino.
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is .
The compound of Formula (I) is not one of the following compounds:
, or .
In some examples, set forth herein is a compound of Formula (I), wherein the
compound has the structure of Formula (PII):
(PII).
In Formula (PII), R3 is selected from –OH, alkyl–C(O)–O–, or RaRbN–aryloxy.
In some of these examples, alkyl–C(O)–O– or RaRbN–aryloxy is ally substituted with
halo. In some es, R3 is –OH. In some examples, R3 is
alkyl–C(O)–O–. In some examples, R3 is RaRbN–aryloxy–. In some examples, R3 is
. In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is . In some examples, R3
is . In some examples, R3 is RaRbN–aryloxy–.
In some examples, in Formula (PII), R4 is ed from the group consisting of
alkyl, aryl, arylalkyl, and an N–containing heterocycloalkyl. In some of these examples, alkyl,
aryl, arylalkyl, or aining heterocycloalkyl are optionally substituted with
–NRaRb. In some examples, R4 is alkyl such as, but not limited to, methyl, ethyl, , butyl,
pentyl, hexyl, heptyl, octyl, or nonyl. In some examples, R4 is methyl. In some examples, R4
is ethyl. In some examples, R4 is n–propyl. In some examples, R4 is i–propyl. In some
examples, R4 is n–butyl. In some examples, R4 is i–butyl. In some examples, R4 is t–butyl. In
some examples, R4 is sec–butyl. In some es, R4 is pentyl. In some examples, R4 is
hexyl. In some examples, R4 is heptyl. In some examples, R4 is octyl, or nonyl. In some
examples, R4 is aryl such as but not limited to phenyl or naphthyl. In some examples, R4 is
phenyl. In some examples, R4 is naphthyl. In some examples, R4 is arylalkyl–such as but not
limited to benzyl. In some examples, R4 is N–containing heterocycloalkyl such as but not
limited to dinyl. In some examples, R4 is 4–amino–phenyl. In some examples, R4 is 4–
aminophenyl optionally tuted with halo.
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
F . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples,
R4 is . In some es, R4 is . In some
examples, R4 is . In some examples, R4 is . In
some examples, R4 is . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
In some examples, R4 is . In some es, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is .
In some examples, R4 is . In some es, R4 is
. In some examples, R4 is . In some
examples, R4 is . In some examples, R4 is . In
some examples, R4 is . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some es,
R4 is .
In some examples, R4 is alkyl substituted with amino such as, but not limited to,
methyl–amino, ethyl–amino, propyl–amino, butyl–amino, pentyl–amino, hexyl–amino, heptyl–
amino, octyl–amino, or nonyl–amino. In some examples, R4 is methyl–amino. In some
examples, R4 is ethyl–amino. In some examples, R4 is yl–amino. In some examples, R4
is i–propyl–amino. In some examples, R4 is n–butyl–amino. In some examples, R4 is i–butyl–
amino. In some examples, R4 is t–butyl–amino. In some es, R4 is pentyl–amino. In
some examples, R4 is hexyl–amino. In some examples, R4 is heptyl–amino. In some
examples, R4 is octyl–amino. In some examples, R4 is nonyl–amino.
In some es, R4 is . In some examples, R4 is
. In some examples, R4 is .
In some examples, herein, Ra and Rb are, independently in each instance,
selected from H or alkyl. In some examples, both Ra and Rb are –H. In some examples, both
Ra and Rb are . In some examples, both Ra and Rb are ethyl. In some es, both
Ra and Rb are propyl. In some examples, one of Ra or Rb is –H and the other is alkyl. In some
examples, one of Ra or Rb is –H and the other is methyl. In some examples, one of Ra or Rb is
–H and the other is ethyl. In some examples, one of Ra or Rb is –H and the other is propyl.
In some examples, in Formula (PII), n is an integer from 0–19. In some
examples, n is 0. In some other examples, n is 1. In n examples, n is 2. In some other
examples, n is 3. In certain examples, n is 4. In some examples, n is 5. In some other
es, n is 6. In certain examples, n is 7. In some other examples, n is 8. In certain
examples, n is 9. In some examples, n is 10. In some other examples, n is 11. In certain
examples, n is 12. In some other examples, n is 13. In certain examples, n is 14. In some
examples, n is 15. In some other examples, n is 16. In certain examples, n is 17. In some
other es, n is 18. In certain examples, n is 19.
In some examples, set forth herein is a nd of Formula (I), wherein the
compound has the structure of Formula (PIIa) or (PIIb):
or .
In some examples, set forth herein is a compound of Formula (PIIa) or (PIIb),
wherein R4 is selected from 4–amino–phenyl, 4–amino–1–methyl–phenyl, 2–amino–ethyl,
piperidinyl, or propyl. In some examples, R4 is 4–amino–phenyl. In some examples, R4 is 4–
amino–1–methyl–phenyl. In some examples, R4 is 2–amino–ethyl. In some examples, R4 is
piperidinyl. In some examples, R4 is propyl. In some examples, R4 is n–propyl. In some
es, R4 is i–propyl.
In some examples, set forth herein is a compound of Formula (PIIa) or (PIIb),
wherein R3 is selected from alkyl–C(O)–O– or RaRbN–aryloxy; wherein alkyl–C(O)–O–, or
RaRbN–aryloxy are optionally substituted with halo.
In some examples, set forth herein is a compound of Formula (PIIa) or (PIIb),
n R3 is .
In some examples, set forth herein is a compound of a (PIIa) or (PIIb),
wherein R3 is .
In some examples, set forth herein is a compound of Formula (PIIa) or (PIIb),
wherein R3 is .
In some examples, set forth herein is a compound of Formula (PIIa) or ,
wherein the compound has the structure of Formula (PIII):
In Formula (PIII), R9 is ed from H or –NRaRb. In some examples, R9 is
H. In some other examples, R9 is –NRaRb, R4, R4, and subscript n are defined as in Formula I
and noted above.
In Formula (PIII), R10 and R11, are each, independently in each instance,
selected from H, F, or –NRaRb.
In some examples, set forth herein is a compound of Formula (III), wherein the
compound has the structure of Formula (PIIIa) or (PIIIb):
or .
In some examples, set forth herein is a compound of a (I), wherein the
compound has the structure of Formula (PIV):
In Formula (PIV), –NRaRb, R4, R5, and subscript n are defined as in Formula I and noted
above.
In some examples, set forth herein is a compound of Formula (I), wherein the
compound has the structure of Formula (PV):
In Formula (PV), R4, R4, and subscript n are defined as in Formula I and noted above.
In some examples, set forth herein is a compound of Formula (PV), wherein the
compound has the structure of Formula (PVa) or (PVb):
or .
In some examples, set forth herein is a compound of Formula (I), n the
compound has the structure of Formula (PVI):
In Formula (PVI) R3 is selected from alkyl–C(O)–O– or RaRbN–aryloxy, wherein alkyl–C(O)–
O–, or –NRaRb–aryloxy are optionally substituted with halo.
In some examples, in Formula (PVI), R4 is selected from –H, –OH, halo, or
alkyl. In some examples, R4 is halo such as but not limited to –F, –Cl, –Br, or –I. In some
examples, R4 is –F. In some examples, R4 is– Cl. In some examples, R4 is –Br. In some
examples, R4 is –I. In some examples, R4 is alkyl such as, but not limited to, methyl, ethyl,
propyl, butyl, pentyl, hexyl, heptyl, octyl, or nonyl. Subscript n is an integer from 0–19. In
some examples, n is 0. In some other examples, n is 1. In certain examples, n is 2. In some
other examples, n is 3. In n examples, n is 4. In some examples, n is 5. In some other
es, n is 6. In certain es, n is 7. In some other examples, n is 8. In n
examples, n is 9. In some examples, n is 10. In some other examples, n is 11. In certain
examples, n is 12. In some other es, n is 13. In certain examples, n is 14. In some
examples, n is 15. In some other examples, n is 16. In certain examples, n is 17. In some
other examples, n is 18. In certain examples, n is 19.
In some examples, in Formula (PVI), R3 is selected from –OH, C(O)–O–
–NRaRb, or NRaRb–aryloxy. In some of these examples, alkyl–C(O)–O– or RaRbN–aryloxy is
optionally tuted with halo. In some examples, R3 is –OH. In some examples, R3 is alkyl–
C(O)–O–. In some examples, R3 is RaRbN–aryloxy.
In some examples, R3 is –NRaRb. In some es, R3 is –NH2. In some
examples, R3 is –NH(CH3).
In some examples, R3 is RaRbN–aryloxy.
In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is . In some
examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is .
In some examples, R3 is . In some examples, R3 is
In some examples, set forth herein is a compound of Formula (I), wherein the
compound has the structure of Formula (PVII):
In Formula (PVII) R3 is selected from alkyl–C(O)–O– or RaRbN–aryloxy, n alkyl–
C(O)–O–, or RaRbN–aryloxy are optionally substituted with halo.
In some es, in Formula (PVII), R4 is ed from –H, –OH, halo, or
alkyl. In some examples, R4 is halo such as but not limited to –F, –Cl, –Br, or –I. In some
examples, R4 is –F. In some examples, R4 is –Cl. In some examples, R4 is –Br. In some
es, R4 is –I. In some examples, R4 is alkyl such as, but not limited to, methyl, ethyl,
, butyl, , hexyl, heptyl, octyl, or nonyl. Subscript n is an integer from 0–19. In
some examples, n is 0. In some other examples, n is 1. In certain examples, n is 2. In some
other examples, n is 3. In certain examples, n is 4. In some examples, n is 5. In some other
es, n is 6. In certain examples, n is 7. In some other examples, n is 8. In certain
es, n is 9. In some examples, n is 10. In some other examples, n is 11. In certain
examples, n is 12. In some other examples, n is 13. In certain examples, n is 14. In some
examples, n is 15. In some other examples, n is 16. In certain examples, n is 17. In some
other es, n is 18. In certain examples, n is 19. In some examples, in Formula (PVII),
R3 is selected from –OH, alkyl–C(O)–O–,
–NRaRb, or RaRbN–aryloxy. In some of these examples, alkyl–C(O)–O– or NRaRb–aryloxy is
optionally substituted with halo. In some examples, R3 is –OH. In some examples, R3 is
alkyl–C(O)–O–. In some examples, R3 is RaRbN–aryloxy.
In some examples, R3 is –NRaRb. In some examples, R3 is –NH2. In some
examples, R3 is 3).
In some examples, R3 is RaRbN–aryloxy–.
In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is . In some
examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is .
In some examples, R3 is .
In some examples, R3 is .
In some examples, set forth herein is a compound of Formula (PVII), wherein
the compound has the structure of Formula (PVIIa):
In some examples, set forth herein is a compound of Formula (PVII), wherein
the compound has the ure of Formula (PVIIb):
(PVIIb).
In some examples, set forth herein is a compound of Formula (PVII), (PVIIa),
or (PVIIb), wherein R3 is or RaRbN–aryloxy– ally substituted with halo.
In some examples, set forth herein is a compound of Formula (PVII), (PVIIa),
or (PVIIb), wherein R3 is .
In some es, set forth herein is a compound of Formula (PVII), (PVIIa),
or (PVIIb), wherein R3 is .
In some examples, set forth herein is a compound of Formula (PVII), wherein
the compound has the structure of Formula (PVIIb–1) or (PVIIb–2):
(PVIIb–1) ( PVIIb–2).
In some examples, set forth herein is a compound of Formula (PVII), ),
(PVIIb), (PVIIb–1), or (PVIIb–2), wherein R3 is alkyl–C(O)–O– or RaRbN–aryloxy.
In some examples, set forth herein is a compound of Formula (I), n the
compound has the structure of Formula (PVIII):
In some examples, of any of the Formula (PI), (PIa), (PIb–1), ), (PIc–
1), (PIc–2), ), (PId–2), (PIe–1), (PIe–2), (PII), (PIIa), (PIIb), (PIIIa), (PIIIb), (PIV),
(PV), (PVa), (PVb), (PVI), (PVII), (PVIIa), (PVIIb), (PVIIb–1), or (PVIIb–2), wherein
halo, when present, is fluoro.
In some examples of the nd of Formula (I), R1 and R2 are,
independently, selected from –H, alkyl, alkyl–C(O)–O–, –OH, or halo. In some other
es, R1 and R2 together form . In certain examples, R1 is –H. In certain other
es, R1 is alkyl. In some examples, R1 is alkyl–C(O)–O–. In some other examples, R1 is
–OH. In certain examples, R1 is halo. In certain other examples, R1 is –F. In some es,
R1 is –Cl. In some other examples, R1 is –Br. In certain es, R1 is –I. In certain other
examples, R2 is –OH. In some examples, R2 is halo. In some other examples, R2 is –F. In
certain examples, R2 is –Cl. In n other examples, R2 is –Br. In some examples, R2 is –I.
In some examples, in Formula (I), R5 is, independently in each instance,
selected from –OH, halo, alkyl, or arylalkyl. In some examples, R5 is –OH. In some
examples, R5 is halo such as but not limited to –F, –Cl, –Br, or –I. In some examples, R5 is
–F. In some examples, R5 is –Cl. In some examples, R5 is –Br. In some examples, R5 is –I. In
some examples, R5 is alkyl such as, but not limited to, methyl, ethyl, propyl, butyl, pentyl,
hexyl, heptyl, octyl, or nonyl. In some examples, R5 is benzyl.
In some examples, in Formula (I), R4 is selected from the group consisting of
alkyl, aryl, arylalkyl, and an aining heterocycloalkyl. In some of these es, alkyl,
aryl, arylalkyl, or N–containing heterocycloalkyl are optionally substituted with
–NRaRb. In some examples, R4 is alkyl such as, but not limited to, methyl, ethyl, propyl, butyl,
pentyl, hexyl, heptyl, octyl, or nonyl. In some examples, R4 is methyl. In some examples, R4
is ethyl. In some examples, R4 is n–propyl. In some examples, R4 is i–propyl. In some
examples, R4 is n–butyl. In some examples, R4 is i–butyl. In some examples, R4 is t–butyl. In
some examples, R4 is sec–butyl. In some examples, R4 is pentyl. In some es, R4 is
hexyl. In some examples, R4 is heptyl. In some examples, R4 is octyl, or nonyl. In some
examples, R4 is aryl such as but not limited to phenyl or naphthyl. In some es, R4 is
. In some examples, R4 is naphthyl. In some examples, R4 is arylalkyl–such as but not
limited to benzyl. In some es, R4 is N–containing heterocycloalkyl such as but not
limited to piperidinyl. In some examples, R4 is 4–amino–phenyl. In some examples, R4 is 4–
aminophenyl optionally substituted with halo.
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is . In some examples, R4 is
F . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
. In some es, R4 is . In some examples,
R4 is . In some examples, R4 is . In some
examples, R4 is . In some examples, R4 is . In
some examples, R4 is . In some examples, R4 is .
In some examples, R4 is . In some examples, R4 is
In some examples, R4 is alkyl tuted with amino such as, but not limited to,
methyl–amino, ethyl–amino, propyl–amino, butyl–amino, pentyl–amino, hexyl–amino, heptyl–
amino, octyl–amino, or nonyl–amino. In some es, R4 is methyl–amino. In some
examples, R4 is ethyl–amino. In some examples, R4 is n–propyl–amino. In some examples, R4
is i–propyl–amino. In some examples, R4 is n–butyl–amino. In some examples, R4 is i–butyl–
amino. In some examples, R4 is t–butyl–amino. In some es, R4 is sec–butyl. In some
es, R4 is pentyl–amino. In some examples, R4 is hexyl–amino. In some examples, R4 is
heptyl–amino. In some examples, R4 is octyl–amino. In some examples, R4 is nonyl–amino.
In some examples, R4 is . In some examples, R4 is
. In some examples, R4 is .
In some examples, , Ra and Rb are, independently in each instance,
selected from H or alkyl. In some examples, both Ra and Rb are H. In some examples, both Ra
and Rb are methyl. In some examples, both Ra and Rb are ethyl. In some examples, both Ra
and Rb are propyl. In some examples, one of Ra or Rb is H and the other is alkyl. In some
examples, one of Ra or Rb is H and the other is methyl. In some examples, one of Ra or Rb is
H and the other is ethyl. In some examples, one of Ra or Rb is H and the other is .
In some examples, in Formula (I), n is an integer from 0–19. In some
examples, n is 0. In some other examples, n is 1. In certain examples, n is 2. In some other
examples, n is 3. In certain examples, n is 4. In some examples, n is 5. In some other
es, n is 6. In certain examples, n is 7. In some other examples, n is 8. In certain
examples, n is 9. In some examples, n is 10. In some other examples, n is 11. In certain
examples, n is 12. In some other examples, n is 13. In certain examples, n is 14. In some
examples, n is 15. In some other examples, n is 16. In certain examples, n is 17. In some
other examples, n is 18. In certain examples, n is 19.
In some es, in a (I), R3 is not –OH when R1 is –OH.
In some examples, in Formula (I), R3 is not –OH when R1 and R2 together form
wherein R4 is a C1–9alkyl or 4–(dimethyl–amino)–phenyl.
In some examples, set forth herein is a compound of Formula (I), wherein R1
and R2 together form . In some of these examples, R4 is alkyl, aryl, arylalkyl, or a N–
containing heterocycloalkyl. In certain examples, alkyl, aryl, arylalkyl, or N–containing
heterocycloalkyl are optionally substituted with –NRaRb. In some of these examples, R4 is
alkyl. In some of these examples, R4 is aryl. In some of these es, R4 is arylalkyl. In
some of these examples, R4 is N–containing heterocycloalkyl. In some examples, R4 is alkyl
such as, but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, or nonyl.
In some es, R4 is methyl. In some examples, R4 is ethyl. In some examples, R4 is n–
propyl. In some examples, R4 is i–propyl. In some examples, R4 is n–butyl. In some
examples, R4 is i–butyl. In some examples, R4 is t–butyl. In some examples, R4 is sec–butyl.
In some examples, R4 is pentyl. In some examples, R4 is hexyl. In some examples, R4 is
heptyl. In some examples, R4 is octyl. In some examples, R4 is nonyl. In some examples, R4
is aryl such as but not limited to phenyl or naphthyl. In some es, R4 is phenyl. In some
examples, R4 is naphthyl. In some examples, R4 is arylalkyl–such as but not limited to benzyl.
In some examples, R4 is N–containing heterocycloalkyl such as but not d to dinyl.
In some es, R4 is o–phenyl. In some es, R4 is 4–aminophenyl optionally
tuted with halo.
In some examples, set forth herein is a compound of Formula (I), wherein R1
and R2 together form wherein R4 is selected from the group consisting of alkyl, aryl,
arylalkyl, and a N–containing heterocycloalkyl; and wherein alkyl, aryl, arylalkyl, or N–
containing heterocycloalkyl are optionally substituted with –NRaRb; and wherein the
stereochemistry of the carbon indicated by * is R.
In some examples, set forth herein is a compound of Formula (I), wherein R1
and R2 together form wherein R4 is selected from the group consisting of alkyl, aryl,
arylalkyl, and a N–containing cycloalkyl; and wherein alkyl, aryl, kyl, or N–
containing heterocycloalkyl are optionally substituted with –NRaRb; and wherein the
stereochemistry of the carbon indicated by * is S.
In Formula (I), R3 is not –OH when R1 is –OH or when R1 and R2 together form
wherein R4 is a C1–9alkyl or 4–(dimethyl–amino)–phenyl.
In some examples, the payload set forth herein is a derivative or analog of
budesonide or diflorasone. In certain examples, the derivative is an amine or aniline
containing molecule which is related in structure to budesonide or diflorasone. As set forth
herein, the payloads set forth herein as well as other steroids can be conjugated to an antibody
or an antigen–binding fragment thereof based on the methods set forth herein. As set forth
, the payloads set forth herein as well as other steroids can be conjugated to an antibody,
or an antigen–binding fragment thereof, and also to a cyclodextrin moiety based on the
s set forth herein. As taught herein, stable linker–payloads can be use with these
methods of conjugation to produce dy–steroid–conjugates. In some es, the
antibody-steroid conjugates also include a cyclodextrin moiety.
In some ments, ed herein are compounds of Formula (I1):
(I1);
or pharmaceutically acceptable salt, solvate, stereoisomer, or derivative thereof,
wherein:
R1 and R2 are, independently, –H, alkyl, alkyl–C(O)–O–, –OH, or halo; or R1 and R2
together form ,
wherein R4 is alkyl, aryl, arylalkyl, or an N–containing heterocycloalkyl,
wherein the alkyl, aryl, arylalkyl, and N–containing heterocycloalkyl
are, independently in each ce, ally substituted with
–NRaRb;
R5 is, independently in each instance, –OH, halo, alkyl, or arylalkyl;
R3 is –OH, alkyl–C(O)–O–, or –X–aryl–NRaRb, wherein X is ed from S, S(O),
S(O)2, SO2NRa, CONRa, C(O), or NRa, wherein the alkyl–C(O)–O– and –X–
aryl–NRaRb are optionally substituted with halo or prodrug.
Ra and Rb are, independently in each ce, H or alkyl, aryl;
Rc is –H or alkyl; and
n is an integer from 0–19;
with the proviso that R3 is not –OH when either (a) or (b): (a) R1 is –OH or (b) R1 and
R2 together form and R4 is a C1–9alkyl or .
In some of these examples, alkyl–C(O)–O– or
–X–aryl–NRaRb is optionally substituted with halo. In some examples, R3 is –OH. In
some examples, R3 is alkyl–C(O)–O–. In some examples, R3 is RaRbN–aryloxy. In
some examples, R3 is . In some examples, R3 is .
In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is .
In some examples, R3 is –X–aryl–NRaRb.
In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is . In some
examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some es, R3 is . In
some examples, R3 is . In some examples, R3 is .
In some examples, R3 is .
In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is . In some
examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is .
In some examples, R3 is .
In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is . In some
examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some es, R3 is . In
some examples, R3 is . In some examples, R3 is .
In some examples, R3 is .
In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is . In some
examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some es, R3 is . In
some examples, R3 is . In some examples, R3 is .
In some examples, R3 is .
In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is . In some
examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is .
In some examples, R3 is .
In some examples, set forth herein is a compound of Formula (I), wherein R3
has a structure selected from one of the following ures:
, , , , or .
In some examples, R3 is . In some examples, R3 is .
In some examples, R3 is . In some examples, R3 is . In some
examples, R3 is . In these examples, q is an integer from 0 to 5.
In some es, set forth herein is a compound of Formula (I), wherein R3
has a structure selected from one of the following structures:
, , , , or .
In some examples, R3 is . In some examples, R3 is
. In some examples, R3 is . In some examples, R3 is .
In some examples, R3 is . In these examples, q is an integer from 0 to 5.
In some examples, set forth herein is a compound of Formula (I), wherein R3
has a structure ed from one of the following structures:
, , , , or
In some examples, R3 is . In some examples, R3 is . In
some examples, R3 is . In some examples, R3 is . In
some examples, R3 is .
In some examples, set forth herein is a compound of Formula (I), n the
compound has the ure of Formula 1000:
or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In Formula 1000, R1
and R2 are, independently, selected from the group consisting of -H, -OH, alkyl,
)-alkyl, and halo; or R1 and R2 together form , wherein R4 is selected from the
group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, -alkylene-NRaRb,
-X-arylene-Y-NRaRb, -X-heteroarylene-Y-NRaRb, and N-containing heterocycloalkyl; wherein
X is absent, -N-, -CH2-, or -O-; wherein Y is absent or -CH2-. R3 is selected from the group
consisting of -OH, -O-C(O)-alkyl, l, -NRaRb, -alkylene-NRaRb, -X-arylene-Y-NRaRb,
-X-heteroarylene-Y-NRaRb, and N-containing heterocycloalkyl; wherein X is absent, -N-,
-CH2-, or -O-; wherein Y is absent or -CH2-. R5 is, independently in each instance, selected
from a substituent in the group consisting of –OH, halo, and alkyl; n is an integer from 0-19;
and each R5 is oned on any ring atom. Ra and Rb are, independently in each ce,
selected from the group consisting of –H and alkyl; or Ra and Rb cyclize to form
cycloheteroalkyl with three to six ring atoms, including one hetero atom, which is the N to
which they are attached. Ra and Rb are, independently in each instance, optionally substituted
with at least one substituent selected from the group consisting of -OH, -PO4H, NH2,
-C(O)OH, and H3.
In ceratain embodiments, provided herein are compounds according to a
1000, wherein R3 is selected from the group consisting of -alkylene-NRaRb,
-X-arylene-Y-NRaRb, -X-heteroarylene-Y-NRaRb, and N-containing heterocycloalkyl; wherein
X is absent, -N-, -CH2-, or -O-; n Y is absent or -CH2-; and R4 is selected from the group
consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, -alkylene-NRaRb,
lene-Y-NRaRb, -X-heteroarylene-Y-NRaRb, and N-containing cycloalkyl; wherein
X is absent, -N-, -CH2-, or -O-; wherein Y is absent or -CH2-.
In ceratain embodiments, provided herein are compounds according to Formula
1000, wherein R3 is selected from the group consisting of -OH, -O-C(O)-alkyl, -O-aryl,
-NRaRb, -alkylene-NRaRb, -X-arylene-Y-NRaRb, -X-heteroarylene-Y-NRaRb, and N-containing
heterocycloalkyl; wherein X is absent, -N-, -CH2-, or -O-; wherein Y is absent or
-CH2-; and R4 is selected from the group consisting of -alkylene-NRaRb, -X-arylene-Y-NRaRb,
-X-heteroarylene-Y-NRaRb, and aining heterocycloalkyl; wherein X is absent, -N-,
-CH2-, or -O-; wherein Y is absent or -CH2-.
In certain embodiments, ed herein are compounds according to Formula
1000, wherein R3 is -NRaRb; and R4 is alkyl. In certain embodiments, R3 is –NH2. In certain
embodiments, R4 is n-propyl. In certain embodiments, R3 is –NH2 and R4 is n-propyl.
In certain embodiments, the compound of a 1000 is according to
Formula 1010, 1020, 1030, or 1040:
1010 1020
1030 1040
or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.
In n embodiments, the compound of Formula 1000 is according to
Formula 1110, 1120, 1130, or 1140:
1110 1120
1130 1140
or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof.
In certain embodiments according to any of Formulas 140, R3 is -OH or
)-alkyl; and R4 is -alkylene-NRaRb, -X-arylene-NRaRb, -X-heteroarylene-NRaRb, or
N-containing heterocycloalkyl; wherein X is absent or -CH2-. In certain embodiments, R4 is
-alkylene-NH2, NH2 or -CH2-C6H5-NH2.
In certain embodiments according to any of Formulas 1000-1140, R3 is -O-aryl,
-NRaRb, -alkylene-NRaRb, -X-arylene-Y-NRaRb, -X-heteroarylene-Y-NRaRb, or N-containing
cycloalkyl; wherein X is absent, -N-, -CH2-, or -O-; wherein Y is absent or
-CH2-; and R4 is alkyl, aryl, alkylaryl, or arylalkyl. In certain embodiments, R3 is
-O-arylene-NRaRb, -O-heteroarylene-NRaRb; wherein aryl or heteroaryl is optionally
substituted with halogen, deuterium, hydroxyl, or methoxyl. In certain embodiments, R3 is
-O-phenyl-NRaRb, -O-heteroarylene-NRaRb; wherein phenyl or heteroaryl is optionally
substituted with halogen or deuterium. In certain ments ing to this paragraph, R4
is n-propyl.
In certain ments, provided herein are compounds according to any of
Formulas 1000-1140, wherein R3 is -NRaRb; and R4 is alkyl. In certain embodiments, R3 is
–NH2. In certain embodiments, R4 is n-propyl. In certain ments, R3 is –NH2 and R4 is
n-propyl.
In any of Formulas 140, R3 can be any specific R3 provided above. In
particular embodiments, R3is -NH2, -N(H)CH3, -N(CH3)2, or . In particular
embodiments, R3 is , , , ,
, , or .
In particular embodiments, R3 is , , or .
In particular embodiments, R3 is .
In any of Formulas 1000-1140, R4 can be any specific R4 provided above. In
particular embodiments, R4 is selected from -CH2-CH2-NH2, ,
, and . In particular ments, R4 is n-propyl.
Set forth herein are also compounds having the ing structures:
O H
O H O
O OH
or a pharmaceutically acceptable salt, e, or stereoisomer thereof.
Included within the scope of this disclosure are pharmaceutically acceptable
salts, es, crystalline forms, amorphous forms, polymorphic forms, regioisomers,
stereoisomers, prodrugs, e.g., phosphatase-prodrugs, glucose-prodrugs, ester prodrugs, etc.,
metabolites, and physiological adducts of the steroid payloads described herein, including
those of Formula (I), (II), and (A1)-(A7).
C. PROTEIN STEROID CONJUGATES
Provided herein are protein conjugates of the steroids described herein. Such
conjugates include proteins, e.g., antibodies or antigen–binding fragments thereof, that are
ntly linked, e.g., via the binding agent linkers described herein, to the compounds
described in Section B above, e.g., the compounds of Formula (A), (A1), (A2), (A3), (A4), (A5),
(A6), (A7), (I), (I1), (PIa), (PIb–1), (PIb–2), PIc–1), (PIc–2), (PId–1), (PId–2), (PIe–1),
(PIe–2), (PII), (PIIa), (PIIb), (PIII), (PIIIa), (PIIIb), (PIV), (PV), (PVa), (PVb), (PVI),
(PVII), (PVIIa), (PVIIb), (PVIIb–1), (PVIIb–2), (PVIII), and (1000)-(1140).
The binding agent linker can be linked to a steroid described herein at any
suitable moiety or position of the steroid, including e.g., through an amide, ether, ester,
carbamate, or amine. For example, the g agent linker can be attached to compounds
through R1, R3, or R4 or hydroxyl group depicted Formula (A1):
(A1).
In certain ments, the steroids bed herein are ed to the binding agent linker
by reacting an amino or yl group of the steroid with a suitable reactive group present on
the linker. In some embodiments, the g agent linker also includes a cyclodextrin moiety.
For example, the cyclodextrin moiety may be bonded to the chemical backbone structure of the
binding agent linker.
In certain embodiments, provided herein are compounds having the structure:
BA-(L-PAY)x
wherien BA is a binding agent as described herein; L is an optional linker as described herein;
PAY is a steroid compound as described herein; and x is an integer from 1-30. In particular
ments, each PAY is a radical obtainable by removal of an atom, for example a
hydrogen atom from a compound according to a Formula selected from the group consisting of
Formulas (A), (A1), (A2), (A3), (A4), (A5), (A6), (A7), (I), (I1), (PIa), ), ), ,
), (PId–1), ), (PIe–1), (PIe–2), (PII), (PIIa), (PIIb), (PIII), (PIIIa), (PIIIb),
(PIV), (PV), (PVa), (PVb), (PVI), (PVII), ), (PVIIb), (PVIIb–1), (PVIIb–2),
(PVIII), and (1000)-(1140). Examples of such compounds are described in detail below.
In certain embodiments, provided herein are compounds having the structure of
Formula (III):
(III);
wherein either (a) or (b):
(a) R3 is –BL–,–BL–X–, or ;
R1 and R2 are each, independently, –H, alkyl, alkyl–C(O)–O–, –OH, or
halo; or R1 and R2 together form , wherein R4 is alkyl, aryl,
arylalkyl, or an aining heterocycloalkyl; wherein the alkyl, aryl,
arylalkyl, and
aining heterocycloalkyl are optionally substituted with –NRaRb;
(b) R3 is –OH, alkyl–C(O)–O–, heteroalkyl, –NRaRb or aryloxy, wherein the
alkyl–C(O)–O–, heteroalkyl, or aryloxy is optionally substituted with
–NRaRb, -NRaRb–aryloxy, or halo, and R1 and R2 er form ,
wherein R4 is –BL–, ,
or –BL–Y, wherein Y is an N–containing divalent heterocycle;
–BL– is a nt binding agent linker;
R5 is, independently in each instance, –OH, halo, alkyl, or arylalkyl;
Ra and Rb are, independently in each instance, –H or alkyl;
RP, independently in each instance, is halo;
BA is a binding agent bonded to –BL–;
X, independently in each ce, is NRa or O;
is aryl or heteroaryl;
t is an integer from 0-2;
x is an integer from 1–30; and
n is an r from 0–19.
In some examples, subscript x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some examples, subscript x is 0.
In some examples, subscript x is 1. In some examples, subscript x is 2. In some examples,
subscript x is 3. In some examples, subscript x is 4. In some examples, ipt x is 5. In
some examples, subscript x is 6. In some examples, subscript x is 7. In some examples,
subscript x is 8. In some examples, subscript x is 9. In some examples, subscript x is 10. In
some examples, ipt x is 11. In some examples, subscript x is 12. In some examples,
subscript x is 13. In some examples, subscript x is 14. In some examples, subscript x is 15. In
some examples, subscript x is 16. In some examples, subscript x is 17. In some es,
ipt x is 18. In some examples, subscript x is 19. In some examples, subscript x is 20. In
some examples, subscript x is 21. In some examples, subscript x is 22. In some examples,
subscript x is 23. In some examples, subscript x is 24. In some examples, subscript x is 25. In
some examples, subscript x is 26. In some examples, subscript x is 27. In some examples,
subscript x is 28. In some examples, subscript x is 29. In some examples, subscript x is 30.
In some examples of Formula (III), R1 and R2 are, each, independently, –H,
alkyl, or –OH. In some es of a (III), one of R1 or R2 is –H, alkyl, or –OH. In
some examples of Formula (III), both R1 and R2 are either –H, alkyl, or –OH.
In some examples of Formula (III), R1 and R2 together form . In some
examples, R4 is –RL. In some es, R4 is RL–NRa–aryl. In some other examples, R4 is
alkyl. In certain examples, R4 is arylalkyl, In some examples, R4 is aryl. In other examples,
R4 is N–containing heterocycloalkyl. In some of these examples, the alkyl, aryl, arylalkyl, or
N–containing heterocycloalkyl is optionally substituted.
In some examples of Formula (III), R5 is –H or halo. In some examples of
a (II), R5 is –H or fluoro. In some examples of Formula (III), one of R5 is –H or halo.
In some examples of Formula (III), R5 is –H or halo and n is 2. In some examples of Formula
(III), R5 is –F and n is 1. In some examples of Formula (II), R5 is –F and n is 2.
In some examples of Formula (III), R3 is BL. In some examples of a
(III), R3 is –aryloxy–. In some other examples of Formula (III), R3 is –OH. In some
other es of Formula (III), R3 is alkyl–C(O)–O–. In some other examples of Formula
(III), R3 is heteroalkyl. In some other examples of Formula (III), R3 is –N–RaRb. In some
other examples of a (III), R3 is aryl. In some other examples of Formula (III), R3 is
aryloxy. In some other examples of a (III), alkyl–C(O)–O–, heteroalkyl, or aryloxy is
ally substituted with
–NRaRb or halo.
In some examples of Formula (II), R3 is –OH. In some examples of Formula (III), R3 is alkyl–
C(O)–O–. In some examples R3 is In some examples of Formula (III), R3 is
heteroalkyl. In some examples R3 is or . In some examples of
Formula (III), R3 is or . In some examples of Formula (III), R3 is
–NRaRb. In some examples, R3 is -NRaRb–aryloxy. In some examples, R3 is . In some
examples R3 is . In some examples, R3 is . In some examples R3 is aryloxy.
In some examples R3 is . In some examples R3 is . In some
examples, R3 is . In some examples R3 is . In some
examples, R3 is . In some examples R3 is . In some
examples, R3 is N
H . In some examples R3 is .
In some examples, R3 is . In some examples R3 is .
In some examples, R3 is .
In Formula (III), subscript n is an integer from 0–19. In some examples, n is 0.
In some other es, n is 1. In certain examples, n is 2. In some other examples, n is 3. In
n examples, n is 4. In some examples, n is 5. In some other examples, n is 6. In n
examples, n is 7. In some other examples, n is 8. In certain examples, n is 9. In some
examples, n is 10. In some other examples, n is 11. In certain examples, n is 12. In some
other examples, n is 13. In certain examples, n is 14. In some examples, n is 15. In some
other examples, n is 16. In certain examples, n is 17. In some other examples, n is 18. In
certain examples, n is 19.
In some examples, set forth herein is a compound having the structure of
Formula (IIIa):
(IIIa);
wherein:
BA is a g agent;
R5 is, independently in each instance, –OH, halo, or alkyl;
R3 is selected from –OH, alkyl–C(O)–O–, heteroalkyl, –NRaRb, -NRaRb–
aryloxy,
or aryloxy, wherein the alkyl–C(O)–O–, heteroalkyl, or aryloxy is ally
substituted with –NRaRb or halo;
BL is a binding agent linker;
Ra and Rb are, independently in each ce, ed from H, alkyl, and
alkyl–C(O);
n is an integer from 0 to 19; and
x is an integer from 1 to 30.
In some examples, set forth herein is a compound having the structure of
Formula (IIIa2):
(IIIa2);
wherein:
BA is a binding agent;
R5 is, independently in each instance, –OH, halo, or alkyl;
R3 is –OH, alkyl–C(O)–O–, heteroalkyl, –NRaRb, -NRaRb–aryloxy, or aryloxy,
n the alkyl–C(O)–O–, heteroalkyl, or aryloxy is optionally substituted
with –NRaRb or halo;
BL is a binding agent ;
Ra and Rb are, independently in each instance, selected from H, alkyl, or
alkyl–C(O);
n is an integer from 0 to 19; and
x is an integer from 0 to 30.
In some examples of Formula (IIIa2), R3 is –OH. In some es of Formula (IIIa2), R3 is
alkyl–C(O)–O–. In some examples R3 is In some examples of a (IIIa2),
R3 is heteroalkyl. In some examples R3 is or . In some
examples of Formula (IIIa2), R3 is –NRaRb. In some examples R3 is . In some
examples R3 is aryloxy. In some examples R3 is . In some examples R3 is
. In some examples R3 is .
In some examples R3 is . In some examples R3 is .
In some examples, R3 is .
In some examples, the compound of a (IIIa2) has the following
structure:
wherein:
BA is a binding agent;
R3 is –OH or alkyl–C(O)–O–;
R5a and R5b are each, independently, –F or H;
BL is a g agent linker; and
x is an integer from 1 to 30.
In some examples, set forth herein is a compound having the structure of
a (IIIb):
(IIIb);
wherein
BA is a binding agent;
R5 is, independently in each instance, –OH, halo, or alkyl;
R4 is selected from alkyl, aryl, arylalkyl, or an N–containing heterocycloalkyl,
wherein the alkyl, aryl, arylalkyl, or N–containing heterocycloalkyl are
optionally
substituted with –NRaRb;
RL is a binding agent linker;
Ra and Rb are, independently in each instance, selected from H, alkyl, and
alkyl–C(O);
n is an integer from 0 to 19; and
x is an integer from 0 to 30.
In some examples of Formula (IIIb), R5 is –H or halo. In some examples of
Formula (IIIb), R5 is fluoro. In some examples of Formula (IIIb), n is at least 2, and two of
R5 is halo. In some es of Formula (IIIb), R5 is –F and n is 1. In some es of
Formula (IIIb), R5 is –F.
In some examples of Formula (IIIb), R4 is alkyl. In some es of Formula
(IIb), R4 is methyl, ethyl, n–propyl, i–propyl, n–butyl, l, t–butyl, i–butyl, a pentyl
moiety, a hexyl moiety, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some
examples of Formula , R4 is n–propyl.
In some examples, the compound of Formula (IIIb) has the following ure:
wherein:
BA is a binding agent;
R4 is alkyl;
R5a and R5b are each, independently, –F or H;
BL is a binding agent linker; and
x is an integer from 1 to 30.
In some examples, set forth herein is a compound having the structure of
Formula (IIIc):
(IIIc);
wherein
BA is a binding agent;
R1 and R2 are, independently, –H, alkyl, alkyl–C(O)–O–, –OH, or halo;
R5 is, independently in each instance, selected from –OH, halo, or alkyl;
BL is a binding agent linker;
n is an integer from 0 to 19; and
x is an r from 1 to 30.
In some examples of Formula (IIIc), R5 is halo. In some examples of Formula
(IIIc), R5 is fluoro. In some examples of Formula (IIIc), one of R5 is halo. In some examples
of a , two of R5 is halo. In some examples of Formula (IIIc), R5 is –F and n is 2.
In some examples of Formula (IIIc), R1 is CH3.
In other examples of Formula (IIIc), R1 is OH.
In some other examples of Formula (IIIc), R1 is H.
In some examples of Formula (IIIc), R2 is CH3.
In other es of Formula (IIIc), R2 is OH.
In some other examples of Formula (IIIc), R2 is H.
In some examples of Formula (IIIc), R1 is CH3 and R2 is CH3.
In other examples of Formula (IIIc), R1 is CH3 and R2 is OH.
In some examples of Formula (IIIc), R1 is CH3 and R2 is H.
In some other examples of Formula (IIIc), R1 is OH and R2 is CH3.
In other examples of Formula (IIIc), R1 is OH and R2 is OH.
In some examples of a (IIIc), R1 is H and R2 is H.
In some other examples of Formula (IIIc), R1 is H and R2 is OH.
In other examples of Formula (IIIc), R1 is H and R2 is H.
In some embodiments, the compound of Formula (IIIc) has the ing
(IIIc)
wherein:
BA is a g agent;
R2 is methyl;
R5a and R5b are each, independently, –F or H;
BL is a binding agent linker; and
x is an integer from 0 to 30.
In some embodiments, the compound of Formula (IIIc) has the following
structure:
O H H
BA RG SP1 PEG AA4 AA5 NH O O H
m OH
HN O
BA is a binding agent;
RG is a reactive group residue;
CD is a cyclodextrin;
SP1 is a spacer group;
AA4 is an amino acid residue;
AA5 is a dipeptide residue;
PEG is polyethylene glycol;
m is an integer from 0 to 4;
x is an integer from 0 to 30;
R4 is alkyl, aryl, arylalkyl, or an N–containing heterocycloalkyl; wherein the alkyl, aryl,
arylalkyl, and aining heterocycloalkyl are optionally substituted with –NRaRb;
Ra and Rb are, independently in each instance, –H or alkyl;
BA is a binding agent bonded to –BL–;
SP1 and SP2 are each, independently in each instance, absent or a spacer group residue, and
wherein SP1 comprises a trivalent linker; AA4 is a trivalent linker sing an amino acid
residue; AA5 is a di-peptide residue; PEG is a polyethylene glycol residue; wherein the
indicates the atom through which the indicated al group is bonded to the adjacent
groups in the formula, CD is, ndently in each instance, absent or a cyclodextrin residue,
n at least one CD is t, subscript m is an integer from 0 to 5; In these examples,
subscript m is 0, 1, 2, 3, 4, or 5. In some examples, ipt m is 0. In some examples,
subscript m is 1. In some examples, subscript m is 2. In some examples, subscript m is 3. In
some examples, subscript m is 4. In some examples, subscript m is 5. In some examples, any
one of AA4 or AA5 ses, ndently in each instance, an amino acid ed from
e, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine,
threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine,
arginine, histidine, or citrulline, a derivative thereof, or a combination f. In certain
embodiments, AA4 is an amino acid selected from alanine, valine, leucine, isoleucine,
methionine, tryptophan, phenylalanine, proline, glycine, serine, ine, cysteine, tyrosine,
asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a
derivative thereof, or a combination f. In certain embodiments, AA4 is lysine. In certain
embodiments, AA4 is lysine or a derivative of lysine. In certain embodiments, the AA5 is
valine-citrulline. In some embodiments, the AA5 is line-valine. In some embodiments, the
AA5 is valine-alanine. In some embodiments, the AA5 is alanine-valine. In some embodiments,
the AA5 is valine-glycine. In some embodiments, the AA5 is glycine-valine. In some
embodiments, the AA5 glutamate-valine-citrulline. In some embodiments, the AA5 is
glutamine-valine-citrulline. In some embodiments, the AA5 is lysine-valine-alanine. In some
embodiments, the AA5 is lysine-valine-citrulline. In some embodiments, the AA5 is glutamatevaline-citrulline.
In some examples, SP1 is independently in each ce, ed from the
group consisting of C1-6 alkylene, -NH-, -C(O)-, (-CH2-CH2-O)e, -NH-CH2-CH2-(-O-CH2-
CH2)e-C(O)-, -C(O)-(CH2)u-C(O)-, -C(O)-NH-(CH2)v-, and combinations thereof, n
subscript e is an integer from 0 to 4, subscript u is an integer from 1 to 8, and subscript v is an
integer from 1 to 8. In some examples, SP2 is independently in each instance, selected from the
group consisting of C1-6 ne, -NH-, -C(O)-, CH2-O)e, -NH-CH2-CH2-(-O-CH2-
CH2)e-C(O)-, -C(O)-(CH2)u-C(O)-, -C(O)-NH-(CH2)v-, and combinations thereof, wherein
subscript e is an integer from 0 to 4, subscript u is an integer from 1 to 8, and subscript v is an
integer from 1 to 8.
Set forth are also compounds of Formula (B2):
wherein n is an integer from 0 to 4, R3 is –OH or RZ-C(O)-O–; wherein RZ is alkyl, BL is a
binding agent , BA is a binding agent, and x is an integer from 1 to 30. In certain
embodiments, n is 0 or 1 and x is an integer from 1-6. In certain embodiments, x is 4.
Set forth are also compounds of Formula (B3):
wherein n is an integer from 1-4, R3 is –OH or RZ-C(O)-O–; wherein RZ is alkyl, BL is a
binding agent linker, BA is a binding agent, and x is an integer from 1-30. In certain
embodiments, n is 2 and x is an r from 1-6. In certain embodiments, x is 4.
Set forth are also compounds of Formula (B4):
wherein R4 is alkyl, wherein Ra is a hydrogen atom or alkyl, BL is a binding agent linker, and
BA is a binding agent. In certain embodiments, R4 is C1-4 alkyl. In some ments, R4 is
propyl. In certain embodiments, R3 is –NH2, –NHCH3, or –N(CH3)2. In certain embodiments,
x is an integer from 1-6. In certain embodiments, x is 4.
Set forth are also compounds of a (B5):
wherein R4 is alkyl, RP1 is halo or a hydrogen atom, and X is NRa or O, wherein Ra is a
en atom or alkyl, BL is a g agent linker, BA is a binding agent, and x is an integer
from 1-30. In some embodiments, R4 is C1-4 alkyl, X is NH, and x is an integer from 1-6. In
certain embodiments, x is 4.
Set forth are also compounds of Formula (B6A):
(B6A)
wherein X is O or NRa, is aryl or heteroaryl, RP is halo, t is an integer from 0-2, Ra is a
hydrogen atom or alkyl, BL is a binding agent linker, BA is a binding agent, and x is an integer
from 1-30, and R4 is alkyl. In some embodiments X is O, R4 is alkyl, and x is an integer from
1-6. In certain embodiments, x is 4.
Set forth herein are also compounds of Formula (B6B)
H H H
R4 F
O O
BA BL NRa O CH3 CH3
(B6B)
wherein Ra is a hydrogen atom or alkyl, BL is a binding agent linker, BA is a binding agent,
and x is an integer from 1-30. In some embodiments, x is an integer from 1-6. In some
embodiments, x is 4.
As used , the phrase “binding agent linker,” or “BL” refers to any divalent
group or moiety that links, connects, or bonds a binding agent (e.g., an antibody or an antigen–
g fragment thereof) with a payload compound set forth herein (e.g., steroid). Generally,
suitable binding agent linkers for the antibody conjugates described herein are those that are
sufficiently stable to exploit the circulating ife of the antibody and, at the same time,
capable of releasing its payload after antigen–mediated internalization of the ate.
Linkers can be cleavable or non–cleavable. Cleavable s are linkers that are d by
intracellular lism following internalization, e.g., ge via hydrolysis, reduction, or
enzymatic reaction. Non–cleavable linkers are linkers that release an attached payload via
lysosomal ation of the antibody following internalization. Suitable linkers include, but
are not limited to, acid–labile linkers, ysis–labile linkers, enzymatically cleavable
linkers, reduction labile linkers, self–immolative linkers, and non–cleavable linkers. Suitable
linkers also include, but are not limited to, those that are or comprise glucuronides,
succinimide–thioethers, polyethylene glycol (PEG) units, ates, hydrazones, mal–
caproyl units, disulfide units (e.g., –S-S–, –S-S- C(R1)(R2) –, wherein R1 and R2 are
independently hydrogen or hydrocarbyl), para-amino-benzyl (PAB) units, phosphate units,
e.g., mono-, bis-, and tris- phosphate units, peptides, e.g., peptide units containing two, three,
four, five, six, seven, eight, or more amino acid units, including but not limited to valine–
line units, valine–alanine units, valine–arginine units, valine–lysine units, -lysine-valine–
citrulline units, and -lysine-valine–alanine units. In some embodiments, the binding agent
linker group of the conjugates described herein are derived from the reaction of a “reactive
linker” group of a linker–payload described herein with a ve portion of an antibody. The
ve linker group (RL) refers to a monovalent group that comprises a reactive group and
linking group, depicted as , wherein RG is the reactive group, L is the g group,
and the wiggly line represents a bond to a payload. The linking group is any divalent moiety
that bridges the reactive group to the payload. The g group may also be any trivalent
moiety that bridges the reactive group, the payload and a extrin moiety. In some
examples, the linking group is trivalent and includes a cyclodextrin moiety bonded to a
trivalent group (e.g., a lysine residue) in the linking group. The reactive s (RL), er
with the payloads to which they are bonded, comprise intermediates (“linker–payloads”) useful
as synthetic precursors for the preparation of the antibody steroid conjugates described herein.
The reactive linker contains a ve group (RG), which is a onal group or moiety that
reacts with a reactive portion of an dy, modified dy, or antigen binding fragment
thereof. The moiety resulting from the reaction of the reactive group (RG) with the antibody,
modified antibody, or antigen binding fragment thereof, er with the linking group (L),
comprise the “binding agent linker” (BL) portion of the conjugate, described herein. Thus, in
some embodiments, BL is has the following structure:
wherein is the bond to the biding agent, RGN is the moiety resulting from the reaction of a
reactive group of a linker–payload with a reactive portion of a binding agent, L is a linking
group, and is a bond to a payload.
In certain embodiments, RGN is derived from the reaction of RG with a cysteine
or lysine residue of an antibody or antigen–binding fragment thereof. In certain embodiments,
RGN is derived from a click chemistry reaction. In some embodiments of said click chemistry
reaction, RGN is derived from a 1,3 cycloaddition reaction between an alkyne and an azide.
Non–limiting examples of such RGNs include those derived from strained alkynes, e.g., those
suitable for –promoted alkyne–azide cycloadditions (SPAAC), cycloalkynes, e.g.,
cyclooctynes, nulated s, and alkynes capable of undergoing 1,3 cycloaddition
reactions with azides in the absence of copper catalysts. Suitable RGNs also include, but are
not limited to those derived from DIBAC, DIBO, BARAC, substituted, e.g., fluorinated
alkynes, aza–cycloalkynes, BCN, and derivatives thereof. Conjugates containing such RGN
groups can be derived from antibodies that have been functionalized with azido groups. Such
functionalized antibodies include antibodies functionalized with azido–polyethylene glycol
groups. In certain embodiments, such functionalized antibody is d by ng an
antibody comprising at least one glutamine residue with a compound according to the formula
H2N–LL–N3, wherein LL is a divalent polyethylene glycol group, in the presence of the
enzyme transglutaminase, e.g., microbial transglutaminase. Suitable glutamine residues of an
antibody include Q295, or those derived by insertion or mutation, e.g., N297Q mutation.
In some embodiments, BA of the conjugates described herein is an antibody or
an antigen-binding nt thereof. In some embodiments, the conjugates described herein
are d from azido-functionalized dies. In certain embodiments, BA of the
conjugates described herein is:
wherein Ab is an antibody or antigen-binding fragment thereof, n is an integer from 1 to 10, w
is the number of linker d moieties, and is a bond to a single binding agent linker (BL),
e.g., bond to a moiety derived from a 1,3-cycloaddition reaction n an alkyne and azide.
In certain embodiments, w is 3. In certain embodiments, w is 2 or 4, i.e., the conjugate
comprises 2 or 4 linker payload es.
In some ments, BL is a divalent moiety of a (BLA);
–RGN–(SP1)q–(A)z–(NRa)s–(B)t–(CH2)u–(O)v–( SP2)w–
(BLA);
wherein RGN is as defined herein;
A is an amino acid or a peptide;
Ra is H or alkyl;
B is aryl, heteroaryl, or heterocycloalkyl, wherein aryl, aryl, or
heterocycloalkyl is optionally substituted with alkyl, –OH, or –NRaRb;
SP1 and SP2 are, independently, a spacer groups; and q, z, s, t, u, v, and w are,
independently in each instance, 0 or 1.
In some other embodiments, BL is a ent moiety of Formula (BLB);
–RGN–(SP1)q–(A)z–(NRa)s–(B)t–(CH2)u–(O)v–( SP2)w–
(BLB);
wherein RGN is as defined herein;
A is tripeptide, wherein at least one of the amino acids in the tripeptide is
bonded directly or indirectly to a cyclodextrin moiety;
Ra is H or alkyl;
B is aryl, aryl, or cycloalkyl, wherein aryl, heteroaryl, or
heterocycloalkyl is ally substituted with alkyl, –OH, or –NRaRb;
SP1 and SP2 are, independently, a spacer groups; and q, z, s, t, u, v, and w are,
independently in each instance, 0 or 1.
In some examples, the cyclodextrin (CD) is bonded directly to an amino acid
e, such as a lysine amino acid residue. This means that the CD is one bond position away
from the lysine amino acid covalent linker. In some of these examples, the covalent linker is
also bonded directly to a payload moiety. This means that the covalent linker is one bond
position away from a payload such as, but not limited to a steroid payload set forth herein. In
some of these examples, the covalent linker is also bonded directly to a CD moiety. This
means that the covalent linker is one bond position away from a CD, such as the CD(s) set
forth herein. In some of these examples, the covalent linker is a lysine amino acid or a
derivative thereof.
In some examples, the CD is bonded ctly to a covalent linker in a linking
group (e.g., a BL). This means that the CD is more than one bond position away from the
covalent linker. This also means that the CD is bonded through r moiety to the covalent
linker. For example, the CD may be bonded to a maleimide group which is bonded to a
polyethylene glycol group which is bonded to the covalent linker. In some of these examples,
the covalent linker is also bonded indirectly to a payload moiety. This means that the covalent
linker is more than one bond position away from a payload such as, but not limited to a steroid
payload set forth herein. This also means that the covalent linker is bonded through another
moiety to the payload. For example, the nt linker may be bonded to a dipeptide, such as
but not limited to Val-Ala or Val-Cit, which may be bonded to para-amino benzoyl which may
be bonded to the payload. In some of these examples, the covalent linker is also bonded
indirectly to a extrin moiety. This means that the covalent linker is more than one bond
position away from a cyclodextrin, such as the cyclodextrins set forth herein. This also means
that the covalent linker is bonded through another moiety to the cyclodextrin. For example, the
covalent linker may be bonded to a polyethylene glycol group which may be bonded to
reactive group which may be bonded to the extrin. In some of these examples, the
covalent linker is a lysine amino acid or a derivative thereof.
In some embodiments, BL is SP1)q–(A)z–. In some ments, BL is
–RGN–(SP1)q–(A)2–. In some embodiments, BL is a moiety of Formula (BLA1)
(BLA1)
wherein RAA1 and RAA2 are each, independently, amino acid side chains. In some examples of
a RLA1, SP1 is a divalent polyethylene glycol group and RGN is a 1,3–cycloaddition
reaction adduct of the reaction between an alkyne and an azide.
In some embodiments, BL is –RGN–(SP1)q–(A)z–. In some embodiments, BL is
–RGN–(SP1)q–(A)2–. In some embodiments, BL is a moiety of Formula (BLB1)
(BLB1)
wherein RAA1 and RAA2 are each, independently, amino acid side . RAA3 is
an amino acid side chain that is bonded directly or ctly to a cyclodextrin moiety. In some
examples of Formula RLB1, SP1 is a divalent polyethylene glycol group and RGN is a 1,3–
cycloaddition reaction adduct of the reaction between an alkyne and an azide.
In some embodiments, BL has the following structure:
–RGN–(SP1)q–Z1–Z2–Z30–1–
wherein:
RGN, SP1, are as defined ;
q is 0 or 1;
Z1 is a polyethylene glycol or caproyl group;
Z2 is a dipeptide or tripeptide; and
Z3 is a PAB group.
In certain embodiments, RGN is derived from a chemistry reactive group and Z1 is a
polyethylene glycol group. In certain embodiments, RGN-(SP1)q-Z1- is:
or mixture thereof; or . In some embodiments,
the ide is valine-citrulline or valine alanine.
In some embodiments, the BL is attached to the payload via tertiary amine. For
example, if the steroid is the following compound, , the RL
can bond to the tertiary amine as follows:
In some examples, set forth is a compound as follows:
wherein:
BL is a binding agent linker as defined above;
Ra and Rb are, independently in each instance, –H or alkyl.
In some examples, herein RGN is derived from a click–chemistry reactive
group. In some examples, RGN is:
, , or mixture thereof;
, , or mixture thereof; n is a bonding to a
binding agent.
In some other examples, herein RGN is selected from a group which reacts with
a cysteine or lysine residue on an antibody or an antigen–binding nt thereof. In some
examples, RGN is ; wherein is a bond to cysteine of a binding agent, e.g.,
antibody. In some examples, RGN is .
In some ments, SP1 is selected from:
, , ,
and .
In some examples, SP1 is . In some other es, SP1 is
. In other examples, SP1 is . In still other
examples, SP1 is . In some other es, SP1is
. In any of the above examples, subscripts a, b, and c are independently, in
each instance, an integer from 1 to 20.
In some embodiments, RAA3 is selected from , wherein CD is a
cyclodextrin moiety. In some embodiments, RAA3 is selected from .
In any of the compounds of Formula (II), (IIa), (IIb), or (IIc), SP1 is selected
from:
, , ,
, ,
and .
In some examples, SP1 is . In some examples, SP1 is
. In some examples,
SP1 is . In some examples, SP1 is
. In some examples, SP1 is .
In some examples, SP1 is . In some examples, SP1 is . In
some examples, SP1 is . In some examples, SP1 is . In some
examples, SP1 is . In some es, SP1 is .
In some embodiments, BL–SP1 is:
, ,
or e thereof;
, ,
or mixture thereof;
, ,
or mixture thereof;
or . In some of these examples, subscripts b, c, and d are independently, in each
instance, an integer from 1 to 20.
In any of the compounds of Formula (II), (IIa), (IIb), or (IIc), BL–SP1is
selected from:
N O O
N O O
O O
NN ,
or mixture thereof;
, ,
or e thereof;
or .
In some embodiments, A is a peptide selected from valine–citrulline, citrulline–
, lysine–phenylalanine, phenylalanine–lysine, valine–asparagine, asparagine–valine,
threonine–asparagine, asparagine–threonine, serine–asparagine, asparagine–serine,
phenylalanine–asparagine, gine–phenylalanine, leucine–asparagine, asparagine–leucine,
isoleucine–asparagine, asparagine–isoleucine, e–asparagine, asparagine–glycine,
glutamic acid–asparagine, asparagine–glutamic acid, citrulline–asparagine, asparagine–
citrulline, alanine–asparagine, or asparagine–alanine.
In some examples, A is valine–citrulline or citrulline–valine.
In some examples, A is valine–alanine or alanine–valine.
In some examples, A is lysine-valine–alanine or alanine–valine-lysine.
In some examples, A is lysine-valine–citrulline or citrulline–valine-lysine.
In some examples, A is valine.
In some examples, A is alanine.
In some examples, A is citrulline.
In some examples, A is . In some of these examples,
RAA1 is an amino acid side chain, and wherein RAA2 is an amino acid side chain.
In some examples, A is . In some of these examples,
RAA1 is an amino acid side chain, RAA2 is an amino acid side chain, and RAA3 is an amino acid
side chain that is bonded directly or indirectly to a cyclodextrin moiety.
In some examples, A is .
In some examples, A is .
In some examples, A is , wherein represents a direct
or ct bond to a cyclodextrin moiety.
In some examples, ing any of the foregoing, CD is, independently in each
instance, selected from
, ,
, ,
, and .
In some es, the CD is .
In some examples, the CD is .
In some es, the CD is .
In some examples, the CD is .
In some examples, the CD is .
In some examples, the CD is .
In some examples, A is .
In some es, Ra is H
In some examples, Ra is alkyl
In some examples, Ra is methyl, ethyl, n–propyl, i–propyl, n–butyl, t–butyl, i–
butyl, or pentyl.
In some embodiments, B is aryl.
In some examples, B is phenyl.
In some examples of compounds of Formula (II), (IIa), (IIb), or (IIc), B is
phenyl or pyridinyl.
In some es herein, B is:
or .
In these examples, R10 is alkyl, alkenyl, alkynyl, alkoxy, aryl, ryl, arylalkyl, halo,
haloalkyl, haloalkoxy, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, , ,
, NRaRb, or azido. In these es, subscripts p and m are independently, in each
instance, selected from an integer from 0 to 4.
In some examples herein, B is: .
In these examples, p is 0, 1, 2, 3 or 4. In some of these examples, R1 is, independently at each
occurrence, alkyl, alkoxy, haloalkyl, or halo. In some examples, R1 is alkyl. In some
examples, R1 is alkoxy. In some examples, R1 is haloalkyl. In some examples, R1 is halo.
In some ments of Formula (BLA), the –(NRa)s–(B)t–(CH2)u–(O)v–( SP2)w
Set forth herein are antibody-steroid conjugates having the following formulas:
x x
x x
H H H H
N O
BA L
O O
O O H
O OH
x x
x x
x x
x x
x x
H H H
O O OH
BA H
L x
x x
L BA
F H
O x
x x
x x
HO O H
O N L
O H O
H O
x x
x x
or a pharmaceutically able salt or e thereof;
wherein BA is a binding agent, and x is an integer from 1-30. In particular embodiments, BA
is an antibody. In some embodiments, x is an integer from 1 to 4. In some ments, x is
4. In some embodiments, x is 2.
Set forth herein are antibody-steroid conjugates according to Formula 1200:
or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein: BA is a
binding agent; each L is an optional linker; BA or L is covalently bonded to R3 or R4; and x is
an integer from 1 to 30. Those of skill will recognize that when L is present, L is bonded to R3
or R4; when L is not present, BA is bonded to R3 or R4. The groups R3 or R4 are described in
detail below. In particular ments, BA is an antibody. In some embodiments, x is an
integer from 1 to 4. In some embodiments, x is 4. In some embodiments, x is 2.
In certain embodiments of Formula 1200, R1 and R2 are, independently,
selected from the group consisting of -H, -OH, alkyl, -O-C(O)-alkyl, and halo; or R1 and R2
together form . In certain ments, R3 is selected from the group consisting of
-alkylene-NRaRb, -X-arylene-Y-NRaRb, -X-heteroarylene-Y-NRaRb, and N-containing
heterocycloalkyl; wherein X is absent, -N-, -CH2-, or -O-; wherein Y is absent or -CH2-; and
R4 is selected from the group consisting of alkyl, aryl, ryl, arylalkyl, heteroaryl,
-alkylene-NRaRb, lene-Y-NRaRb, -X-heteroarylene-Y-NRaRb, and N-containing
heterocycloalkyl; wherein X is absent, -N-, -CH2-, or -O-; wherein Y is absent or -CH2-.
In certain embodiments of Formula 1200, R3 is selected from the group
consisting of -OH, -O-C(O)-alkyl, -O-aryl, -NRaRb, -alkylene-NRaRb, -X-arylene-Y-NRaRb,
-X-heteroarylene-Y-NRaRb, and N-containing heterocycloalkyl; wherein X is absent, -N-,
-CH2-, or -O-; wherein Y is absent or -CH2-; and R4 is selected from the group ting of
-alkylene-NRaRb, lene-Y-NRaRb, -X-heteroarylene-Y-NRaRb, and N-containing
heterocycloalkyl; n X is absent, -N-, -CH2-, or -O-; wherein Y is absent or -CH2-.
In certain embodiments of Formula 1200, R3 is -NRaRb; and R4 is alkyl.
In each embodiment of Formula 1200, BA or L is bonded to a functional group
in R3 or R4. For instance, if R3 or R4 comprises a amino group, BA or L can be bonded to the
amino group, substituting for a hydrogen atom. In each embodiment, R5 is, ndently in
each instance, ed from a substituent in the group consisting of –OH, halo, and alkyl; n is
an integer from 0-19; and each R5 is positioned on any ring atom. In each embodiment, Ra and
Rb are, independently in each instance, selected from the group consisting of -H and alkyl; or
Ra and Rb cyclize to form cycloheteroalkyl with three to six ring atoms, including one hetero
atom, which is the N to which they are attached. In particular embodiments, BA is an antibody.
In some embodiments, x is an integer from 1 to 4. In some embodiments, x is 4. In some
embodiments, x is 2.
Set forth herein are dy-steroid conjugates according to according to
Formula 1210, 1220, 1230, or 1240:
R H
1 F
R H O
BA O
3 OH
1210 1220
1230 1240
or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof; wherein R3 is covalently
bonded to L or BA.
In certain embodiments of Formula 1210, 1220, 1230, or 1240, R1 and R2 are,
ndently, ed from the group consisting of -H, -OH, alkyl, -O-C(O)-alkyl, and halo;
or R1 and R2 together form . In certain embodiments, R3 is selected from the group
consisting of -alkylene-NRaRb, -X-arylene-Y-NRaRb, -X-heteroarylene-Y-NRaRb, and N-
containing heterocycloalkyl; wherein X is absent, -N-, -CH2-, or -O-; wherein Y is absent or
-CH2-; and R4 is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl,
heteroaryl, -alkylene-NRaRb, lene-Y-NRaRb, -X-heteroarylene-Y-NRaRb, and N-
containing heterocycloalkyl; wherein X is absent, -N-, -CH2-, or -O-; wherein Y is absent or
-CH2-. In certain embodiments, R3 is -NRaRb; and R4 is alkyl. In each embodiment, BA or L
is bonded to an amino group in R3, for instance, substituting for a hydrogen atom. In each
embodiment, Ra and Rb are, independently in each ce, selected from the group consisting
of -H and alkyl; or Ra and Rb cyclize to form cycloheteroalkyl with three to six ring atoms,
including one hetero atom, which is the N to which they are attached. In particular
embodiments, BA is an antibody. In some embodiments, x is an integer from 1 to 4. In some
embodiments, x is 4. In some embodiments, x is 2.
Set forth herein are antibody-steroid conjugates according to ing to
Formula 1310, 1320, 1330, or 1340:
1310 1320
1330 1340
or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof; wherein R4 is covalently
bonded to L or BA.
In certain ments of Formula 1310, 1320, 1330, or 1340, R3 is ed
from the group consisting of -OH, -O-C(O)-alkyl, -O-aryl, -NRaRb, -alkylene-NRaRb, -X-
e-Y-NRaRb, -X-heteroarylene-Y-NRaRb, and N-containing heterocycloalkyl; n X
is absent, -N-, -CH2-, or -O-; wherein Y is absent or -CH2-; and R4 is selected from the group
consisting of -alkylene-NRaRb, -X-arylene-Y-NRaRb, -X-heteroarylene-Y-NRaRb, and N-
containing heterocycloalkyl; wherein X is absent, -N-, -CH2-, or -O-; wherein Y is absent or
-CH2-. In each embodiment, BA or L is bonded to an amino group in R4, for instance,
substituting for a hydrogen atom. In each embodiment, Ra and Rb are, independently in each
instance, selected from the group consisting of -H and alkyl; or Ra and Rb cyclize to form
cycloheteroalkyl with three to six ring atoms, including one hetero atom, which is the N to
which they are attached. In particular embodiments, BA is an antibody. In some embodiments,
x is an r from 1 to 4. In some embodiments, x is 4. In some embodiments, x is 2.
Set forth herein are also antibody-steroid conjugates having the following
formulas:
or mixture f;
or mixture thereof;
or e thereof;
or mixture thereof;
or mixture thereof;
or e thereof;
or mixture thereof;
or e thereof;
or mixture thereof;
or mixture thereof;
or mixture thereof;
wherein Ab is an antibody and x is an integer from 1-30. In some embodiments, x is an integer
from 1 to 4. In some ments, x is 4. In some embodiments, x is 2.
Set forth herein are also antibody-steroid conjugates having the following
formulas:
or mixtures thereof.
In particular embodiments, Ab is an antibody and x is an integer from 1-30. In some
embodiments, x is an integer from 1 to 4. In some embodiments, x is 4. In some
embodiments, x is 2.
Set forth herein are also antibody-steroid conjugates having the following
formula:
Set forth herein are also antibody-steroid conjugates having the ing
formulas:
O H
O O O N
H H F
N N O
O N N
O O N H H F
N O N O O
H H2 O H
N N O
O N NH2
O O O H
H O
N O H
O O O O N
H H F
Ab H
N N O
O N N
N H H
N O N O F
H2 O H
N O
N NH2
or mixtures thereof;
or es thereof;
or mixtures thereof;
or es thereof;
or es thereof;
or es thereof;
or mixtures thereof;
or es thereof;
or mixtures f;
O O F
N H H H O
O O
Ab F
N O O OH
N O O O O
N N O O N
N O O N N
H H H
O O F
or mixtures thereof;
O O
H F
N O O
N F
N O O O O
H N O O
N O O N H
N N O
H H OH
O O
O N O O
O NH2
or mixtures thereof.
In particular embodiments, Ab is an antibody and x is an integer from 1-30. In some
ments, x is an integer from 1 to 4. In some embodiments, x is 4. In some
embodiments, x is 2.
ed herein are also g agent conjugates of budesonide or diflorasone.
Suitable binding agents for any of the conjugates provided in the instant
disclosure include, but are not limited to, antibodies, lymphokines, hormones, growth s,
viral receptors, interleukins, or any other cell binding or peptide binding molecules or
substances.
In some embodiments, the g agent is an antibody. The term "antibody",
as used herein, means any antigen-binding molecule or molecular complex comprising at least
one complementarity determining region (CDR) that specifically binds to or interacts with a
particular antigen. The term ody" includes globulin molecules comprising four
polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide
bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain
variable region (abbreviated herein as HCVR or VH) and a heavy chain nt region. The
heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain
comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain
constant region. The light chain constant region comprises one domain (CL1). The VH and VL
regions can be further subdivided into regions of hypervariability, termed complementarity
determining regions (CDRs), interspersed with regions that are more conserved, termed
framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to y-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3, FR4. In some embodiments, the FRs of the antibody (or antigen-binding portion
thereof) may be identical to the human germline sequences, or may be naturally or artificially
modified. An amino acid consensus sequence may be defined based on a side-by-side analysis
of two or more CDRs.
The term "antibody", as used herein, also includes antigen-binding fragments of
full dy molecules. The terms "antigen-binding portion" of an antibody, en-binding
nt" of an antibody, and the like, as used herein, include any naturally occurring,
enzymatically obtainable, synthetic, or genetically engineered polypeptide or rotein that
specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody
may be derived, e.g., from full antibody molecules using any suitable standard techniques such
as proteolytic digestion or recombinant genetic engineering ques involving the
manipulation and sion of DNA encoding antibody variable and optionally constant
domains. Such DNA is known and/or is readily available from, e.g., commercial sources,
DNA ies (including, e.g., phage-antibody libraries), or can be synthesized. The DNA
may be sequenced and manipulated chemically or by using molecular y techniques, for
example, to arrange one or more variable and/or constant domains into a suitable
configuration, or to introduce codons, create cysteine residues, modify, add or delete amino
acids, etc.
Non-limiting examples of antigen-binding fragments include: (i) Fab
fragments; (ii) F(ab')2 fragments; (iii) Fd nts; (iv) Fv fragments; (v) single-chain Fv
(scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the
amino acid residues that mimic the ariable region of an antibody (e.g., an isolated
complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3-
CDR3-FR4 peptide. Other ered molecules, such as domain-specific antibodies, single
domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted
dies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent
nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs),
and shark variable IgNAR domains, are also encompassed within the expression "antigenbinding
fragment," as used herein.
An n-binding fragment of an antibody will typically comprise at least one
variable domain. The variable domain may be of any size or amino acid ition and will
generally comprise at least one CDR which is adjacent to or in frame with one or more
framework sequences. In antigen-binding fragments having a VH domain associated with a VL
domain, the VH and VL domains may be situated relative to one another in any suitable
ement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or
VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a
monomeric VH or VL domain.
In certain ments, an antigen-binding fragment of an antibody may
n at least one variable domain covalently linked to at least one constant domain. Nonlimiting
, exemplary configurations of variable and constant domains that may be found within
an antigen-binding fragment of an antibody of the t disclosure include: (i) VH-CH1; (ii)
VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) -CH3; (vii) VH-CL;
(viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VLCH2-CH3
; and (xiv) VL-CL. In any configuration of variable and constant domains, including
any of the exemplary configurations listed above, the variable and constant domains may be
either directly linked to one another or may be linked by a full or partial hinge or linker region.
A hinge region may t of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which
result in a flexible or semi-flexible e between nt variable and/or constant domains
in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody of the
present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of
the variable and constant domain configurations listed above in non-covalent association with
one another and/or with one or more monomeric VH or VL domain (e.g., by disulfide bond(s)).
As with full antibody molecules, antigen-binding nts may be
monospecific or multispecific (e.g., bispecific). A multispecific antigen-binding fragment of
an antibody will typically comprise at least two different variable domains, wherein each
variable domain is capable of specifically g to a te antigen or to a different epitope
on the same antigen. Any multispecific antibody format, including the exemplary bispecific
dy formats disclosed herein, may be adapted for use in the context of an antigen-binding
fragment of an antibody of the present disclosure using routine techniques available in the art.
The antibodies of the present disclosure may function through complementdependent
cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity .
"Complement-dependent cytotoxicity" (CDC) refers to lysis of n-expressing cells by an
antibody of the instant disclosure in the ce of ment. "Antibody-dependent cellmediated
cytotoxicity" (ADCC) refers to a cell-mediated reaction in which nonspecific
cytotoxic cells that express Fc receptors (FcRs) (e.g., l Killer (NK) cells, neutrophils,
and macrophages) recognize bound antibody on a target cell and thereby lead to lysis of the
target cell. CDC and ADCC can be measured using assays that are well known and available
in the art. (See, e.g., U.S. Patent Nos 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc.
Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important in the
ability of an antibody to fix complement and e cell-dependent cytotoxicity. Thus, the
isotype of an antibody may be selected on the basis of whether it is desirable for the antibody
to mediate cytotoxicity.
The antibodies useful for the nds herein include human antibodies.
The term "human dy", as used herein, is ed to include antibodies having variable
and constant regions derived from human germline immunoglobulin sequences. The human
antibodies can include amino acid residues not d by human ne immunoglobulin
sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by
somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the
term "human antibody", as used herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species, such as a mouse, have
been grafted onto human framework sequences. The term ''human antibody'' does not include
lly occurring molecules that normally exist without modification or human
intervention/manipulation, in a lly occurring, unmodified living organism.
The antibodies can, in some embodiments, be recombinant human antibodies.
The term "recombinant human antibody", as used herein, is intended to include all human
antibodies that are prepared, expressed, created or isolated by inant means, such as
antibodies expressed using a recombinant expression vector transfected into a host cell
(described further below), antibodies isolated from a recombinant, combinatorial human
antibody y (described further below), antibodies isolated from an animal (e.g., a mouse)
that is transgenic for human immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids
Res. 20:6287-6295) or antibodies prepared, expressed, d or isolated by any other means
that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
Such recombinant human antibodies have variable and constant regions derived from human
germline immunoglobulin sequences. In certain embodiments, however, such recombinant
human dies are subjected to in vitro nesis (or, when an animal transgenic for
human Ig ces is used, in vivo somatic mutagenesis) and thus the amino acid sequences
of the VH and VL regions of the recombinant antibodies are sequences that, while derived from
and related to human germline VH and VL sequences, may not naturally exist within the human
dy germline repertoire in vivo.
Human antibodies can exist in two forms that are associated with hinge
heterogeneity. In one form, an globulin molecule comprises a stable four chain
construct of approximately 150-160 kDa in which the dimers are held together by an interchain
heavy chain disulfide bond. In a second form, the dimers are not linked via inter-chain
disulfide bonds and a molecule of about 75-80 kDa is formed composed of a covalently
coupled light and heavy chain (half-antibody). These forms have been extremely ult to
separate, even after affinity purification.
The ncy of appearance of the second form in various intact IgG isotypes
is due to, but not d to, structural differences associated with the hinge region isotype of
the antibody. A single amino acid substitution in the hinge region of the human IgG4 hinge
can icantly reduce the appearance of the second form (Angal et al. (1993) Molecular
Immunology 30:105) to levels typically observed using a human IgG1 hinge. The instant
disclosure asses antibodies having one or more mutations in the hinge, CH2 or CH3
region which may be desirable, for example, in production, to improve the yield of the desired
antibody form.
The antibodies useful for the compounds herein can be isolated antibodies. An
"isolated antibody," as used herein, means an antibody that has been identified and separated
and/or recovered from at least one component of its l environment. For example, an
antibody that has been ted or removed from at least one component of an organism, or
from a tissue or cell in which the antibody naturally exists or is naturally produced, is an
ted antibody" for purposes of the instant disclosure. An isolated antibody also includes
an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been
subjected to at least one purification or ion step. According to certain embodiments, an
isolated antibody may be substantially free of other cellular material and/or chemicals.
The antibodies useful for the compounds disclosed herein may comprise one or
more amino acid substitutions, insertions and/or deletions in the framework and/or CDR
regions of the heavy and light chain variable domains as ed to the ponding
ne sequences from which the antibodies were derived. Such mutations can be readily
ascertained by comparing the amino acid sequences disclosed herein to germline sequences
available from, for example, public antibody ce databases. The t disclosure
includes antibodies, and antigen-binding fragments thereof, which are derived from any of the
amino acid sequences disclosed herein, wherein one or more amino acids within one or more
framework and/or CDR regions are mutated to the corresponding residue(s) of the ne
sequence from which the antibody was derived, or to the corresponding residue(s) of another
human germline sequence, or to a conservative amino acid substitution of the corresponding
germline residue(s) (such sequence changes are ed to herein collectively as "germline
mutations"). A person of ordinary skill in the art, starting with the heavy and light chain
variable region sequences disclosed herein, can easily produce numerous antibodies and
antigen-binding fragments which se one or more dual germline ons or
combinations thereof. In certain embodiments, all of the framework and/or CDR residues
within the VH and/or VL domains are d back to the residues found in the original
germline sequence from which the antibody was derived. In other embodiments, only certain
residues are mutated back to the original germline sequence, e.g., only the mutated residues
found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the
d residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of
the framework and/or CDR residue(s) are d to the corresponding residue(s) of a
different ne sequence (i.e., a germline sequence that is different from the ne
ce from which the dy was originally derived). Furthermore, the antibodies of the
present disclosure may contain any combination of two or more germline mutations within the
framework and/or CDR regions, e.g., wherein certain individual residues are mutated to the
corresponding residue of a particular germline sequence while certain other es that differ
from the original germline ce are ined or are mutated to the corresponding
residue of a different germline sequence. Once obtained, antibodies and antigen-binding
fragments that contain one or more germline mutations can be easily tested for one or more
desired property such as, improved binding specificity, increased binding affinity, improved or
enhanced antagonistic or agonistic biological properties (as the case may be), reduced
immunogenicity, etc.
In some embodiments, the antibody is a monoclonal antibody, polyclonal
antibody, antibody fragment (Fab, Fab’, and F(ab)2, minibody, diabody, tribody, and the like),
or bispecific dy. Antibodies herein can be humanized using methods described in US
Patent No. 6,596,541 and US Publication No. 2012/0096572, each incorporated by reference in
their entirety.
Where the binding agent is an antibody, it binds to an antigen g partner
that is a polypeptide and may be a transmembrane molecule (e.g., receptor) or a growth factor
that might be glycosylated or phosphorylated.
Suitable targets to which the binding agent binds include any target to which
d delivery is desirable. In some embodiments, the binding agent is an antibody, modified
antibody, or antigen binding fragment there of that binds a target selected from: AXL, BAFFR,
BCMA, BCR–list components, BDCA2, BDCA4, BTLA, BTNL2 BTNL3, BTNL8, BTNL9,
C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137,
CD138, CD14, CD168, CD177, CD19, CD20, CD209, CD209L, CD22, CD226, CD248,
CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, CD40,
CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69,
CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2, CD96, CLEC12A, CLEC12B,
CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1/2, CXCR4, CXCR5,
DDR1, DDR2, DEC–205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, Fire, GITR, HHLA2,
HLA class II, HVEM, ICOSLG, IFNLR1, IL10R1, , IL12R, IL13RA1, IL13RA2,
IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA,
IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R,
ins, LAG3, LIFR, MAG/Siglec–4, MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46,
PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec–1, Siglec–10,
Siglec–5, Siglec–6, Siglec–7, Siglec–8, Siglec–9, SIRPA, , TACI, TCR–list
components/assoc, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT,
TLR2, TLR4, TROY, TSLPR, TYRO, VLDLR, VSIG4, and VTCN1.
The binding agent linkers can be bonded to the binding agent, e.g., antibody or
antigen–binding molecule, through an attachment at a particular amino acid within the
antibody or antigen–binding le. Exemplary amino acid ments that can be used in
the context of this aspect of the disclosure e, e.g., lysine (see, e.g., US 5,208,020;
US 2010/0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358–361;
e.g., US 258987;
(see, e.g.,
12456), formyl glycine (see, e.g., Carrico et al., Nat. Chem. Biol., 2007, 3:321–322; Agarwal
et al., Proc. Natl. Acad. Sci., USA, 2013, 110:46–51, and Rabuka et al., Nat. Protocols, 2012,
:1052–1067), non–natural amino acids (see, e.g.,
and acidic amino acids (see, e.g.,
via transglutaminase–based chemo–enzymatic conjugation (see, e.g., Dennler et al.,
Bioconjugate Chem. 2014, 25, 569–578). Linkers can also be conjugated to an antigen–
binding protein via attachment to carbohydrates (see, e.g., US 2008/0305497,
disulfide linkers (see, e.g.,
the binding agent is an antibody, and the antibody is bonded to the linker through a lysine
e. In some embodiments, the dy is bonded to the linker through a cysteine residue.
D. METHODS OF PREPARING COMPOUNDS
The conjugates described herein can be synthesized by coupling the linker–
payloads described herein with a binding agent, e.g., antibody under standard ation
conditions (see, e.g., Drug Deliv. 2016 Jun;23(5):1662-6; AAPS Journal, Vol. 17, No. 2,
March 2015; and Int. J. Mol. Sci. 2016, 17, 561, the ties of which are incorporated herein
by reference). Linker-payloads are tic intermediates comprising the payload of interest
and linking moiety that ultimately serves as the moiety (or portion thereof) that connects the
binding agent with the payload. Linker-payloads comprise a reactive group that reacts with the
binding agent to form the conjugates bed herein. When the binding agent is an antibody,
the antibody can be coupled to a linker-payload via one or more ne, lysine, or other
e of the antibody. Linker payloads can be coupled to cysteine residues, for example, by
ting the antibody to a reducing agent, e.g., dithiotheritol, to cleave the disulfide bonds of
the antibody, purifying the reduced dy, e.g., by gel filtration, and subsequently reacting
the antibody with a linker-payload containing a ve moiety, e.g., a maleimido group.
Suitable solvents include, but are not limited to water, DMA, DMF, and DMSO. Linkerpayloads
ning a reactive group, e.g., activated ester or acid halide group, can be coupled
to lysine residues. Suitable solvents include, but are not limited to water, DMA, DMF, and
DMSO. Conjugates can be ed using known protein techniques, including, for example,
size exclusion chromatography, dialysis, and ultrafiltration/diafiltration.
Binding agents, e.g., antibodies, can also be conjugated via click chemistry
reaction. In some embodiments of said click chemistry reaction, the linker–payload ses
a reactive group, e.g., alkyne that is e of undergoing a 1,3 ddition reaction with an
azide. Such suitable reactive groups include, but are not limited to, strained alkynes, e.g.,
those suitable for strain–promoted alkyne–azide dditions (SPAAC), cycloalkynes, e.g.,
cyclooctynes, benzannulated alkynes, and alkynes capable of undergoing 1,3 cycloaddition
reactions with azides in the absence of copper catalysts. Suitable alkynes also include, but are
not limited to, DIBAC, DIBO, BARAC, DIFO, substituted, e.g., fluorinated alkynes, aza–
cycloalkynes, BCN, and derivatives thereof. Linker–payloads comprising such reactive groups
are useful for conjugating antibodies that have been functionalized with azido groups. Such
functionalized antibodies include antibodies functionalized with azido–polyethylene glycol
groups. In certain embodiments, such functionalized antibody is derived by reacting an
antibody comprising at least one glutamine residue, e.g., heavy chain Q295, with a compound
according to the formula H2N–LL–N3, wherein LL is a divalent polyethylene glycol group, in
the presence of the enzyme transglutaminase. For convenience, in n Formulas herein, the
antibody Ab is a modified antibody with one or more covalently linked –LL–N3 groups, or
residues thereof. Preferably, each –LL–N3 is covalently bonded to an amino acid side chain of
a glutamine residue of the antibody. Also preferably, the –LL–N3 is or can be reacted with a
reactive group RG to form a covalent bond to a linker-payload. Again for convenience, in
certain Formulas herein, the -LL–N3 groups are expressly drawn.
Set forth here are methods of synthesizing the conjugates described herein
comprising contacting a binding agent, e.g., dy, with a linker-payload described herein.
In certain embodiments, the linker-payload es a extrin moiety.
In some ments, the linker d is a compound of Formula (II):
(a) R3 is RL–, RL–X–, or ;
R1 and R2 are each, ndently, –H, alkyl, alkyl–C(O)–O–, –OH, or
halo; or R1 and R2 together form , wherein R4 is alkyl, aryl,
arylalkyl, or an N–containing heterocycloalkyl; wherein the alkyl, aryl,
arylalkyl, and N–containing cycloalkyl are optionally substituted
with ; or
(b) R3 is –OH, alkyl–C(O)–O–, heteroalkyl, –NRaRb or aryloxy, wherein the
alkyl–C(O)–O–, heteroalkyl, or aryloxy is optionally substituted with
–NRaRb or halo, and R1 and R2 together form , wherein R4 is
–RL–, , or RL–Y, n Y
is an N–containing divalent heterocycle;
RL is a reactive linker;
R5 is, ndently in each instance, –OH, halo, alkyl, or kyl;
Ra and Rb are, independently in each instance, –H or alkyl;
RP, independently in each instance, is halo;
X, independently in each instance, is NRa or O;
is aryl or heteroaryl; and
n is an integer from 0–19.
Compounds of Formula (II) are linker–payloads that are useful as synthetic
intermediates in the synthesis of the conjugates described herein. These linker–payloads
comprise a reactive group that can react with an dy to form the ates described
herein.
In some examples of Formula (II), R1 and R2 are, each, independently, –H,
alkyl, or –OH. In some examples of Formula (II), one of R1 or R2 is –H, alkyl, or –OH. In
some examples of Formula (II), both R1 and R2 are either –H, alkyl, or –OH.
In some examples of Formula (II), R1 and R2 together form . In some
examples, R4 is –RL. In some examples, R4 is RL–NRa–aryl. In some other examples, R4 is
alkyl. In certain examples, R4 is arylalkyl, In some examples, R4 is aryl. In other examples,
R4 is N–containing heterocycloalkyl. In some of these examples, the alkyl, aryl, arylalkyl, or
aining heterocycloalkyl is optionally substituted.
In some examples of Formula (II), R5 is halo. In some examples of Formula
(II), R5 is fluoro. In some examples of Formula (II), one of R5 is halo. In some es of
Formula (II), R5 is halo and n is 2. In some es of Formula (II), R5 is –F and n is 1. In
some examples of Formula (II), R5 is –F and n is 2.
In some examples of Formula (II), R3 is RL. In some examples of Formula
(II), R3 is RL–NRa–aryloxy–. In some other examples of Formula (II), R3 is –OH. In some
other examples of Formula (II), R3 is alkyl–C(O)–O–. In some other examples of Formula
(II), R3 is heteroalkyl. In some other examples of a (II), R3 is –N–RaRb. In some other
examples of Formula (II), R3 is aryl. In some other examples of Formula (II), R3 is aryloxy.
In some other examples of a (II), alkyl–C(O)–O–, heteroalkyl, or aryloxy is ally
substituted with –NRaRb or halo.
In some examples of Formula (II), R3 is –OH. In some examples of Formula
(II), R3 is alkyl–C(O)–O–. In some examples of Formula (II), R3 is In some
es of a (II), R3 is heteroalkyl. In some examples of Formula (II), R3 is
or . In some examples of Formula (II), R3 is or . In some es
of Formula (II), R3 is –NRaRb. In some examples of Formula (II), R3 is . In some
examples of Formula (II), R3 is . In some examples of Formula (II), R3 is .
In some examples of Formula (II), R3 is aryloxy. In some examples of Formula (II), R3 is
. In some examples of Formula (II), R3 is . In some examples
of Formula (II), R3 is . In some examples of Formula (II), R3 is
F O
. In some examples of Formula (II), R3 is H . In some
examples of Formula (II), R3 is . In some examples of Formula (II), R3 is
. In some examples of Formula (II), R3 is . In some
examples of a (II), R3 is .
In some examples of Formula (II), R3 is .
In Formula (II), subscript n is an integer from 0–19. In some examples, n is 0.
In some other examples, n is 1. In certain examples, n is 2. In some other examples, n is 3. In
certain examples, n is 4. In some examples, n is 5. In some other examples, n is 6. In certain
examples, n is 7. In some other examples, n is 8. In certain examples, n is 9. In some
examples, n is 10. In some other examples, n is 11. In certain examples, n is 12. In some
other es, n is 13. In certain examples, n is 14. In some examples, n is 15. In some
other examples, n is 16. In n examples, n is 17. In some other examples, n is 18. In
certain examples, n is 19.
In some examples, set forth herein is a compound having the structure of
Formula (IIa):
wherein:
R5 is, independently in each instance, –OH, halo, or alkyl;
R3 is selected from –OH, alkyl–C(O)–O–, heteroalkyl, –NRaRb or aryloxy,
wherein
the alkyl–C(O)–O–, heteroalkyl, or aryloxy is optionally tuted with –
NRaRb or
halo;
RL is a reactive linker;
Ra and Rb are, independently in each instance, selected from H, alkyl, and
C(O); and
n is an integer from 0–19.
In some examples, set forth herein is a compound having the structure of
Formula (IIa2):
(IIa2);
R5 is, independently in each instance, –OH, halo, or alkyl;
R3 is –OH, alkyl–C(O)–O–, heteroalkyl, –NRaRb or aryloxy, wherein the
alkyl–C(O)–O–, heteroalkyl, or aryloxy is optionally substituted with –NRaRb or
halo;
RL is a reactive linker;
Ra and Rb are, independently in each instance, selected from H, alkyl, or
alkyl–C(O); and
n is an integer from 0–19.
In some examples of Formula (IIa2), R3 is –OH. In some examples of Formula (IIa2), R3 is
alkyl–C(O)–O–. In some examples R3 is In some examples of Formula (IIa2), R3
is heteroalkyl. In some es R3 is or. In some examples of a (IIa2),
R3 is –NRaRb. In some examples R3 is . In some examples of Formula (IIa2), R3
is y. In some examples of Formula (IIa2), R3 is . In some examples of
Formula (IIa2), R3 is . In some examples of a (IIa2), R3 is
. In some examples of Formula (IIa2), R3 is .
In some es of a (IIa2), R3 is .
In some examples, the compound of Formula (IIa2) has the following structure:
wherein:
R3 is –OH or alkyl–C(O)–O–;
R5a and R5b are each, ndently, –F or H; and
RL is a reactive linker.
In some examples, set forth herein is a compound having the structure of
Formula (IIb):
(IIb);
wherein
R5 is, independently in each instance, –OH, halo, or alkyl;
R4 is selected from alkyl, aryl, arylalkyl, or an N–containing heterocycloalkyl,
wherein the alkyl, aryl, arylalkyl, or N–containing heterocycloalkyl are
optionally
substituted with –NRaRb;
RL is a reactive linker;
Ra and Rb are, independently in each instance, selected from H, alkyl, and
alkyl–C(O); and
n is an integer from 0–19.
In some examples of Formula (IIb), R5 is halo. In some examples of Formula
(IIb), R5 is fluoro. In some examples of Formula (IIb), n is at least 2, and two of R5 is halo.
In some examples of a (IIb), R5 is F and n is 1. In some examples of Formula (IIb), R5
is –F.
In some examples of Formula (IIb), R4 is alkyl. In some examples of Formula
(IIb), R4 is methyl, ethyl, n–propyl, i–propyl, n–butyl, s–butyl, t–butyl, i–butyl, a pentyl
, a hexyl moiety, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. In some
examples of Formula (IIb), R4 is n–propyl.
In some examples, the compound of Formula (IIb) has the following structure:
R4 is alkyl;
R5a and R5b are each, independently, –F or H; and
RL is a reactive linker.
In some es, set forth herein is a compound having the structure of
Formula (IIc):
(IIc);
wherein
R1 and R2 are, independently, –H, alkyl, C(O)–O–, –OH, or halo;
R5 is, independently in each instance, selected from –OH, halo, or alkyl;
RL is a reactive linker; and
n is an integer from 0–19.
In some examples of Formula (IIc), R5 is –halo. In some examples of Formula
(IIc), R5 is fluoro. In some examples of Formula (IIc), one of R5 is halo. In some examples
of Formula (IIc), two of R5 is halo. In some examples of Formula (IIc), R5 is –F and n is 2.
In some examples of Formula (IIc), R1 is CH3.
In other examples of Formula (IIc), R1 is OH.
In some other examples of Formula (IIc), R1 is H.
In some examples of Formula (IIc), R2 is CH3.
In other es of Formula (IIc), R2 is OH.
In some other examples of Formula (IIc), R2 is H.
In some examples of Formula (IIc), R1 is CH3 and R2 is CH3.
In other examples of Formula (IIc), R1 is CH3 and R2 is OH.
In some examples of Formula (IIc), R1 is CH3 and R2 is H.
In some other examples of Formula (IIc), R1 is OH and R2 is CH3.
In other examples of Formula (IIc), R1 is OH and R2 is OH.
In some examples of Formula (IIc), R1 is H and R2 is H.
In some other examples of Formula (IIc), R1 is H and R2 is OH.
In other examples of Formula (IIc), R1 is H and R2 is H.
In some ments, the compound of Formula (IIc) has the ing
structure:
(IIc)
wherein:
R2 is methyl;
R5a and R5b are each, independently, –F or H; and
RL is a ve linker.
In certain embodiments, set forth herein is a compound having the structure of
Formula (III-R):
(III-R);
wherein:
R3 is ;
R1 and R2 are each, independently, –H, alkyl, alkyl–C(O)–O–, –OH, or
halo; or R1 and R2 together form , wherein R4 is alkyl, aryl,
arylalkyl, or an N–containing cycloalkyl; wherein the alkyl, aryl,
arylalkyl, and
aining heterocycloalkyl are optionally substituted with –NRaRb;
R5 is, independently in each instance, –OH, halo, alkyl, or arylalkyl;
Ra and Rb are, ndently in each instance, –H or alkyl;
RP, independently in each instance, is halo;
is aryl or heteroaryl;
t is an integer from 0-2;
x is an integer from 1–30; and wherein
RL is a reactive linker, defined below; SP1 and SP2 are each, independently in each
instance, absent or a spacer group residue, and wherein SP1 comprises a trivalent
linker; AA1 is a trivalent linker sing an amino acid residue; AA2 is a di-peptide
residue; PEG is a polyethylene glycol e; wherein the indicates the atom
through which the indicated chemical group is bonded to the adjacent groups in the
formula, CD is, independently in each ce, absent or a cyclodextrin residue,
wherein at least one CD is present, subscript m is an integer from 0 to 5; In these
examples, subscript m is 0, 1, 2, 3, 4, or 5. In some examples, subscript m is 0. In some
examples, subscript m is 1. In some examples, subscript m is 2. In some examples,
subscript m is 3. In some es, subscript m is 4. In some examples, subscript m is
. In some examples, any one of AA1 or AA2 comprises, independently in each
instance, an amino acid selected from alanine, valine, leucine, cine, methionine,
tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine,
glutamine, aspartic acid, ic acid, lysine, arginine, histidine, or citrulline, a
derivative thereof, or a combination thereof. In certain embodiments, AA1 is an amino
acid selected from alanine, valine, leucine, isoleucine, methionine, tryptophan,
phenylalanine, proline, glycine, , threonine, cysteine, tyrosine, asparagine,
ine, aspartic acid, ic acid, lysine, arginine, histidine, or citrulline, a
derivative thereof, or a combination f. In certain embodiments, AA1 is lysine. In
certain embodiments, AA1 is lysine or a derivative of lysine. In certain embodiments,
the AA2 is valine-citrulline. In some ments, the AA2 is citrulline-valine. In some
embodiments, the AA2 is valine-alanine. In some embodiments, the AA2 is alaninevaline.
In some embodiments, the AA2 is valine-glycine. In some embodiments, the
AA2 is e-valine. In some embodiments, the AA2 glutamate-valine-citrulline. In
some embodiments, the AA2 is glutamine-valine-citrulline. In some embodiments, the
AA2 is lysine-valine-alanine. In some embodiments, the AA2 is lysine-valine-citrulline.
In some embodiments, the AA2 is glutamate-valine-citrulline. In some examples, SP1 is
independently in each instance, selected from the group consisting of C1-6 alkylene,
-NH-, , (-CH2-CH2-O)e, -NH-CH2-CH2-(-O-CH2-CH2)e-C(O)-, -C(O)-(CH2)u-
C(O)-, -C(O)-NH-(CH2)v-, and combinations thereof, wherein subscript e is an integer
from 0 to 4, subscript u is an integer from 1 to 8, and subscript v is an integer from 1 to
8. In some examples, SP2 is independently in each instance, selected from the group
consisting of C1-6 alkylene, -NH-, -C(O)-, (-CH2-CH2-O)e, -NH-CH2-CH2-(-O-CH2-
CH2)e-C(O)-, -C(O)-(CH2)u-C(O)-, -C(O)-NH-(CH2)v-, and combinations thereof,
wherein subscript e is an integer from 0 to 4, subscript u is an integer from 1 to 8, and
subscript v is an r from 1 to 8.
In certain embodiments, set forth herein is a compound having the ure of
Formula (IIIc-R):
Formula (IIIc-R)
RL is a reactive linker;
CD is a cyclodextrin;
SP1 is a spacer group;
AA4 is an amino acid residue;
AA5 is a dipeptide residue;
PEG is polyethylene glycol;
m is an integer from 0 to 4;
x is an integer from 0 to 30;
R4 is alkyl, aryl, arylalkyl, or an N–containing heterocycloalkyl; wherein the alkyl, aryl,
arylalkyl, and N–containing heterocycloalkyl are optionally substituted with –NRaRb;
Ra and Rb are, independently in each instance, –H or alkyl;
SP1 and SP2 are each, independently in each ce, absent or a spacer group residue, and
wherein SP1 comprises a trivalent linker; AA4 is a trivalent linker comprising an amino acid
residue; AA5 is a di-peptide e; PEG is a polyethylene glycol residue; wherein the
indicates the atom through which the indicated chemical group is bonded to the adjacent
groups in the formula, CD is, independently in each instance, absent or a extrin residue,
wherein at least one CD is present, subscript m is an integer from 0 to 5; In these examples,
subscript m is 0, 1, 2, 3, 4, or 5. In some examples, subscript m is 0. In some examples,
subscript m is 1. In some es, subscript m is 2. In some examples, ipt m is 3. In
some examples, subscript m is 4. In some examples, ipt m is 5. In some examples, any
one of AA4 or AA5 comprises, independently in each instance, an amino acid ed from
alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, e, serine,
ine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine,
arginine, histidine, or citrulline, a derivative f, or a combination thereof. In certain
embodiments, AA4 is an amino acid selected from alanine, valine, leucine, isoleucine,
methionine, tryptophan, phenylalanine, proline, glycine, serine, threonine, cysteine, tyrosine,
asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, or citrulline, a
derivative thereof, or a combination thereof. In certain ments, AA4 is lysine. In certain
embodiments, AA4 is lysine or a derivative of lysine. In certain embodiments, the AA5 is
valine-citrulline. In some embodiments, the AA5 is citrulline-valine. In some ments, the
AA5 is valine-alanine. In some embodiments, the AA5 is alanine-valine. In some embodiments,
the AA5 is valine-glycine. In some embodiments, the AA5 is glycine-valine. In some
embodiments, the AA5 glutamate-valine-citrulline. In some embodiments, the AA5 is
glutamine-valine-citrulline. In some embodiments, the AA5 is lysine-valine-alanine. In some
embodiments, the AA5 is lysine-valine-citrulline. In some embodiments, the AA5 is glutamatevaline-citrulline.
In some examples, SP1 is ndently in each instance, selected from the
group consisting of C1-6 alkylene, -NH-, -C(O)-, (-CH2-CH2-O)e, -NH-CH2-CH2-(
-O-CH2-CH2)e-C(O)-, -C(O)-(CH2)u-C(O)-, -C(O)-NH-(CH2)v-, and combinations thereof,
wherein ipt e is an integer from 0 to 4, subscript u is an integer from 1 to 8, and
subscript v is an integer from 1 to 8. In some examples, SP2 is ndently in each instance,
selected from the group consisting of C1-6 alkylene, -NH-, -C(O)-, (-CH2-CH2-O)e,
-NH-CH2-CH2-(-O-CH2-CH2)e-C(O)-, -C(O)-(CH2)u-C(O)-, -C(O)-NH-(CH2)v-, and
ations thereof, wherein subscript e is an integer from 0 to 4, subscript u is an integer
from 1 to 8, and subscript v is an integer from 1 to 8.
As used herein, the phrase “reactive linker,” or the abbreviation “RL” refers to a
monovalent group that comprises a reactive group and linking group, depicted as ,
wherein RG is the reactive group and L is the linking group. The linking group is any divalent
moiety that s the reactive group to a d. The linking group also includes any
trivalent moiety that bridges the reactive group, a cyclodextrin moiety, and a payload. The
reactive s (RL), together with the payloads to which they are bonded, comprise
intermediates (“linker–payloads”) useful as synthetic precursors for the preparation of the
antibody d conjugates described . The reactive linker contains a reactive group
(“RG”), which is a functional group or moiety that reacts with a reactive portion of an
antibody, modified dy, or antigen binding nt thereof. The moiety resulting from
the reaction of the reactive group with the antibody, modified antibody, or antigen binding
fragment thereof, together with the linking group, comprise the “binding agent linker” (“BL”)
portion of the conjugate, bed herein. In certain embodiments, the “reactive group” is a
onal group or moiety (e.g., maleimide or NHS ester) that reacts with a cysteine or lysine
residue of an dy or antigen–binding fragment thereof. In certain embodiments, the
“reactive group” is a functional group or moiety that is capable of undergoing a click try
reaction. In some embodiments of said click chemistry reaction, the reactive group is an
alkyne that is e of oing a 1,3 cycloaddition on with an azide. Such suitable
reactive groups include, but are not limited to, strained s, e.g., those suitable for –
promoted alkyne–azide cycloadditions (SPAAC), cycloalkynes, e.g., cyclooctynes,
benzannulated alkynes, and alkynes capable of undergoing 1,3 cycloaddition reactions with
alkynes in the absence of copper catalysts. le alkynes also include, but are not limited
to, DIBAC, DIBO, BARAC, substituted, e.g., fluorinated alkynes, aza–cycloalkynes, BCN,
and derivatives thereof. Linker–payloads comprising such reactive groups are useful for
conjugating dies that have been functionalized with azido . Such functionalized
antibodies include antibodies functionalized with azido–polyethylene glycol groups. In certain
embodiments, such functionalized antibody is derived by reacting an antibody comprising at
least one glutamine residue, e.g., heavy chain Q295, with a compound according to the formula
–N3, wherein LL is, for example, a divalent polyethylene glycol group, or wherein LL
is a trivalent group which includes polyethylene glycol and a cyclodextrin moiety, in the
presence of the enzyme transglutaminase. In some embodiments, the antibody is a
functionalized antibody having the following structure:
wherein Ab is an antibody, R is hydrocarbyl, n is an integer from 1 to 10, w is an integer from
1-10. In certain embodiments, R is ethylene. In certain embodiments, n is 3. In certain
embodiments, w is 2 or 4.
In some examples, the reactive group is an , e.g., , which can react via click
chemistry with an azide, e.g., , to form a click chemistry product, e.g.,
, its regioisomer, or a mixture thereof. In some examples, the reactive group is
an , e.g., , which can react via click chemistry with an azide, e.g.,
to form a click chemistry product, e.g., . In some examples, the
reactive group is an alkyne, e.g., , which can react via click chemistry with an azide,
e.g., , to form a click chemistry product, e.g., , its regioisomer, or a
mixture thereof. In some examples, the reactive group is a onal group, e.g.,
,which reacts with a cysteine residue on an antibody or antigen–binding fragment thereof, to
form a bond thereto, e.g., , wherein Ab refers to an antibody or antigen–binding
fragment thereof and S refers to the S atom on a cysteine residue through which the functional
group bonds to the Ab. In some examples, the reactive group is a functional group, e.g.,
,which reacts with a lysine e on an dy or n–binding fragment
thereof, to form a bond thereto, e.g., , wherein Ab refers to an antibody or
antigen–binding fragment thereof and –NH- refers to the end of the lysine residue through
which the functional group bonds to the Ab. In some examples, this N atom on a lysine
residue through which the functional group bonds is indicated herein as the letter N above a
bond, e.g., .
In some embodiments, RL is a monovalent moiety of a (RLA);
1)q–(A)z–(NRa)s–(B)t–(CH2)u–(O)v–( SP2)w–(RLA);
n RG is a reactive group;
A is an amino acid or a peptide;
Ra is H or alkyl;
B is aryl, heteroaryl, or heterocycloalkyl, wherein aryl, heteroaryl, or
heterocycloalkyl is optionally substituted with alkyl, –OH, or –N–RaRb;
SP1 and SP2 are, independently, a spacer groups; and q, z, s, t, u, v, and w are,
independently in each instance, 0 or 1.
In some embodiments, RL is RG–(SP1)q–(A)z–. In some embodiments, RL is
RG–(SP1)q–(A)2–. In some embodiments, RL is a moiety of Formula (RLA1)
(RLA1)
wherein RAA1 and RAA2 are each, independently, amino acid side chains. In some examples of
Formula RLA1, SP1 is a divalent polyethylene glycol group and RG is a group sing an
alkyne that is capable of undergoing a 1,3–cycloaddition reaction with an azide.
In some ments, RL has the following structure:
RG–(SP1)q–Z1–Z2–Z30–1–
wherein:
RG, SP1, and q are as defined herein;
Z1 is a polyethylene glycol or caproyl group;
Z2 is a dipeptide; and
Z3 is a PAB group.
In some other embodiments, BL is a trivalent moiety of Formula (BLB);
–RGN–(SP1)q–(A)z–(NRa)s–(B)t–(CH2)u–(O)v–( SP2)w–(BLB);
wherein RGN is as defined herein;
A is tripeptide, wherein at least one of the amino acids in the tripeptide is
bonded directly or indirectly to a cyclodextrin moiety;
Ra is H or alkyl;
B is aryl, heteroaryl, or heterocycloalkyl, wherein aryl, aryl, or
heterocycloalkyl is optionally substituted with alkyl, –OH, or –NRaRb;
SP1 and SP2 are, independently, a spacer groups; and q, z, s, t, u, v, and w are,
independently in each instance, 0 or 1.
In some examples, the cyclodextrin (CD) is bonded directly to an amino acid
residue, such as a lysine amino acid residue. This means that the CD is one bond position away
from the lysine amino acid covalent linker. In some of these examples, the covalent linker is
also bonded ly to a payload moiety. This means that the covalent linker is one bond
position away from a payload such as, but not limited to a steroid payload set forth herein. In
some of these examples, the covalent linker is also bonded directly to a CD moiety. This
means that the covalent linker is one bond position away from a CD, such as the CD(s) set
forth herein. In some of these examples, the covalent linker is a lysine amino acid or a
derivative thereof.
In some examples, the CD is bonded ctly to a covalent linker in a linking
group (e.g., a BL). This means that the CD is more than one bond position away from the
covalent linker. This also means that the CD is bonded through another moiety to the covalent
linker. For example, the CD may be bonded to a ide group which is bonded to a
polyethylene glycol group which is bonded to the covalent . In some of these examples,
the covalent linker is also bonded indirectly to a payload moiety. This means that the covalent
linker is more than one bond position away from a payload such as, but not limited to a steroid
payload set forth herein. This also means that the covalent linker is bonded through another
moiety to the payload. For example, the covalent linker may be bonded to a dipeptide, such as
but not limited to a or Val-Cit, which may be bonded to para-amino benzoyl which may
be bonded to the payload. In some of these examples, the nt linker is also bonded
indirectly to a cyclodextrin moiety. This means that the covalent linker is more than one bond
position away from a cyclodextrin, such as the cyclodextrins set forth herein. This also means
that the covalent linker is bonded through another moiety to the cyclodextrin. For example, the
covalent linker may be bonded to a polyethylene glycol group which may be bonded to
ve group which may be bonded to the cyclodextrin. In some of these examples, the
covalent linker is a lysine amino acid or a derivative thereof.
In some embodiments, BL is –RGN–(SP1)q–(A)z–. In some ments, BL is
–RGN–(SP1)q–(A)2–. In some ments, BL is a moiety of Formula (BLB1)
(BLB1)
wherein RAA1 and RAA2 are each, ndently, amino acid side chains. RAA3 is an amino acid
side chain that is bonded directly or indirectly to a cyclodextrin moiety. In some examples of
Formula RLB1, SP1 is a divalent polyethylene glycol group and RGN is a cloaddition
reaction adduct of the reaction between an alkyne and an azide.
In some examples, A is . In some of these examples,
RAA1 is an amino acid side chain, RAA2 is an amino acid side chain, and RAA3 is an amino acid
side chain that is bonded directly or indirectly to a cyclodextrin .
In some examples, A is , wherein represents a direct
or indirect bond to a cyclodextrin moiety.
In some examples, including any of the foregoing, CD is, independently in each
instance, selected from
, ,
, ,
, and .
In some es, the CD is .
In some examples, the CD is .
In some es, the CD is .
In some examples, the CD is .
In some examples, the CD is .
In some examples, the CD is .
In some examples, A is .
In some embodiments, the RL attaches to a tertiary amine. For example, if the
steroid is the following compound, , the RL may bond to the
tertiary amine as follows:
In some examples, set forth is a compound as follows:
wherein:
RL is a reactive linker as defined above;
Ra and Rb are, independently in each instance, –H or alkyl.
In some es, herein RG is selected from a click–chemistry reactive group.
In some other examples, herein RG is ed from a group which reacts with a
cysteine or lysine residue on an antibody or an n–binding fragment thereof.
In some embodiments, RG is
, , , , , or .
In some examples, RG is . In other examples, RG is . In some other
examples, RG is . In some examples, RG is . In other examples, RG is
. In other examples, RG is .
In some embodiments, SP1 may be selected from:
, , ,
or .
In some examples, SP1 is . In some other examples, SP1 is
. In other examples, SP1 is . In still other
examples, SP1 is . In some other examples, SP1is
In any of the above examples, ipts a, b, and c are independently, in each
instance, an integer from 1 to 20.
In any of the compounds of Formula (II), (IIa), (IIb), or (IIc), SP1 may be
selected from:
, , ,
, , or
In some examples, SP1 is . In some examples,
SP1 is , . In some
examples, SP1 is . In some examples,
SP1 is . In some examples, SP1 is .
In some examples, SP1 is . In some examples, SP1 is . In
some examples, SP1 is . In some examples, SP1 is . In some
examples, SP1 is . In some examples, SP1 is .
In some ments, RL–SP1 may be selected from the group consisting of:
, ,
, ,
or . In some of these examples, subscripts b,
c, and d are independently, in each instance, an r from 1 to 20.
In some examples RL–SP1– is . In some
examples RL–SP1 is . In some examples RL–SP1
is . In some examples RL–SP1 is
. In some examples RL–SP1 is
. In some examples RL–SP1is .
In any of the compounds of Formula (II), (IIa), (IIb), or (IIc), RL–SP1is
selected from:
, or .
In some embodiments, A is a peptide selected from valine–citrulline, citrulline–
, lysine–phenylalanine, phenylalanine–lysine, valine–asparagine, asparagine–valine,
threonine–asparagine, asparagine–threonine, serine–asparagine, asparagine–serine,
phenylalanine–asparagine, asparagine–phenylalanine, leucine–asparagine, asparagine–leucine,
isoleucine–asparagine, asparagine–isoleucine, glycine–asparagine, asparagine–glycine,
glutamic sparagine, asparagine–glutamic acid, citrulline–asparagine, asparagine–
citrulline, alanine–asparagine, or asparagine–alanine.
In some es, A is valine–citrulline or citrulline–valine.
In some examples, A is valine–alanine or alanine–valine.
In some examples, A is valine.
In some examples, A is alanine.
In some es, A is citrulline.
In some examples, A is . In some of these examples,
RAA1 is an amino acid side chain, and wherein RAA2 is an amino acid side chain.
In some examples, A is .
In some examples, A is .
In some examples, Ra is H
In some examples, Ra is alkyl
In some es, Ra is methyl, ethyl, n–propyl, i–propyl, n–butyl, t–butyl, i–
butyl, or pentyl.
In some embodiments, B is aryl.
In some examples, B is phenyl.
In some examples of compounds of Formula (II), (IIa), (IIb), or (IIc), B is
phenyl or pyridinyl.
In some examples herein, B is:
or .
In these examples, R10 is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkylaryl,
kyl, halo, haloalkyl, haloalkoxy, heteroaryl, heterocycloalkyl, yl, cyano, nitro,
, , , NRaRb, or azido. In these examples, subscripts p and m are
independently, in each instance, selected from an integer from 0 to 4.In some examples herein,
B is: .
In these examples, p is 0, 1, 2, 3 or 4. In some of these es, R1 is, independently at each
occurrence, alkyl, alkoxy, haloalkyl, or halo. In some examples, R1 is alkyl. In some
examples, R1 is alkoxy. In some examples, R1 is haloalkyl. In some examples, R1 is halo.
In some ments of Formula (RLA), the –(NRa)s–(B)t–(CH2)u–(O)v–( SP2)w
Provided herein are also linker–payloads of budesonide or diflorasone. In some
embodiments, provided herein is a linker–payload having the ing structure:
wherein RL is a reactive linker.
Examples of linker-payloads include, but are not limited to:
NWHWO/VOWO/VOW
CANNOWONOWOVLHH/O Z]:
\: j
0 o o
A H
O /\/O\/\o/\JJ\N N, (KN
H H
O o o M
GAOANNOWONOwOVkN H
N’“ N@0
H H H
—153—
H H H O
O OH
O O
H O
H O
N O
N O N
O N
O N N
O H
O H
HN O
O H O O
OH O
N O OH
N OH
O OH
OH HO
O O
HO O
O OH OH
JL /
0/ Wm ”
(37: H
NH2 0
o o o
/ NOWONO\/\(NH H ©floku
u Ngku/
o o o ‘
, m Hg
HN\¢O /\ ‘
$ka Ho 0H9+6§9 OH
O x
N_\ NS
N 2/ 0“
0 fi
\ O
0 K
O\ ‘\OH
HO \g OHOHOM
0:HO/
\j (0
—155—
/’“x<
KH H H
w/Om/N\/\O/\/OM\O/\/OWN
O OH
/\ O
O 0 /©/ 0
H H 9 H
‘ H NYAH
o O O 2
‘ \/ NWNwO/VOWONOWN HO
/ \
‘ O
\ / K
HN¥\O o GHQ-1O WWO
O \N
NCN’ OH 0H0
O HO\\
OH / O/
O HO)/ \ROH
O OHOHO [O
—156—
and salts f.
E. PHARMACEUTICAL COMPOSITIONS AND S OF TREATMENT
The present sure includes methods of treating diseases, conditions, or
disorders e.g., inflammatory diseases and autoimmune disorders, or managing ms
thereof, comprising administering a therapeutically ive amount of one or more of the
compounds disclosed herein. Included are any diseases, disorders, or conditions associated
with the glucocorticoid receptor, orticoid binding, and/or glucocorticoid receptor
signaling. Such methods comprise administering a steroid payload or protein conjugate thereof
described herein to a patient. Thus, included in this disclosure are methods of treating a
disease, disorder, or condition associated with the glucocorticoid receptor comprising
administering a nd of Formula (I), (I)1, or n conjugate thereof, e.g., compound of
a (III) to a patient having said disease, disorder, or condition. Set forth herein are
s of treating a disease, disorder, or condition ated with the glucocorticoid receptor
comprising administering a protein conjugate of a compound of Formula ed from the
group consisting of (A), (A1), (A2), (A3), (A4), (A5), (A6), (A7), (I), (I1), (PIa), (PIb–1), (PIb–
2), PIc–1), ), (PId–1), (PId–2), (PIe–1), (PIe–2), (PII), (PIIa), (PIIb), (PIII), (PIIIa),
(PIIIb), (PIV), (PV), (PVa), (PVb), (PVI), (PVII), (PVIIa), (PVIIb), –1), (PVIIb–2),
(PVIII), and combinations thereof.
In some embodiments, the disease, disorder, or condition is allergic state,
including but not limited to asthma, atopic dermatitis, contact dermatitis, drug hypersensitivity
reactions, ial or seasonal allergic rhinitis, and serum ss; dermatologic diseases,
including but not limited to bullous dermatitis herpetiformis, exfoliative erythroderma,
mycosis fungoides, pemphigus, and severe ma multiforme (Stevens-Johnson syndrome);
endocrine disorders, including but not limited to primary or secondary adrenocortical
insufficiency, congenital adrenal hyperplasia, alcemia associated with cancer, and
nonsuppurative thyroiditis; gastrointestinal diseases; hematologic disorders, including but not
limited to acquired (autoimmune) tic anemia, congenital (erythroid) hypoplastic anemia
(Diamond-Blackfan anemia), idiopathic thrombocytopenic purpura in adults, pure red cell
aplasia, and secondary thrombocytopenia; trichinosis; tuberculous meningitis with
subarachnoid block or impending block; neoplastic es, including but not limited to
leukemias and lymphomas; s system disorders, including but not limited to acute
exacerbations of le sclerosis, cerebral edema associated with primary or metastatic brain
tumor, craniotomy, or head injury; ophthalmic diseases, including but not limited to
sympathetic ophthalmia, temporal arteritis, uveitis, and ocular inflammatory conditions
unresponsive to topical corticosteroids; renal diseases, including but not limited to for inducing
a diuresis or remission of proteinuria in idiopathic tic me or that due to lupus
erythematosus; respiratory diseases, including but not limited to berylliosis, ating or
disseminated pulmonary tuberculosis when used concurrently with appropriate antituberculous
chemotherapy, idiopathic eosinophilic pneumonias, symptomatic dosis; and Rheumatic
disorders, ing but not limited to use as adjunctive y for short-term administration
(to tide the patient over an acute episode or bation) in acute gouty arthritis, acute
rheumatic carditis, ankylosing spondylitis, psoriaticarthritis, rheumatoid arthritis, including
juvenile rheumatoid arthritis, and for use in dermatomyositis, polymyositis, and systemic lupus
erythematosus.
In some es, set forth herein is a method for treating a e, er,
or condition selected from an autoimmune disease, an allergy, tis, asthma, a breathing
disorder, a blood disorder, a cancer, a collagen disease, a connective tissue disorders, a
dermatological disease, an eye disease, an endocrine problem, an immunological disease, an
inflammatory disease, an intestinal ers, a gastrointestinal disease, a neurological
disorder, an organ transplant condition, a rheumatoid disorder, a skin disorder, a swelling
condition, a wound healing condition, and a combination thereof comprising administering a
steroid payload or conjugate thereof described herein.
In some es, the autoimmune disorder is selected from multiple sclerosis,
autoimmune hepatitis, shingles, systemic lupus erythematosus (i.e., lupus), myasthenia gravis,
Duchenne ar phy, and sarcoidosis. In some examples, the breathing disorder is
selected from asthma, chronic obstructive pulmonary disease, bronchial mation, and
acute itis. In some examples, the cancer is ed from leukemia, lymphoblastic
leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin’s
lymphoma, Non–Hodgkin’s lymphoma (NHL), and multiple myeloma. In some examples, the
collagen disease is systemic lupus erythematosus. In some examples, the eye e is
keratitis. In some examples, the endocrine problem is selected from Addison's Disease, adrenal
insufficiency, adrenocortical, and congenital adrenal hyperplasia. In some examples, the
inflammatory disease is selected from joint inflammation, tendon inflammation, bursitis,
epicondylitis, Crohn's disease, inflammatory bowels disease, lipid pneumonitis thyroiditis,
urticaria (hives), pericarditis, nephrotic syndrome, and uveitis. In some examples, the
inal disorder is selected from ulcerative colitis, Crohn’s disease, and inflammatory
bowels disease. In some examples, the rheumatoid disorder is selected from rheumatoid
arthritis, polymyalgia rheumatic, psoriatic arthritis, ankylosing spondylitis, and systemic lupus
erythematosus. In some examples, the skin disorder is selected from psoriasis, eczema, and
poison ivy. In some examples, the neurological disorder is chronic inflammatory
demyelinating polyradiculoneuropathy.
In some embodiments, the compounds bed herein are administered to a
patient to treat an acute inflammatory event, including but not d to shock, brain edema,
and graft-vs-host disease. In some embodiments, the compounds described herein are
administered to treat lympholytic s, including but not limited to those associated with
hematological ancies, e.g., leukemias, lymphomas, and myelomas.
In some examples, set forth herein is a method for reducing inflammation in a
t in need thereof, comprising administering to a subject in need thereof a therapeutically
effective amount of a steroid or conjugate thereof described herein. In some examples, set
forth herein is a method for modulating the immune system in a subject in need thereof,
comprising administering to a subject in need thereof a therapeutically effective amount of a
steroid or ate thereof described herein. In some examples, set forth herein is a method
for modulating cortisol levels in a subject in need thereof, comprising administering to a
subject in need thereof a therapeutically effective amount of a steroid or ate thereof
described . In some examples, set forth herein is a method of reducing lymphocyte
migration in a t in need thereof, comprising administering to a subject in need thereof a
therapeutically effective amount of a steroid or conjugate f described herein. In some
examples, set forth herein is a method of treating hypercalcemia due to cancer, Meniere's
disease, a migraine headache, a cluster he, a severe aphthous ulcer, laryngitis, severe
ulosis, a Herxheimer reaction to syphilis, a ensated heart failure, allergic is
or nasal polyps, comprising administering to a subject in need thereof a steroid payload or
conjugate thereof described herein. In some examples, the compounds disclosed herein can be
used for treating inflammatory bowel disease, Crohn's disease, or ulcerative colitis. In some
examples, the disease, disorder, or condition is a chronic inflammatory condition, including but
not limited to asthma, skin infections, and ocular infections. In some es, compounds
described herein are used for immunosuppression in ts undergoing organ transplantation.
In some embodiments, the steroid payloads and conjugates thereof described
herein are administered to a patient to treat a s er associated with GR signaling,
including but not limited to psychiatric disorders such as schizophrenia, drug addiction, posttraumatic
stress disorder (PTSD), and mood disorders, substance abuse, stress, and anxiety.
In some embodiments, the steroid payloads and conjugates thereof described herein are
administered to a patient to treat a visual system disorder, including but not limited to ocular
inflammation (e.g., ctivitis, keratitis, s), r edema, and macular
degeneration. In some embodiments, the steroid payloads and conjugates thereof described
herein are administered to a patient to treat a cardiovascular disorder. In some embodiments,
the steroid payloads and conjugates thereof described herein are administered to a patient to
treat a e and/or liver metabolism disorder. In some embodiments, the steroid payloads
and conjugates thereof described herein are administered to a patient to treat a musculoskeletal
system disorder. In some embodiments, the steroid payloads and conjugates thereof described
herein are administered to a patient to treat a cutaneous inflammatory condition, such as
eczema and psoriasis.
The protein conjugates described herein provide a means for ed ry
of its steroid payload to particular cells or organ systems, thereby reducing or preventing side
effects that result from administration of the free unconjugated d d. Thus,
provided herein are methods for treating a disease, disorder, or condition associated with the
glucocorticoid receptor comprising administering a ate of Formula (I) or (I)1, to a
patient having said disease, disorder, or condition, wherein the side effects ated with
administration of the free steroid payload of said conjugate is reduced. Furthermore, provided
herein are methods of delivering a compound of Formula (I) or (I)1 to a cell sing
ting said cell with a protein conjugate the compound of Formula (I) or (I)1, wherein the
protein conjugate comprises an antibody or antigen binding fragment thereof that binds a
surface antigen of said cell.
The compounds described herein can be administered alone or together with
one or more onal therapeutic agents. The one or more additional therapeutic agents can
be administered just prior to, concurrent with, or shortly after the administration of the
compounds described herein. The t disclosure also includes pharmaceutical
compositions comprising any of the compounds described herein in combination with one or
more additional eutic agents, and methods of treatment comprising administering such
combinations to subjects in need thereof.
Suitable additional therapeutic agents include, but are not limited to: a second
glucocorticoid, an autoimmune therapeutic agent, a hormone, a biologic, or a monoclonal
antibody. Suitable therapeutic agents also include, but are not limited to any ceutically
acceptable salts, acids or derivatives of a compound set forth herein.
The compounds described herein can also be administered and/or co–
formulated in combination with antivirals, antibiotics, analgesics, corticosteroids, steroids,
oxygen, antioxidants, COX inhibitors, cardioprotectants, metal chelators, IFN–gamma, and/or
NSAIDs.
In some embodiments of the methods described , le doses of a
compound described herein (or a pharmaceutical composition comprising a ation of an
nd described herein and any of the additional therapeutic agents mentioned herein)
may be administered to a subject over a defined time course. The methods according to this
aspect of the disclosure comprise tially administering to a t multiple doses of a
compound described herein. As used herein, ntially administering” means that each
dose of the compound is administered to the subject at a different point in time, e.g., on
different days ted by a predetermined interval (e.g., hours, days, weeks or months).
The present disclosure es methods which comprise sequentially administering to the
patient a single initial dose of a compound described herein, followed by one or more
secondary doses of the compound, and optionally followed by one or more tertiary doses of the
compound.
The terms "initial dose, " "secondary doses, " and "tertiary doses, " refer to the
temporal sequence of administration of the compounds described herein. Thus, the "initial
dose" is the dose which is administered at the beginning of the treatment regimen (also referred
to as the “baseline dose”); the "secondary doses" are the doses which are administered after the
initial dose; and the “tertiary doses” are the doses which are administered after the secondary
doses. The initial, secondary, and tertiary doses can all contain the same amount the
nd described herein, but generally can differ from one another in terms of frequency of
administration. In certain embodiments, the amount of the compound contained in the initial,
secondary and/or ry doses varies from one another (e.g., adjusted up or down as
appropriate) during the course of treatment. In certain embodiments, two or more (e.g., 2, 3, 4,
or 5) doses are administered at the beginning of the treatment regimen as "loading doses"
followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance
doses”).
In certain exemplary embodiments of the present disclosure, each ary
and/or tertiary dose is administered 1 to 26 (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7,
7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½, 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½,
18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½, or more)
weeks after the immediately preceding dose. The phrase “the immediately preceding dose,” as
used herein, means, in a ce of multiple administrations, the dose the compound which is
administered to a patient prior to the administration of the very next dose in the sequence with
no intervening doses.
The methods according to this aspect of the disclosure may comprise
administering to a patient any number of secondary and/or tertiary doses of the compound.
For example, in certain embodiments, only a single secondary dose is administered to the
patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses
are administered to the patient. Likewise, in certain embodiments, only a single ry dose is
administered to the t. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or
more) tertiary doses are administered to the patient. The administration regimen may be
carried out indefinitely over the lifetime of a ular subject, or until such treatment is no
longer therapeutically needed or advantageous.
In embodiments involving multiple secondary doses, each secondary dose may
be administered at the same frequency as the other secondary doses. For example, each
ary dose may be administered to the patient 1 to 2 weeks or 1 to 2 months after the
immediately preceding dose. Similarly, in embodiments involving multiple tertiary doses,
each tertiary dose may be stered at the same frequency as the other tertiary doses.
For example, each tertiary dose may be administered to the patient 2 to 12 weeks after the
immediately preceding dose. In certain embodiments of the disclosure, the frequency at which
the secondary and/or tertiary doses are administered to a patient can vary over the course of the
treatment regimen. The frequency of stration may also be adjusted during the course of
treatment by a physician depending on the needs of the dual patient ing clinical
examination.
The present disclosure es administration regimens in which 2 to 6 loading
doses are stered to a patient at a first frequency (e.g., once a week, once every two
weeks, once every three weeks, once a month, once every two months, etc.), followed by
administration of two or more maintenance doses to the patient on a less nt basis.
For example, according to this aspect of the sure, if the loading doses are administered at
a frequency of once a month, then the maintenance doses may be administered to the patient
once every six weeks, once every two , once every three months, etc.
The present disclosure includes pharmaceutical compositions of the compounds
and/or conjugates described herein, e.g., the compounds of Formula (I) and (II), e.g.,
compositions comprising a compound described herein, a salt, stereoisomer, polymorph
thereof, and a pharmaceutically acceptable carrier, diluent, and/or excipient. Examples of
suitable carriers, diluents and excipients include, but are not d to: buffers for maintenance
of proper composition pH (e.g., citrate buffers, succinate buffers, e buffers, phosphate
buffers, lactate buffers, oxalate buffers and the like), carrier proteins (e.g., human serum
albumin), nanoparticles, saline, polyols (e.g., trehalose, sucrose, xylitol, sorbitol, and the like),
tants (e.g., polysorbate 20, polysorbate 80, olate, and the like), antimicrobials, and
antioxidants.
In some examples, set forth herein is a method of treating a disease, disorder or
condition including administering to a patient having said disorder a therapeutically effective
amount of a compound of Formula I, III, or a ceutical composition f.
In some examples, set forth herein is a method of treating a disease, disorder or
ion including administering to a patient having said disorder a eutically effective
amount of a compound set forth herein, or a pharmaceutical ition f.
In some examples, set forth herein is a method of treating a disease, disorder or
condition selected from the group consisting of an immunological disease, autoimmune
disease, inflammation, asthma, or an inflammatory bowel disorder, Crohn's disease, ulcerative
colitis.
In some examples, set forth herein is a method of treating a disease, disorder or
condition by ing an antigen, e.g., cell–surface expressing antigen, to which steroid
delivery can achieve a therapeutic benefit comprising administering the conjugates described
herein. In some embodiments, the antigen is AXL, BAFFR, BCMA, BCR–list components,
BDCA2, BDCA4, BTLA, BTNL2 BTNL3, BTNL8,BTNL9, C10orf54, CCR1, CCR3, CCR4,
CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD168, CD177, CD19,
CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30,
, CD33, CD37, CD38, CD4, CD40, CD44, CD45, CD47, CD46, CD48, CD5, CD52,
CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80,
CD86, CD90.2, CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM,
CSF1R, CTLA4, CXCR1/2, CXCR4, CXCR5, DDR1, DDR2, DEC–205, DLL4, DR6, FAP,
FCamR, FCMR, FcR's, Fire, GITR, HHLA2, HLA class II, HVEM, ICOSLG, IFNLR1,
IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, ,
IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R,
IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, Integrins, LAG3, LIFR, MAG/Siglec–4, MMR,
MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PROKR1, PVR, PVRIG, PVRL2,
PVRL3, RELT, , Siglec–1, Siglec–10, Siglec–5, Siglec–6, Siglec–7, Siglec–8,
Siglec–9, SIRPA, SLAMF7, TACI, TCR–list components/assoc, PTCRA, TCRb, CD3z, CD3,
TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, TROY, TSLPR, TYRO, VLDLR,
VSIG4, or VTCN1. In some embodiments, the antigen is IL2R–γ.
In some examples, set forth herein is a method for ng a disease, disorder,
or condition selected from an immunological disease, an autoimmune disease, an inflammatory
disease, a dermatological disease, or a gastrointestinal disease.
In some examples, the disease is s disease, ulcerative colitis, Cushing's
me, adrenal insufficiency, or congenital adrenal hyperplasia.
In some examples, the e is inflammation, asthma, or an inflammatory
bowel disorder.
In some examples, the disease is an autoimmune diseases selected from
multiple sis, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis,
sis, or eczema.
In some examples, set forth herein is a method for reducing or ameliorating the
side effects of chemotherapy, wherein the method es administering to a patient having
said disorder a therapeutically effective amount of a compound or a composition described
herein.
In some examples, set forth herein is a method for reducing or ameliorating the
side s of immunosuppressive therapy, wherein the method includes administering to a
patient having said disorder a therapeutically effective amount of a compound or a composition
described herein.
In some examples, set forth herein is a method for treating cancer, wherein the
method includes administering to a patient having said disorder a therapeutically effective
amount of a compound or a composition described . In some examples, the cancer is
selected from acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin’s
lymphoma, dgkin’s ma (NHL), or multiple myeloma, as well as others.
F. ES
Certain embodiments are illustrated by the following non–limiting examples.
ts and solvents were obtained from commercial sources such as
Sinopharm Chemical Reagent Co. (SCRC), Sigma–Aldrich, Alfa, or other vendors, unless
explicitly stated otherwise.
1H NMR and other NMR spectra were recorded on a Bruker AVIII 400 or
Bruker AVIII 500. The data were processed with Nuts software or MestReNova software,
measuring proton shifts in parts per million (ppm) downfield from an internal standard
tetramethyl silane.
HPLC–MS measurements were run on an Agilent 1200 HPLC/6100 SQ System
using the follow ions:
Method A for HPLC–MS measurement included, as the Mobile Phase: A:
Water (0.01% trifluoroacetic acid TFA) and B: acetonitrile (0.01% TFA). The Gradient Phase
was 5% of B that was increased to 95% of B over a time period of 15 minutes (min) and at a
flow rate of 1.0 mL/min. The column used was a SunFire C18, 4.6x50 mm, 3.5 µm. The
column temperature was 50 ºC. The detectors ed an Analog to Digital Converter ELSD
(Evaporative Light–scattering Detector, hereinafter “ADC ELSD”), DAD (Diode array
detector, 214 nm and 254 nm), and Electrospray Ionization-Atmospheric re tion
(ES–API).
Method B for HPLC–MS measurements included, as the Mobile Phase: A:
Water (10mM NH4HCO3) and B: acetonitrile. The Gradient Phase was 5% of B that was
increased to 95% of B over a time period of 15 min and a flow rate of 1.0 . The column
used was a XBridge C18, 4.6x50 mm, 3.5 µm. The column ature was 50 ºC. The
detectors included an ADC ELSD, DAD (214 nm and 254 nm), and a mass-selcetive detector
(MSD ES–API).
LC–MS measurement was run on an Agilent 1200 HPLC/6100 SQ System
using the follow conditions:
Method A for LC–MS ement was performed on a WATERS 2767
instrument. The column was a Shimadzu Shim–Pack, PRC–ODS, 20x250mm, 15µm, two
connected in series. The Mobile Phase was A: Water (0.01% TFA ) and B: acetonitrile
(0.01% TFA). The nt Phase was 5% of B that was increased to 95% of B over a time
period of 3 min and at a flow rate of 1.8 – 2.3 mL/min. The column used was a SunFire C18,
4.6x50 mm, 3.5 µm. The column temperature was 50 ºC. The detectors included an Analog to
l Converter ELSD (Evaporative–Light Scattering Detector), DAD (Diode Array
Detector) (214 nm and 254 nm), and ES–API.
Method B for LC–MS measurement was performed on a Gilson GX–281
instrument. The column was an Xbridge Prep C18 10 um OBD, 19x250 mm. The Mobile
Phase was A: Water (10mM NH4HCO3) and B: Acetonitrile. The Gradient Phase was 5% of B
that was increased to 95% of B over a time period of 3 min and at a flow rate of1.8 – 2.3
mL/min. The column used was an e C18, 4.6x50 mm, 3.5 µm. The column temperature
was 50 ºC. The detectors included ADC ELSD, DAD (214 nm and 254 nm), and Mass
Selective Detector (MSD) (ES–API).
Preparative high–pressure liquid chromatography (Prep–HPLC) was med
on a Gilson GX–281 instrument. Two solvent systems were used, one acidic and one basic.
The acidic solvent system included a Waters SunFire 10 µm C18 column (100 Å, 250 x 19
mm). Solvent A for prep–HPLC was 0.05% TFA in water and solvent B was acetonitrile. The
elution condition was a linear gradient that increased solvent B from 5% to 100% over a time
period of 20 s and at a flow rate of 30 mL/min. The basic solvent system included a
Waters Xbridge 10 µm C18 column (100 Ǻ, 250 x 19 mm). Solvent A for prep–HPLC was
mM ammonium bicarbonate (NH4HCO3) in water and solvent B was acetonitrile. The
elution condition was a linear gradient that increased t B from 5% to 100% over a time
period of 20 minutes and at a flow rate of 30 mL/min.
Flash chromatography was performed on a Biotage ment, with Agela
Flash Column –CS. Reversed phase flash chromatography was performed on Biotage
instrument, with Boston ODS or Agela C18, unless explicitly ted otherwise.
The following abbreviations are used in the Examples and throughout the
specification:
Abbreviation Term
ADC Antibody–drug conjugate
Aglycosylated antibody Antibody that does not have any glycan residues
API Atmospheric pressure tion
aq Aqueous
Boc N–tert–butoxycarbonyl
Thermo Scientific Prod# 28372, containing 100 mM sodium
BupHTM
phosphate and 150 mM sodium chloride, potassium free, pH was
Abbreviation Term
adjusted from 7.2 to 7.6–7.8 MQ, unless ise noted.
CD Cyclodextrin
COT Cyclooctynol
Da Dalton
DAD Diode array detector
DAR Drug to antibody ratio
DCM Dichloromethane
Dibenzocyclooctyne; or 11,12–didehydro–5,6–dihydro–
DIBAC Dibenz[b,f]azocine; or Dibenz[b,f]azocine–5(6H)–butanoic acid,
11,12–didehydro
11,12–didehydro–5,6–dihydro–Dibenz[b,f]azocine succinamic
DIBAC–Suc
{4–[(2S)–2–[(2S)–2–[1–(4–{2–azatricyclo[10.4.0.04,9]hexadeca–
1(12),4(9),5,7,13,15–hexaen–10–yn–2–yl}–4–oxobutanamido)–
DIBAC–Suc–PEG4–VC–
3,6,9,12–tetraoxapentadecan–15–amido]–3–methylbutanamido]–
–(carbamoylamino)pentanamido]phenyl}methyl 4–nitrophenyl
carbonate
3H–Benzo[c]–1,2,3–triazolo[4,5–e][1]benzazocine, 8,9–dihydro– ;
DIBACT
or Dibenzocyclooctyne triazole
DIPEA Diisopropylethylamine
DMF N,N–dimethylformamide
DMSO Dimethylsulfoxide
EC Enzyme commission
ELSD Evaporative light scattering detector
ESI Electrospray tion
Fmoc Fluorenylmethyloxycarbonyl chloride
N–Fmoc–L–valine–L–citrulline–p–aminobenzyl alcohol p–
Fmoc–vcPAB–PNP
nitrophenyl carbonate
g Gram
za–1H–benzotriazole–1–yl)–1,1,3,3–tetramethyluronium
hexafluorophosphate
Abbreviation Term
HC Heavy chain of immunoglobulin
HEK Human embryonic kidney (cells)
HPLC High performance liquid chromatography
hr or hrs Hours
LC Liquid chromatography
HPLC High-pressure Liquid chromatography
MALDI Matrix–assisted laser desorption/ionization
MC Maleimidocaproyl
mg milligrams
min minutes
mL milliliters
mmh myc–myc–hexahistidine tag
µL microliters
mM olar
µM micromolar
MMAE Monomethyl auristatin E
MS Mass spectrometry
MsCl Methanesulfonyl chloride
MSD Mass–selective detector
Microbial lutaminase (MTG EC 2.3.2.13, Zedira, Darmstadt,
MW Molecular weight
ncADC Non–Cytotoxic antibody drug conjugation
NHS N–hydroxy succinimide
nM nanomolar
NMR Nuclear ic resonance
NOESY Nuclear Overhauser effect spectroscopy
PAB Para–amino-benzyl alcohol
Abbreviation Term
Para–aminobenzyloxy(carbonyl)
PBS 10 mM sodium phosphate buffer and 150 mM sodium chloride
PBSg 10 mM phosphate, 150 mM sodium chloride, 5% glycerol
PEG Polyethyleneglycol
PNP p–nitrophenyl
MC–VC–PAB–PNP
ppm Parts per million (chemical shift)
RP Reversed phase
RT Room temperature
SDS–PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis
SEC Size exclusion chromatography
Suc Succinic acid
TCEP Tris(2–carboxyethyl)phosphine hloride
TEA Triethylamine
TFA Trifluoroacetic acid
TG Transglutaminase
THF Tetrahydrofuran
TOF Time–of–flight
UPLC Ultra Performance Liquid Chromatography
UV iolet
VA –alanine
VC Valine–citrulline
VC–PABC Valine–citrulline–para–aminobenzyloxy(carbonyl)
Abbreviation Term
CD extrin
2–(7–Aza–1H–benzotriazole–1–yl)–1,1,3,3–tetramethyluronium
hexafluorophosphate
MC Maleimidocaproyl
COT Cyclooctynol
SFC Supercritical fluid chromatography
Abbreviation IUPAC name Structure
Boc-vcPAB-PNP tert-butyl (S)methyl((S)(4-
(L2a) (((4-
nitrophenoxy)carbonyloxy)methyl)
phenylamino)oxo
pentanylamino)
oxobutanylcarbamate
Fmoc-vcPAB-PNP (9H-fluorenyl)methyl (S)
(L2b) methyl((S)(4-(((4-
nitrophenoxy)carbonyloxy)methyl)
phenylamino)oxo
ureidopentanylamino)
oxobutanylcarbamate
Boc-Val-Ala-OH (S)((S)(tert-
(L3a) butoxycarbonylamino)
butanamido)propanoic acid
Fmoc-Val-Ala-OH (S)((S)(((9H-fluoren
(L3b) yl)methoxy)carbonylamino)
methylbutanamido)propanoic acid
Boc-Val-Cit-OH (6S,9S)aminoisopropyl-13,13-
(L3c) dimethyl-1,8,11-trioxooxa-
2,7,10-triazatetradecane
carboxylic acid
Abbreviation IUPAC name Structure
Fmoc-D-Lys-COT (2R)(((9H-fluoren
(L5) yl)methoxy)carbonylamino)(2-
(cyclooct
ynyloxy)acetamido)hexanoic acid
CD-N3 (L7a) 5-(azidomethyl)-10,15,20,25,30-
pentakis(hydroxymethyl)-
2,4,7,9,12,14,17,19,22,24,27,29-
dodecaoxaheptacyclo[26.2.2.2³,⁶.2⁸,¹
¹.2¹³,¹⁶.2¹⁸,²¹.2²³,²⁶]dotetracontane-
31,32,33,34,35,36,37,38,39,40,41,4
N3-PEG4- 1-azidooxo-3,6,9,12-tetraoxa-
CONHCH2CH2SO3H 16-azaoctadecanesulfonic acid
(L7b)
BCN-PEG4-acid (Endo)(bicyclo[6.1.0]nonyn-
(L9a) 9-yl)oxo-2,7,10,13,16-pentaoxa-
4-azanonadecanoic acid
DIBAC-PEG4-acid 1-(4-{2-
(L9b) cyclo[10.4.0.0⁴,⁹]hexadeca-
1(12),4(9),5,7,13,15-hexaenyn-
2-yl}oxobutanamido)-3,6,9,12-
xapentadecanoic acid
BCN-PEG4-NHS (Endo)-2,5-dioxopyrrolidinyl 1-
(L10a) (bicyclo[6.1.0]nonynyl)
oxo-2,7,10,13,16-pentaoxa
azanonadecanoate
DIBAC-PEG4-NHS 2,5-dioxopyrrolidinyl 1-(4-{2-
(L10b) azatricyclo[10.4.0.0⁴,⁹]hexadeca-
4(9),5,7,13,15-hexaenyn-
2-yl}oxobutanamido)-3,6,9,12-
tetraoxapentadecanoate
Abbreviation IUPAC name Structure
MAL-PEG4-NHS 2,5-dioxopyrrolidinyl 1-(2,5-
(L10c) dioxo-2,5-dihydro-1H-pyrrolyl)-
3,6,9,12-tetraoxapentadecan
DIBAC-PEG4- {4-[(2S)[(2S)[1-(4-{2-
vcPAB-PNP (L11) azatricyclo[10.4.0.0⁴,⁹]hexadeca-
4(9),5,7,13,15-hexaenyn-
2-yl}oxobutanamido)-3,6,9,12-
tetraoxapentadecanamido]
methylbutanamido]
(carbamoylamino)pentanamido]phe
nyl}methyl 4-nitrophenyl ate
Lk-DIBAC -
Lk-BCN -
Lk-MAL -
Lk-CCK -
aCDCCK -
SulCCK -
Abbreviation IUPAC name Structure
dualSulCCK -
PREPARATION METHODS
EXAMPLE 1
This example demonstrates one method for making chemical derivatives of
de with stereochemical control at the C22–position. In FIGs. 1 and 2, the C22 position is
identified for compounds 7, 8 and 11 with an sk, i.e., *. The synthesis of steroids with
stereochemical control at the C22–position was performed following the synthetic route
depicted in FIGs. 1 and 2.
Desonide (1), which is a generic name for (1S,2S,4R,8S,9S,11S,12S,13R)–11–
hydroxy–8–(2–hydroxyacetyl)–6,6,9,13–tetramethyl–5,7–
dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–16–one, was reacted with isobutyric
anhydride (compound 2) to produce intermediate 3 by esterification at the y alcohol
position of compound 1. Compound 3 was reacted with a series of aldehydes (4-1; 4-2; 4-3;
and 4-4, each differing with respect to the R-CHO group illustrated to the right of these
ical labels) by transacetalation under strong acid HClO4 condition to e alcohols 5
and esters 6. As indicated in these des differed from each other with respect to
the R group ted in
Alcohols 5 and ester 6 were separated by column chromatography.
Each alcohol 5 or ester 6 was individually, d with diethylamine to remove
Fmoc-group or with Fe/NH4Cl to reduce nitro to provide epimer compounds 7 and 8 having
both R/S stereochemistry at C22, respectively.
As detailed below, R and S epimers were separated and their R–and S–
configurations were identified. The R–epimers of, for example, compounds 7 and 8 in
were isolated and confirmed to be the majority stereoisomer by greater 90% by 1H NMR. The
C22 configuration of each epimer was determined by 2D–NOESY oscopic studies.
Table 1 below presents steroids made using the methods described herein.
Table 1 – Structure and Chemical-Physical Properties of Compounds
MW HPLC
Cpd. MS cLog
C22 Structure MF (Cal. purity
No (M+H) P
) (%)
7-1 S NO6 479.6 480.2 96 2.53
7-1 R C28H33NO6 479.6 480.3 100 2.53
8-1 R C32H39NO7 549.7 550.3 96 4.22
H H H
7-2 R/S O C29H35NO6 493.6 494.3 98 2.59
O O
H2N HO O OH
8-2 R/S C33H41NO7 563.7 564.3 98 4.28
8-3 R/S C27H37NO6 471.6 472.2 96 1.63
7-4 R C27H37NO6 471.6 472.2 96 1.63
11-1 R/S C25H35NO5 429.6 429.9 100 2.63
11-2 R/S C26H37NO5 443.6 444.2 96 3.06
11-3 R/S C27H39NO5 457.6 458.2 100 3.44
11-4 R/S NO5 497.3 498.2 94 4.29
11-5 R/S C31H39NO6 521.6 522.3 100 4.24
11-5 S C31H39NO6 521.6 522.2 99.8 4.24
11-5 R C31H39NO6 521.6 522.2 99.1 4.24
11-6 S C31H38FNO6 539.6 540.3 98 4.38
11-6 R C31H38FNO6 539.6 540.3 100 4.38
11-7 R C31H38FNO6 539.6 540.2 100 4.38
518.2
11-8 R C32H41NO6 535.7 (M+H 100 4.54
-H2O)
11-10 R/S C31H37FO6 524.3 525.3 100 5.21
11-11 R/S C33H41NO7 563.7 564.4 100 4.31
11-12 R/S C31H37F2NO6 557.6 558.3 97 3.94
11-12 R C31H37F2NO6 557.6 558.2 100
11-13 R C31H37F2NO6 557.6 558.2 100 3.94
11-14 R/S C31H36F3NO6 575.6 576.2 100 4.09
11-15 R/S F2NO6 554.2 555.2 100 3.90
11-16 R/S C31H38O7 522.3 523.5 100 4.76
C30H36F2N2O
11-17 R/S 558.6 559.2 100 3.91
11-19 R/S 98.5
C25H33F2NO5 465.2 466.2 2.33
11-19 R 100
11-20 R/S C34H43F2NO6 599.2 600.3 100 4.71
11-21 R/S C26H35F2NO5 479.3 480.2 100 2.76
14-2 C26H35F2NO5 479.6 480.2 98 2.81
-5 C26H35F2NO5 479.6 480.2 98 2.81
16-5 C28H33F2NO5 483.6 484 98 2.85
Table 2 below presents steroids made using the methods described herein.
Table 2 – Structure and Chemical-Physical Properties of Compounds
MS Highes
Cpd. HPLC purity
Structure MF (m/z) t m/z
No (%)
100% peak
C25H33F2NO5. 466.2 466.2
4b 98.5
C2HF3O2 (M+H) (M+H)
392.2 392.2
4c F4NO6 > 99
(M+H) (M+H)
410.2 410.2
4d C24H30F5NO6 98
(M+H) (M+H)
C21H28FNO5.C2 394.2 394.2
4e > 99
HF3O2 (M+H) (M+H)
374.3 374.3
4f C22H31NO4 > 99
(M+H) (M+H)
MS Highes
Cpd. HPLC purity
Structure MF (m/z) t m/z
No (%)
100% peak
C25H34FNO5.C2 448.2 448.2
4h > 99
HF3O2 (M+H) (M+H)
C31H38F2N2O5. 557.1 557.1
-I > 99
C2HF3O2 (M+H) (M+H)
522.3 522.3
6-I F2NO6 97
(M+H) (M+H)
522.2 522.2
RI C31H37F2NO6 > 99
(M+H) (M+H)
522.2 522.2
SI C31H37F2NO6 97
(M+H) (M+H)
297.6 558.2
6-I D C31H37F2NO6 (M/2+H (M+H) 98.4
) (10%)
C32H39F2NO7.C 558.3 558.3
6-II > 99
2HF3O2 (M+H) (M+H)
MS Highes
Cpd. HPLC purity
Structure MF (m/z) t m/z
No (%)
100% peak
558.3 558.3
6-III C31H36F3NO6 > 99
(M+H) (M+H)
576.2 576.2
6-VI C31H39NO6 > 99
(M+H) (M+H)
R 588.3 588.3
C31H39NO6 > 99
VI (M+H) (M+H)
665.2
S 587.2 (M+Na
C31H39NO6 > 99
VI (M-55) )
(25%)
587.3 587.3
6-VII C28H35NO5 > 99
(M+H) (M+H)
Table 3 below presents linker ds made using the methods described
herein.
Table 3. Examples of Linker–Payloads
LP No. ures of Linker-Payloads
LP No. ures of Linker-Payloads
LP No. ures of Linker-Payloads
H H O
O OH
O O
H O
H O
N O
N O N
O N
O N N
O H
O H
LP16 O
O H O O
OH O
N N O OH
N OH
O OH
OH HO
O O
HO O
O OH OH
Table 4 below presents linker payloads made using the methods described
herein.
Table 4. es of Linker–Payloads
Structure
LP101
LP102
LP103
LP104
LP105
LP108
LP110
Structure
LP112
LP113
LP114
LP115
LP116
This example demonstrates methods for making chemical derivatives of
budesonide, dexamethasone, and flumethasone. These methods are illustrated, generally, as
shown in FIGs. 2, 3, and 4.
As shown in mesylate analogs of Budesonide (9) or its difluoro-analog
(9B) were reacted with alkyl amines or substituted phenols (10) to yield aniline– or amine–
including compounds (11), such as compounds 11-1 to 11-23 in
As shown in mesylate analogs of Dexamethasone (12), were reacted
with alkyl amines or substituted s (10) to yield aniline– or amine–including compounds
(14) or(15) in
As shown in mesylate analogs of Flumethasone (13), were reacted with
alkyl amines or substituted s (10) to yield aniline– or amine–including compounds (16)
in
As detailed below, stereochemically pure epimers of 11-5S and 11-5R in Table
1 were obtained by chiral separation from a mixture of their corresponding R/S isomers. The
absolute chemistry for each compound was ined by 2D–NOESY. The 2D–
NOESY spectra showed that H22 and H18 were correlated in 11-5R, and that there was no
correlation n H22 and H18 in 11-5S. Similarly, the chiral centers at C22–position were
identified for compounds 7-1S, 7-1R, 7-4R, 8-1R, 11-6S, 11-6R, 11-7R, 11-8R, 11-12R, 11-
13R, and 11-19R in Table 1 by 2D–NOESY.
EXAMPLE 3
This example demonstrates a method for making nds 7-1S and 7-1R in
Table 1. This example refers to the compounds numbered in
2–[(1S,2S,4R,8S,9S,11S,12S,13R)–11–Hydroxy–9,13–dimethyl–6–(4–
henyl)–16–oxo–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–8–yl]–2–
oxoethyl 2–methylpropanoate (5–1) and
(1S,2S,4R,8S,9S,11S,12S,13R)–11–Hydroxy–8–(2–hydroxyacetyl)–9,13–
dimethyl–6–(4–nitrophenyl)–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–16–
one (6–1).
Step 1: Compound 3 was synthesized according to the procedures in
US2007/135398, the entire contents of which are herein incorporated by reference in its
entirety for all purpose, by reacting desonide (1) with isobutyric acid in acetone.
Step 2: To a solution of compound 3 (320 mg, 0.657 mmol) in nitropropane (20
mL) was added aqueous oric acid (70%, 1.90 g, 1.33 mmol) dropwise at 0oC, followed
by the addition of 4–nitrobenzaldehyde (4–1, 151 mg, 1.00 mmol). The resulting mixture was
stirred at RT overnight, and was then diluted with ethyl acetate (80 mL). The resulting mixture
was washed with saturated aqueous sodium bicarbonate solution (30 mL x 3) and then brine
(30 mL x 2). The resulting solution was then dried over sodium sulfate and trated in
vacuo. The residue was then purified by flash chromatography eluting with 0–35% ethyl
acetate in petroleum–ether to yield compound (5–1) as a yellow solid (120 mg, yield 32%),
which was a mixture of 5R/5S epimers in a ratio 3/1 based on 1H NMR, and further eluting
with 60–70% ethyl acetate in petroleum ether to yield compound (6–1) as a yellow solid (150
mg, yield 36%), which was a mixture of 6R/6S epimers in a ratio 5/1 based on 1H NMR (R/S
not determined).
nd (5–1): ESI m/z: 580 (M + H)+. 1H NMR (CDCl3, 400 MHz, epimers
A and B with ratio = 3) δ 8.27 and 8.25 (d, J = 8.8 Hz, 2H), 7.62 and 7.55 (d, J = 8.8 Hz, 2H),
7.28–7.21 (m, 1H), 6.33–6.23 (m, 1H), 6.03 and 6.05 (s, 1H), 5.62 and 6.16 (s, 1H), 5.12 and
.43 (d, J = 5.4 Hz, 1H), 4.97 and 4.77 (d, J = 17.6 Hz, 1H), 4.88 and 4.33 (d, J = 17.6 Hz,
1H), 4.52 (br s, 1H), 2.80–2.50 (m, 2H), 2.44–2.29 (m, 1H), 2.29–2.05 (m, 3H), 2.01–1.84 (m,
2H), 1.80–1.67 (m, 2H), 1.51 and 1.59 (br s, 1H), 1.46 and 1.48 (s, 3H), 1.29–1.07 (m, 7H),
1.03 and 1.05 (s, 3H) ppm.
Compound 6–1: ESI m/z: 510 (M + H)+. 1H NMR (DMSOd6, 400 MHz, epimers
A and B with ratio = 5) δ 8.26 and 8.24 (d, J = 8.8 Hz, 2H), 7.77 and 7.57 (d, J = 8.8 Hz, 2H),
7.32 (d, J = 10.0 Hz, 1H), 6.17 and 6.18 (dd, J = 10.0 Hz, 1.8 Hz, 1H), 5.93 and 5.95 (s, 1H),
.63 and 6.28 (s, 1H), 5.14 and 5.03 (t, J = 6.0 Hz, 1H), 4.99 and 5.35 (d, J = 6.3 Hz, 1H), 4.82
(d, J = 3.2 Hz, 1H), 4.64–4.13 (m, 3H), 2.64–2.51 (m, 1H), 2.37–2.24 (m, 1H), 2.20–1.99 (m,
2H), 1.94–1.57 (m, 5H), 1.40 (s, 3H), 1.14–0.98 (m, 2H), 0.88 (s, 3H) ppm.
Step 3: Making (1S,2S,4R,6R,8S,9S,11S,12S,13R)–6–(4–Aminophenyl)–11–
hydroxy–8–(2–hydroxyacetyl)–9,13–dimethyl–5,7–
dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–16–one (7-1R) in Table 1) and
(1S,2S,4R,6S,8S,9S,11S,12S,13R)–6–(4–Aminophenyl)–11–hydroxy–8–(2–hydroxyacetyl)–
9,13–dimethyl–5,7–dioxapentacyclo[10.8.0. 02,9.04,8.013,18]icosa–14,17–dien–16–one (7-1S) in
Table 1).
Iron powder (56.0 mg, 1.00 mmol) and um de (53.5 mg, 1.00
mmol) were simultaneously added to a solution of compound 5–1 (51.0 mg, 0.100 mmol) in a
combined solution of ethanol (3 mL) and water (0.5 mL). The sion was stirred at 80ºC
for an hour and was filtered h Celite to remove the solid. The filtrate was concentrated in
vacuo and the residue was purified by prep–HPLC (method B) to yield compound 7-1R (30
mg, yield 63%) as a white solid and compound 7-1S (8 mg, yield 17%) as a white solid.
2D–NOESY spectroscopy was used to determine the stereochemical
urations of the chiral centers of compound 7-1R and nd 7-1S. The 2D–NOESY
spectra confirmed that there is a correlation between H22 and H21 in compound 7-1R, which
indicates that it has an R configuration chiral center. No correlation was observed between H22
and H21 in compound 7-1S, ting it has an S configuration chiral center. The NMR study
also showed that the shift of H22 in compound 7-1R (5.33 ppm) was much higher than that of
compound 7-1S (6.01 ppm), ting H22 of nd 7-1R was more hindered. The 2DNOESY
spectra of compound 722R and compound 722S are shown in FIGs. 5 and 6.
Compound 7-1R in Table 1: ESI m/z: 480 (M + H)+. 1H NMR (MeODd4, 400
MHz) δ 7.46 (d, J = 10.1 Hz, 1H), 7.17 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 6.27 (dd,
J = 10.1, 1.8 Hz, 1H), 6.04 (s, 1H), 5.33 (s, 1H), 5.00 (d, J = 5.4 Hz, 1H), 4.61 (d, J = 19.4 Hz,
1H), 4.50–4.39 (m, 1H), 4.31 (d, J = 19.4 Hz, 1H), 2.78–2.61 (m, 1H), 2.47–2.35 (m, 1H),
2.35–2.22 (m, 1H), 2.22–2.10 (m, 1H), 2.04–1.94 (m, 1H), 1.91–1.66 (m, 4H), 1.51 (s, 3H),
1.25–1.11 (m, 1H), 1.07 (dd, J = 11.2 Hz, 3.5 Hz, 1H), 0.99 (s, 3H) ppm.
Compound 7-1S in Table 1: ESI m/z: 480 (M + H)+. 1H NMR (MeODd4, 400
MHz) δ 7.47 (d, J = 10.1 Hz, 1H), 7.02 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 8.5 Hz, 2H), 6.27 (dd,
J = 10.1, 1.8 Hz, 1H), 6.03 (s, 1H), 6.01 (s, 1H), 5.36 (d, J = 6.2 Hz, 1H), 4.46–4.31 (m, 2H),
4.12 (d, J = 19.2 Hz, 1H), 2.75–2.61 (m, 1H), 2.47–2.35 (m, 1H), 2.27–2.11 (m, 2H), 2.08–
1.97 (m, 1H), .73 (m, 4H), 1.51 (s, 3H), 1.33–1.17 (m, 2H), 1.17–1.09 (m, 1H), 1.01 (s,
3H) ppm.
This example demonstrates a method for making compounds (8–1R/S) and
compound (8–1R) in Table 1. This example refers to the compound numbering in
2–[(1S,2S,4R,8S,9S,11S,12S,13R)–6–(4–Aminophenyl)–11–hydroxy–9,13–
dimethyl–16–oxo–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–8–yl]–2–
oxoethyl 2–methylpropanoate (8–1R).
Iron powder (56.0 mg, 1.00 mmol) and ammonium chloride (53.5 mg, 1.00
mmol) were simultaneously added to a solution of compound (6–1) (58.0 mg, 0.100 mmol) in
a combined solution of ethanol (3 mL) and water (1 mL). The resulting sion was stirred
at 80oC for an hour and was filtered through Celite to remove the solid. The filtrate was
concentrated in vacuo and the residue was purified by prep–HPLC (method B) to yield
compound (8–1R) and its enantiomer (i.e., S stereochemistry at C22) (26 mg, yield 45%) as a
white solid. The ratio of the R epimer to the S– epimer is 4:1 by HPLC and 1H NMR. ESI m/z:
550 (M + H)+.
The R–epimer was further isolated and the configuration was determined by 2D
Compound (8–1R): ESI m/z: 550 (M + H)+. 1H NMR (MeODd4, 500 MHz) δ
7.46 (d, J = 10.0 Hz, 1H), 7.19 (d, J = 8.5 Hz, 2H), 6.69 (d, J = 8.4 Hz, 2H), 6.27 (dd, J = 10.0
Hz, 2.0 Hz, 1H), 6.05 (s, 1H), 5.44 (s, 1H), 5.07 (d, J = 17.5 Hz, 1H), 4.96 (d, J = 5.5 Hz, 1H),
4.88 (d, J = 17.5 Hz, 1H), .44 (m, 1H), .64 (m, 2H), 2.42–2.39 (m, 1H), 2.32–
2.24 (m, 1H), 2.19–2.15 (m, 1H), .99 (m, 1H), 1.95–1.92 (m, 1H), 1.90–1.83 (m, 2H),
1.76–1.69 (m, 1H), 1.52 (s, 3H), 1.27–1.12 (m, 7H), .05 (m, 1H), 1.02 (s, 3H) ppm.
EXAMPLE 5
This example demonstrates a method for making nd (7–2R/S) in Table
1. This example refers to the compound numbering in
Step 1: 1S,2S,4R,8S,9S,11S,12S,13R)–11–Hydroxy–8–(2–hydroxyacetyl)–
9,13–dimethyl–6–[(4–nitrophenyl)methyl]–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–
14,17–dien–16–one (5–2).
To a solution of compound (3) (226 mg, 0.464 mmol) in nitropropane (10 mL)
was added aqueous perchloric acid (70%, 985 mg, 6.90 mmol) dropwise at 0 oC, followed by
the addition of 2–(4–nitrophenyl)acetaldehyde (4–2, 115 mg, 0.696 mmol) according to the
synthesis in Synthesis, 2011, 18, 2935–2940, the entire contents of which are herein
incorporated by reference in their entirety for all purposes. The resulting mixture was stirred at
RT ght, and was then diluted with ethyl acetate (60 mL). The mixture was washed with
saturated aqueous sodium bicarbonate solution (50 mL x 3), then brine (50 mL x 3), and then
dried over sodium sulfate and concentrated in vacuo. The residue was ed by flash
chromatography eluting with 0–35% ethyl acetate in petroleum ether to yield compound (6–2)
as a brown solid (95 mg, yield 34%, including 22R/S s in a ratio >10/1 by 1H NMR),
and further eluting with 60–70% ethyl acetate in petroleum ether to yield compound (5–2) (145
mg, yield 60%) as a brown solid.
Compound (5–2): ESI m/z: 524 (M + H)+. 1H NMR (CDCl3, 400 MHz) δ 8.09
(d, J = 8.7 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H), 7.17 (d, J = 10.1 Hz, 1H), 6.31 (dd, J = 10.1 Hz,
1.8 Hz, 1H), 6.02 (s, 1H), 4.92 (d, J = 5.3 Hz, 1H), 4.86 (t, J = 3.6 Hz, 1H), 4.52–4.39 (m, 2H),
4.28–4.17 (m, 1H), 3.08 (d, J = 3.5 Hz, 2H), 2.96 (t, J = 4.9 Hz, 1H), 2.53–2.40 (m, 1H), 2.32–
2.19 (m, 1H), 2.04–1.95 (m, 1H), 1.95–1.82 (m, 2H), 1.60–1.46 (m, 3H), 1.38 (s, 3H), 1.34 (br
s, 1H), 0.91–0.77 (m, 4H), 0.76–0.62 (m, 2H) ppm.
Step 2: (1S,2S,4R,8S,9S,11S,12S,13R)–6–[(4–Aminophenyl)methyl]–11–
hydroxy–8–(2–hydroxyacetyl)–9,13–dimethyl–5,7–
dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–16–one (7–2R/S)
Iron powder (78.0 mg, 1.40 mmol) and ammonium chloride (75.0 mg, 1.40
mmol) were simultaneously added to a solution of nd (5–2) (75.0 mg, 0.143 mmol) in
a combined solution of ethanol (4 mL) and water (0.5 mL). The suspension was stirred at
80ºC for 1.5 hours and was filtered through Celite to remove the solid. The filtrate was
concentrated in vacuo and the e was purified by prep–HPLC (method B) to yield
compound (7–2R/S) (26 mg, yield 37%) as a white solid. ESI m/z: 494 (M + H)+. 1H NMR
(MeODd4, 400 MHz) δ 7.44 (d, J = 10.1 Hz, 1H), 6.93 (d, J = 8.3 Hz, 2H), 6.48 (d, J = 8.3 Hz,
2H), 6.30 (dd, J = 10.1 Hz, 1.9 Hz, 1H), 6.07 (s, 1H), 4.85–4.77 (m, 2H), 4.51 (d, J = 19.4 Hz,
1H), 4.35–4.29 (m, 1H), 4.24 (d, J = 19.4 Hz, 1H), .72 (m, 2H), 2.62–2.47 (m, 1H),
2.38–2.28 (m, 1H), .93 (m, 1H), 1.90–1.78 (m, 2H), 1.67–1.58 (m, 1H), 1.53–1.37 (m,
5H), 0.91–0.77 (m, 5H), 0.74 (dd, J = 11.2 Hz, 3.4 Hz, 1H) ppm.
This example demonstrates a method for making compound S) in Table
1. This example refers to the compound numbering in
Step 1: 2–[(1S,2S,4R,8S,9S,11S,12S,13R)–11–Hydroxy–9,13–dimethyl–6–[(4–
nitrophenyl)methyl]–16–oxo–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–8–
yl]–2–oxoethyl 2–methylpropanoate (6–2)
The synthesis of compound 6-2 was described in EXAMPLE 5, above.
Compound 6–2: ESI m/z: 594 (M + H)+. 1H NMR (CDCl3, 400 MHz) δ 8.15 (d, J = 8.7 Hz,
0.1H) and 8.09 (d, J = 8.7 Hz, 1.9H), 7.40 (d, J = 8.6 Hz, 2H), 7.20 (d, J = 10.1 Hz, 1H), 6.31
(dd, J = 10.1 Hz, 1.8 Hz, 1H), 6.02 (s, 1H), 4.94 (t, J = 3.6 Hz, 1H), 4.87 (d, J = 5.1 Hz, 1H),
4.81 (d, J = 17.6 Hz, 1H), 4.71 (d, J = 17.6 Hz, 1H), 4.46 (s, 1H), 3.09 (d, J = 3.5 Hz, 2H),
.61 (m, 1H), 2.53–2.41 (m, 1H), 2.31–2.21 (m, 1H), 2.07–1.96 (m, 1H), 1.94–1.84 (m,
2H), 1.84–1.76 (m, 1H), 1.63–1.43 (m, 3H), 1.39 (s, 3H), 1.22 (t, J = 7.0 Hz, 6H), 0.92–0.82
(m, 4H), 0.76–0.61 (m, 2H) ppm.
Step 2: 2–[(1S,2S,4R,8S,9S,11S,12S,13R)–6–[(4–Aminophenyl)methyl]–11–
hydroxy–9,13–dimethyl–16–oxo–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–
8–yl]–2–oxoethyl 2–methylpropanoate (8–2R/S)
To a solution of nd 6–2 (65.0 mg, 0.109 mmol) in a combined solution
of ethanol (5 mL) and water (1 mL) were simultaneously added iron powder (61.0 mg, 1.09
mmol) and ammonium chloride (58.4 mg, 1.09 mmol). The suspension was stirred at 80 ºC for
an hour and was filtered through Celite to remove the solid. The filtrate was concentrated in
vacuo and the residue was purified by prep–HPLC (method B) to yield compound (8–2R/S)
(30 mg, yield 49%) as a white solid. ESI m/z: 564 (M + H)+. 1H NMR (CDCl3, 400 MHz) δ
7.25 (d, J = 10.2 Hz, 1H), 6.95 (d, J = 8.3 Hz, 2H), 6.44 (d, J = 8.3 Hz, 2H), 6.31 (dd, J = 10.1,
1.8 Hz, 1H), 6.05 (s, 1H), 4.92–4.84 (m, 2H), 4.80 (d, J = 5.2 Hz, 1H), 4.73 (d, J = 17.7 Hz,
1H), 4.41 (s, 1H), 3.48 (br s, 1H), 2.85 (d, J = 2.7 Hz, 2H), 2.75–2.62 (m, 1H), 2.56–2.41 (m,
1H), 2.31–2.19 (m, 1H), 2.05–1.91 (m, 2H), 1.88–1.80 (m, 1H), 1.77–1.70 (m, 1H), 1.55–1.41
(m, 3H), 1.39 (s, 3H), 1.29–1.18 (m, 8H), 0.91–0.74 (m, 5H) ppm.
This example trates a method for making compound S) in Table
1. This example refers to the compound numbering in
Step 1: 2–[(1S,2S,4R,8S,9S,11S,12S,13R)–6–(2–{[(9H–Fluoren–9–
oxy)carbonyl]amino}ethyl)–11–hydroxy–9,13–dimethyl–16–oxo–5,7–
dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–8–yl]–2–oxoethyl 2–
methylpropanoate (6–3)
To a solution of nd 3 (240 mg, 0.493 mmol) in nitropropane (5 mL) was
added aqueous oric acid (70%, 214 mg, 1.49 mmol) dropwise at 0 oC, followed by the
addition of Fmoc–3–amino–1–propanal (4–3, 236 mg, 0.799 mmol) according to the synthesis
in J. Am. Chem. Soc., 2006, 128 (12), 4023–4034, the entire contents of which are herein
incorporated by reference in their entirety for all purposes. The resulting mixture was stirred at
RT overnight, and was then diluted with ethyl acetate (80 mL). The mixture was washed with
saturated aqueous sodium bicarbonate solution (50 mL x 3), then water (50 mL x 2) then brine
(50 mL), and then dried over sodium sulfate and concentrated in vacuo. The residue was
purified by prep–TLC (silica gel, methanol / methylene chloride, v/v = 1/25) to yield
compound (6–3) (200 mg, yield 56%, 6R/6S epimers) as an ite solid. ESI m/z: 724 (M +
H)+. 1H NMR (CDCl3, 400 MHz) δ 7.76 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 7.2 Hz, 2H), 7.40 (d,
J = 7.2 Hz, 1H), 7.32–7.20 (m, 3H), 6.28–6.25 (m, 2H), 6.00 (s, 1H), 5.28–5.04 (m, 2H), 4.87–
4.76 (m, 1H), 4.46–4.35 (m, 3H), 4.18 (t, J = 6.8 Hz, 1H), 3.49 (s, 1H), 3.39–3.24 (m, 2H),
.49 (m, 2H), 2.37–2.26 (m, 1H), 2.23–1.96 (m, 3H), 1.96–1.47 (m, 6H), 1.45–1.41 (m,
3H), 1.28–1.06 (m, 10H), 1.02–0.94 (m, 3H) ppm.
Step 2: 2–[(1S,2S,4R,8S,9S,11S,12S,13R)–6–(2–Aminoethyl)–11–hydroxy–
9,13–dimethyl–16–oxo–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–8–yl]–2–
oxoethyl 2–methylpropanoate (8–3R/S)
A solution of compound (6–3) (40.0 mg, 55.3 µmol) in lamine (1 mL)
and methylene chloride (1 mL) was stirred at RT overnight. The volatiles were removed in
vacuo and the residue was purified by prep–HPLC (method B) followed by prep–TLC (thin
layer chromatography) (silica gel, methylene chloride / ol, v/v = 75/10) to yield
compound (8–3R/S) (3 mg, yield 11%) as an ite solid. ESI m/z: 502 (M + H)+. 1H NMR
(MeODd4, 400 MHz) δ 7.36 (d, J = 10.1 Hz, 1H), 6.16 (dd, J = 10.1 Hz, 1.8 Hz, 1H), 5.91 (s,
1H), 5.23 (t, J = 4.4 Hz, 1H), 5.08–4.90 (m, 1H), 4.75–4.65 (m, 1H), 4.38–4.28 (m, 1H), 2.83–
2.50 (m, 2H), 2.33–2.23 (m, 1H), 2.13–2.00 (m, 2H), 1.90–1.46 (m, 6H), 1.39 (s, 3H), 1.24–
1.12 (m, 2H), 1.23–0.78 (m, 11H) ppm.
EXAMPLE 8
This example demonstrates a method for making compound 7–4R in Table 1.
This example refers to the nd numbering in
(1S,2S,4R,6R,8S,9S,11S,12S,13R)–11–Hydroxy–8–(2–hydroxyacetyl)–9,13–
dimethyl–6–(piperidin–4–yl)–5,7–dioxapentacyclo[10.8.0.002,9.04,8.013,18]icosa–14,17–dien–
16–one (7–4R).
To a solution of desonide (1, 0.10 g, 0.25 mmol) in nitropropane (5 mL) was
added aqueous perchloric acid (70%, 0.11 g, 0.75 mmol) dropwise at 0 oC, followed by the
addition of 1–Boc–4–piperidinecarboxaldehyde (4–4, 64 mg, 0.30 mmol). After being stirred
at RT overnight, the suspension was concentrated in vacuo. The e was basified by the
on of a solution in methanol (7 M, 10 mL). The resulting mixture was
concentrated in vacuo and the crude product was purified by prep–HPLC twice (method B) to
yield compound 7–4R (15 mg, yield 13%) as a white solid. ESI m/z: 472 (M + H)+. 1H NMR
(MeODd4, 500 MHz) δ 7.47 (d, J = 10.0 Hz, 1H), 6.27 (dd, J = 10.0 Hz, 2.0 Hz, 1H), 6.03 (s,
1H), 4.90 (d, J = 4.0 Hz, 1H), 4.50 (d, J = 19.0 Hz, 1H), 4.46–4.43 (m, 1H), 4.41 (d, J = 4.0
Hz, 1H), 4.29 (d, J = 19.0 Hz, 1H), 3.13–3.09 (m, 2H), .60 (m, 3H), 2.42–2.38 (m, 1H),
2.27–2.13 (m, 2H), 1.99–1.96 (m, 1H), 1.85–1.64 (m, 7H), 1.52 (s, 3H), 1.51–1.38 (m, 2H),
1.14–0.99 (m, 2H), 0.96 (s, 3H) ppm. The stereochemical R-configuration for nd 7–4R
was determined by 2D NMR.
EXAMPLE 9
This example demonstrates a method for making compound (11-1R/S) in Table
1. The method is illustrated, generally, as shown in
Step 1: 2–[(1S,2S,4R,8S,9S,11S,12S,13R)–11–Hydroxy–9,13–dimethyl–16–
oxo–6–propyl–5,7–dioxapentacyclo[10.8. 002,9.04,8.013,18]icosa–14,17–dien–8–yl]–2–oxoethyl
methanesulfonate (9)
l procedure A for the synthesis of mesylates from its alcohol: To a
solution of the alcohol (1.0 equiv.) in DCM (10 mL per gram of the starting material) were
added triethylamine or 4-dimethylaminopyridine (2 equiv.) and methanesulfonyl chloride (1.2
equiv.). After stirred at 0 oC for half an hour or until the starting material was consumed
according to TLC, the on mixture was added silica gel (100-200 mesh) and concentrated
in vacuo. The residue with silica gel was purified by silica gel column chromatography (0-50%
ethyl acetate in petroleum ether) to give the mesylate product. Alternatively, the e was
washed with diluted aq. hydrochloride (1 N) and brine, dried over sodium sulfate and
concentrated. The crude product was purified by flash chromatography (0-2% MeOH in DCM)
to give the corresponding mesylate product.
Alternative method to make compound 9: to a on of Budesonide (0.28 mg,
0.65 mmol) in pyridine (5 mL) was added 4–dimethylaminopyridine (0.16 g, 1.3 mmol) and
then methanesulfonyl chloride (0.11 g, 0.97 mmol) was added dropwise at 0oC. After being
stirred at RT for 2 hours, the resulting e was poured into ethyl acetate (100 mL). The
mixture was washed with diluted aq. hydrochloride (1N) and then brine, dried over sodium
e and concentrated. The crude product was purified by flash chromatography (0–1%
methanol in methylene chloride) to yield compound (9) (0.26 g, yield 85%) as a white solid.
ESI m/z: 509 (M + H)+. 1H NMR (CDCl3, 400 MHz) (which epimers) δ 7.25 and 7.22 (d, J =
2.0 Hz, 1H), 6.30–6.27 (m, 1H), 6.03–6.02 (m, 1H), .11 (m, 1.5H), 5.06–4.96 (m, 1.5
H), 4.87–4.86 (m, 0.5 H), 4.59 (d, J = 4.5 Hz, 0.5 H), .50 (m, 1 H), 3.24 (s, 3H), 2.60–
2.53 (m, 1H), .33 (m, 1H), 2.24–2.00 (m, 3H), 1.86–1.62 (m, 4H), 1.53–1.33 (m, 8H),
1.21–1.09 (m, 2 H), 1.02–0.96 (m, 3H), 0.94–0.91 (m, 3H) ppm.
Step 2: (1S,2S,4R,8S,9S,11S,12S,13R)–8–(2–Aminoacetyl)–11–hydroxy–9,13–
dimethyl–6–propyl–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–16–one (11-
1R/S)
To a solution of ammonia in MeOH (7 M, 15 mL) at RT was added compound
9 (0.10 g, 0.20 mmol). The solution was sealed and stirred at 40oC overnight. The volatiles
were removed in vacuo and the crude product was purified by prep–HPLC (method B) to yield
compound (11-1R/S) (8.0 mg, 9% yield) as an off–white solid. ESI m/z: 429.9 (M + H)+. 1H
NMR (MeODd4, 400 MHz) δ 7.46 (d, J = 10.0 Hz, 1H), 6.26 (d, J = 10.0 Hz, 1H), 6.02 (s, 1H),
.22–5.15 (m, 1.5 H), 4.88 (m, 0.6H), 4.58 (m, 0.5H), 4.42 (m, 1H), .81 (m, 0.7H),
3.50–3.41 (m, 0.7H), 2.70–2.63 (m, 1H), 2.40–2.37 (m, 1H), 2.22–1.94 (m, 3H), 1.87–1.25 (m,
11H), 1.17–0.80 (8H) ppm. Anal. HPLC: > 95%, Retention time: 7.63 min (method B).
EXAMPLE 10
This e trates a method for making compound 11-2R/S in Table 1.
This example refers to the compound numbering in
(1S,2S,4R,8S,9S,11S,12S,13R)–11–hydroxy–9,13–dimethyl–8–[2–
(methylamino)acetyl]–6–propyl–5,7–dioxapentacyclo[10.8.0.0²,⁹.0⁴,⁸.0¹³,¹⁸]icosa–14,17–dien–
16–one (11-2R/S)
A solution of compound 9 (51 mg, 0.10 mmol) in methylamine (2 M solution in
THF, 0.5 mL) in a sealed tube was stirred at 20–25oC for 4 hours, and was then stirred at 40oC
overnight. The volatiles were d in vacuo and the residue was purified by PLC
(method A) and then prep–HPLC (method B) to yield compound /S) (15 mg, 33%
yield) as a white solid. ESI m/z: 444.3 (M + H)+. 1H NMR (CDCl3, 400 MHz) δ 7.26–7.23 (d,
J = 10.8 Hz, 1H), .26 (m, 1H), 6.03–6.02 (m, 1H), 5.20–5.16 (m, 1H), 4.90–4.89 (d, J =
4.8 Hz, 0.5 H), 4.69–4.66 (t, J = 4.8 Hz, 0.5H), .51 (m, 1H), 3.50–3.29 (m, 2H), 2.61–
2.52 (m, 1H), 2.37–2.32 (m, 1H), 2.17–2.16 (d, J = 3.6 Hz, 3H), 2.14–2.08 (m, 3H), 1.86–1.74
(m, 3H), 1.59–1.48 (m, 2H), 1.45 (s, 3H), 1.42–0.89 (m, 12H) ppm.
EXAMPLE 11
This example demonstrates a method for making compound 11-3R/S in Table 1.
This example refers to the compound numbering in
(1S,2S,4R,8S,9S,11S,12S,13R)–8–[2–(Dimethylamino)acetyl]–11–hydroxy–
9,13–dimethyl–6–propyl–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–16–one
(11-3R/S)
To a solution of compound 9 (51 mg, 0.10 mmol) in THF (3 mL) was added
dropwise a solution of dimethylamine in THF (2 M, 0.75 mL, 1.5 mmol) at RT. The reaction
mixture was stirred at 50oC overnight. The reaction mixture was concentrated, and the crude
product was purified by prep–HPLC d B) to yield compound 11-3R/S (15 mg, 33%
yield) as a white solid. ESI m/z: 458.2 (M + H)+. 1H NMR (MeODd4, 400 MHz) δ 7.46 (d, J =
.4 Hz, 1H), 6.26 (d, J = 10.0 Hz, 1H), 6.02 (s, 1H), 5.21 (t, J = 4.8 Hz, 0.6H), 5.17 (d, J =
7.2 Hz, 0.6H), 4.58 (d, J = 4.4 Hz, 0.4H), 4.44–4.41 (m, 1H), 3.80–3.57 (m, 1H), 3.26 (d, J =
18.8 Hz, 0.7H), 3.08–2.91 (m, 0.7H), 2.70–2.61 (m, 1H), 2.49–2.33 (m, 7H), 2.26–2.11 (m,
2H), .95 (m, 1H), 1.85–1.55 (m, 5H), 1.49 (s, 3H), 1.49–1.30 (m, 3H), 1.09–1.00 (m,
2H), 0.98–0.90 (m, 6H) ppm. Anal. HPLC: > 95%, Retention time: 8.34 min (method B).
EXAMPLE 12
This example demonstrates a method for making compound S in Table 1.
This example refers to the compound numbering in
(1S,2S,4R,8S,9S,11S,12S,13R)–8–[2–(4–Aminophenoxy)acetyl]–11–hydroxy–
9,13–dimethyl–6–propyl–5,7–dioxapentacyclo[10.8. 002,9.04,8.013,18]icosa–14,17–dien–16–one
(11-5R/S)
General procedure B for making substituted phenol ether from its mesylate
precursor: To hot acetonitrile or acetone (60-65 oC) were added mesylate precursor (1 ),
substituted phenol (2.0-2.5 equiv.), and potassium carbonate or cesium carbonate (2.0-3.0
equiv.). The resulting suspension was ed for 2-3 hours, and the reaction was monitored
by LCMS and/or TLC. After the reaction was cooled to RT, the volatiles were removed in
vacuo and to the e was added water. The aqueous mixture was extracted with ethyl
acetate. The combined organic solution was washed with water and brine, dried over sodium
e and concentrated in vacuo. The crude product was used for the next step ly or
purified by flash chromatography or PLC.
Step 1: A mixture of compound 9 (0.13 g, 0.26 mmol), 4–nitrophenol (10–5,
72 mg, 0.52 mmol) and potassium carbonate (72 mg, 0.52 mmol) in acetone (10 mL) was
refluxed (60 °C) overnight. After tion to remove the solids, the filtrate was concentrated in
vacuo. The crude product was purified by flash chromatography (0–1% methanol in methylene
chloride) to yield a nitro–intermediate (0.11 g, yield 77%) as brown oil. ESI m/z: 552 (M +
H)+. 1H NMR (CDCl3, 500 MHz) (with epimers) δ 8.23–8.15 (m, 2.4H), 7.26–7.23 (m, 1H),
6.97–6.91 (m, 2.4H), 6.31–6.28 (m, 1H), .04 (m, 1H), 5.22–5.18 (m, 1.4H), 5.10–5.07
(m, 0.6H), 4.93 (d, J = 5.0 Hz, 0.6H), 4.83–4.77 (m, 1H), 4.67 (d, J = 5.0 Hz, 0.6H), 4.56–4.53
(m, 1H), 2.62–2.55 (m, 1H), 2.38–2.5 (m, 1H), 2.24–2.07 (m, 3H), 1.88–1.56 (m, 5H), 1.46–
1.40 (m, 6H), 1.20–1.13 (m, 2H), 1.05–0.99 (m, 3H), 0.97–0.94 (m, 3H) ppm.
Step 2: Iron powder (0.10 g, 1.9 mmol) and um chloride (0.10 g, 1.9
mmol) were simultaneously added to a solution of the nitro–intermediate (0.10 g, 0.19 mmol)
in a combined solution of ethanol (20 mL) and water (2 mL). The suspension was stirred at
80oC for 2 hours and was filtered through Celite to remove nic salts. The filtrate was
concentrated in vacuo and the residue was purified by PLC (method B) to yield
compound (11-5R/S) (50 mg, yield 50%) as a white solid. ESI m/z: 522 (M + H)+. 1H NMR
(MeODd4, 500 MHz) (with epimers) δ 7.47 (d, J = 10.0 Hz, 1H), 6.78–6.70 (m, 4H), 6.29–6.26
(m, 1H), 6.04 (br s, 1H), 5.25 (t, J = 5.0 Hz, 0.4H), 5.20 (d, J = 7.0 Hz, 0.4H), 5.06 (d, J =18.0
Hz, 0.4H), 4.98 (d, J =18.0 Hz, 0.6H), 4.90–4.87 (m, 0.6 H), 4.75–4.66 (m, 1.6H), 4.46–4.44
(m, 1H), 2.71–2.64 (m, 1H), 2.42–2.38 (m, 1H), 2.28–2.18 (m, 2H), 2.06–2.00 (m, 1H), 1.87–
1.83 (m, 1H), 1.76–1.73 (m, 1H), 1.69–1.61 (m, 3H), .38 (m, 3H), 1.51 (s, 3H), 1.20–
1.02 (m, 3H), 0.98–0.92 (m, 5H) ppm.
A mixture of two s of compound 11-5R and compound 11-5S from Table
1 (0.30 g, 0.58 mmol) were isolated by chiral HPLC (Instrument: Gilson–281, : OZ–H
*250mm, 10um ), using mobile phase: hexane (0.1% DEA)/ Ethanol (0.1%
DEA)=70/30 at flow rate of 60 mL/min, detected at 214nm. The resultant solution was
concentrated to afford compound 11-5S (30 mg, 10% yield) and compound 11-5R (50 mg,
17% yield) as white solids, separately. The structures of compound 11-5S and compound
11-5R were determined by 2D–NOESY.
(1S,2S,4R,8S,9S,11S,12S,13R)–8–[2–(4–Aminophenoxy)acetyl]–11–hydroxy–
9,13–dimethyl–6–propyl–5,7–dioxapentacyclo[10.8.0.0²,⁹.0⁴,⁸.0¹³,¹⁸]icosa–14,17–dien–16–one
(11-5S): First peak on HPLC; ESI m/z: 522 (M + H)+. Retention time in HPLC (method A):
7.54 min; chiral SFC (CC4): Retention time 4.71 min, 99.5d.e.%; 1H NMR (400 MHz, CDCl3)
δ 7.21 (d, J = 10.1 Hz, 1H), 6.77 (d, J = 8.8 Hz, 2H), 6.63 (d, J = 8.8 Hz, 2H), 6.24 (dd, J =
.1, 1.6 Hz, 1H), 6.02 (s, 1H), 5.20 (d, J = 6.8 Hz, 1H), 5.18 (t, J = 4.8 Hz, 1H), 4.99 (d, J =
–17.9 Hz, 1H), 4.61 (d, J = –17.9 Hz, 1H), 4.43 (s, 1H), 3.46 (s, 2H), 2.57 (td, J = 13.2, 4.4 Hz,
1H), 2.34 (dd, J = 13.4, 3.2 Hz, 1H), 2.16–2.01 (m, 4H), 1.85–1.68 (m, 3H), 1.59–1.49 (m,
3H), 1.44 (s, 3H), 1.44–1.26 (m, 2H), 1.18–1.09 (2H), 1.00 (s, 3H), 0.91 (t, J = 7.3 Hz, 3H)
ppm. 13C NMR (100 MHz, CDCl3) δ 204.0, 186.7, 170.0, 156.3, 151.4, 141.0, 127.9, 122.6,
116.5, 116.4, 108.4, 98.6, 83.2, 72.6, 69.8, 55.3, 53.0, 47.2, 44.2, 41.5, 37.3, 34.1, 33.0, 32.0,
31.1, 21.2, 17.9, 17.7, 14.1 ppm.
(1S,2S,4R,8S,9S,11S,12S,13R)–8–[2–(4–Aminophenoxy)acetyl]–11–hydroxy–
9,13–dimethyl–6–propyl–5,7–dioxapentacyclo[10.8.0.0²,⁹.0⁴,⁸.0¹³,¹⁸]icosa–14,17–dien–16–one
(11-5R): Second peak on HPLC; ESI m/z: 522 (M + H)+; Retention time in HPLC (method A):
7.58 min; chiral SFC (CC4): Retention time 3.80 min, 98.1d.e.%; 1H NMR (400 MHz, CDCl3)
δ 7.23 (d, J = 10.1 Hz, 1H), 6.79 (dd, J = 8.8 Hz, 2H), 6.65 (d, J = 8.8 Hz, 2H), 6.27 (dd, J =
.1, 1.7 Hz, 1H), 6.04 (s, 1H), 4.94 (d, J = 4.4 Hz, 1H), 4.89 (d, J = –18.0 Hz, 1H), 4.65 (d, J
= –18.0 Hz, 1H), 4.61 (t, J = 4.4 Hz, 1H), 4.48 (d, J = 2.1 Hz, 1H), 3.51 (s, 2H), 2.58 (td, J =
13.3, 4.9 Hz, 1H), 2.35 (dd, J = 13.4, 2.8 Hz, 1H), 2.23–1.99 (m, 4H), 1.79–1.61 (m, 6H),
1.46–1.38 (m, 2H), 1.44 (s, 3H), 1.23–1.09 (m, 2H), 0.95 (s, 3H), 0.93 (t, J = 7.3 Hz, 3H) ppm.
13C NMR (101 MHz, CDCl
3) δ 204.9, 186.6, 170.0, 156.2, 151.2, 141.0, 127.9, 122.5, 116.3,
116.3, 104.5, 97.6, 81.9, 72.6, 69.9, 55.1, 49.8, 45.7, 44.0, 41.1, 35.0, 34.0, 33.3, 31.9, 30.3,
21.1, 17.5, 17.1, 14.0 ppm.
EXAMPLE 13
This example demonstrates a method for making compounds 11-5S and (11-5R)
in Table 1. This example refers to the compound numbering in
Compound 9R was prepared from (R)-budesonide and compound 9S was
prepared from (S)-Budesonide, respectively, according to the General procedure A in
Example 9. Using the same method described in E 12, compound ) was
obtained from the reaction of compound (9S) with compound (10–12), and compound (11-5R)
was ed from the on of compound (9R) with compound (10–9), respectively. A
representative procedure is following. To a solution of compound (9R) or compound (9S (100
mg) in acetone (10 mL) was simultaneously added compound 10–9 (2eq.) and Cs2CO3 (2eq.).
The mixture was ed for 2 hours, and the crude was worked up by ng the acetone
in vacuo, extracting the crude with ethyl acetate, washing the nic salts with water, and
purifying the resulting product by chromatography (0–50% ethyl e in petroleum ether) to
provide compound 11-5R or compound 11-5S (25-60% yield) as a pale yellow solid. ESI m/z:
522 (M + H)+. Anal. HPLC: 98%. The 2D-NOESY spectra of compound 11-5R and compound
11-5S were shown in FIGs. 7 and 8.
EXAMPLE 14
This example demonstrates a method for making compound 11-6S and 11-6R
from Table 1. This example refers to the compound numbering in
(1S,2S,4R,6S,8S,9S,11S,12S,13R)–8–[2–(4–Amino–3–fluorophenoxy)acetyl]–
11–hydroxyl–9,13–dimethyl–6–propyl–5,7–dioxapentacyclo[10.8.0.0²,⁹.0⁴,⁸.0¹³,¹⁸]icosa–14,17–
dien–16–one (11-6S) and (1S,2S,4R,6R,8S,9S,11S,12S,13R)–8–[2–(4–Amino–3–
phenoxy)acetyl]–11–hydroxyl–9,13–dimethyl–6–propyl–5,7–
dioxapentacyclo[10.8.0.0²,⁹.0⁴,⁸.0¹³,¹⁸]icosa–14,17–dien–16–one (11-6R).
A racemic mixture of compounds 11–6R/S were prepared according to the
method set forth in Example 12. The racemic products were separated by chiral SFC (see
details in Section 2.3) to yield compound 11-6S (second peak) and compound 11-6R (first
peak) as off–white .
Compound 11-6S (30 mg, 7.9% . ESI m/z: 540.2 (M + H)+. 1H NMR (500
MHz, DMSOd6) δ 7.32 (d, J = 10.1 Hz, 1H), 6.71–6.62 (m, 2H), 6.49 (dd, J = 8.5, 2.0 Hz, 1H),
6.19–6.16 (m, 1H), 5.93 (s, 1H), 5.21 (t, J = 4.8 Hz, 1H), 5.10 (d, J = 7.3 Hz, 1H), 5.02 (d, J =
18.1 Hz, 1H), 4.69 (dd, J = 58.9, 28.6 Hz, 4H), 4.31 (s, 1H), .51 (m, 1H), 2.29 (d, J =
.6 Hz, 1H), 2.06–1.97 (m, 3H), 1.89 (s, 2H), 1.79–1.72 (m, 1H), 1.30 (m, 10H), 0.88–0.85
(m, 6H) ppm. Retention time: 2.94 min, 98% in chiral SFC (AD). Anal. HPLC: > 96.94%,
Retention time: 7.94 min (method B).
Compound 11-6R (28 mg, 7.4% yield). ESI m/z: 540.3 (M + H)+. 1H NMR (500
MHz, DMSOd6) δ 7.32 (d, J = 10.1 Hz, 1H), 6.72–6.68 (m, 2H), 6.52 (dd, J = 8.6, 2.1 Hz, 1H),
6.18 (d, J = 10.1 Hz, 1H), 5.93 (s, 1H), 5.01 (d, J = 18.3 Hz, 1H), 4.77 (dd, J = 12.9, 3.3 Hz,
2H), 4.71 (s, 2H), 4.65 (t, J = 4.3 Hz, 1H), 4.32 (s, 1H), 3.17 (d, J = 5.2 Hz, 1H), 2.57–2.51 (m,
1H), 2.30 (d, J = 10.5 Hz, 1H), 2.10 (d, J = 7.2 Hz, 1H), 2.01–1.99 (m, 1H), 1.84 (s, 2H), 1.62–
1.52 (m, 5H), .33 (m, 5H), 1.23 (s, 1H), 1.02–0.95 (m, 2H), 0.87 (t, J = 7.4 Hz, 3H),
0.83 (s, 3H) ppm. Retention time: 2.25 min, 100% in chiral SFC (AD). Anal. HPLC: > 98.50%,
Retention time: 8.01 min (method B).
EXAMPLE 15
This example demonstrates a method for making compound 11-7R in Table 1.
This example refers to the compound numbering in
(1S,2S,4R,8S,9S,11S,12S,13R)[2-(4-Aminofluorophenoxy)acetyl]
hydroxyl-9,13-dimethylpropyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-dien-
16-one (11-7S and 11-7R)
A racemic e of steroids 11–7–22R/S were prepared according to the
method set forth in Example 12. The racemic products were separated by chiral SFC (see
details in Section 2.3) to yield compound 11-7S (second peak) and compound 11-7R (first
peak).
Compound 11-7R: ESI m/z: 540.2 (M + H)+. 1H NMR (500 MHz, CDCl3)
δ 7.25 (d, J = 10.1 Hz, 1H), 6.87 (dt, J = 15.5, 7.7 Hz, 1H), 6.47 (dd, J = 12.8, 2.4 Hz, 1H),
6.37 (d, J = 8.7 Hz, 1H), 6.29 (dd, J = 9.9, 4.4 Hz, 1H), 6.04 (s, 1H), 5.22–4.49 (m, 5H), 3.61
(s, 2H), 2.58 (td, J = 13.5, 4.9 Hz, 1H), 2.36 (d, J = 10.3 Hz, 1H), .03 (m, 3H), 1.87–
1.72 (m, 2H), 1.67–1.55 (m, 3H), 1.51–1.33 (m, 7H), 1.21–1.11 (m, 2H), 1.00–0.90 (m, 6H).
Anal. HPLC: > 62.24%, 36.49%, Retention time: 7.78, 7.86 min (method B).
EXAMPLE 16
This example demonstrates a method for making compound 11-8R in Table 1.
This e refers to the compound numbering in
(1S,2S,4R,6R,8S,9S,11S,12S,13R)–11–Hydroxyl–9,13–dimethyl–8–{2–[4–
(methylamino)phenoxy]acetyl}–6–propyl–5,7–dioxapentacyclo–[10.8.0.0²,⁹.0⁴,⁸.0¹³,¹⁸]icosa–
14,17–dien–16–one (11-8R)
Steroid 11–8 was prepared according to the method set forth in Example 13.
Compound ) was obtained as a white solid (14 mg, 54% yield). ESI m/z:
525.3 (M + H)+. 1H NMR (500 MHz, ) δ 7.47 (d, J = 10.1 Hz, 1H), 6.83–6.80 (m, 2H),
6.65–6.62 (m, 2H), 6.28 (dd, J = 10.1, 1.9 Hz, 1H), 6.04 (s, 1H), 4.99 (d, J = 18.2 Hz, 1H),
4.90 (d, J = 4.8 Hz, 1H), 4.74 (d, J = 18.1 Hz, 1H), 4.66 (t, J = 4.5 Hz, 1H), 4.46 (d, J = 3.0
Hz, 1H), 2.75 (s, 3H), 2.67 (td, J = 13.6, 5.2 Hz, 1H), 2.40 (dd, J = 13.5, 2.7 Hz, 1H), 2.30–
2.22 (m, 1H), 2.16–2.12 (m, 1H), 2.02 (dd, J = 13.7, 3.3 Hz, 1H), 1.85 (dd, J = 13.7, 2.6 Hz,
1H), 1.76 (d, J = 6.9 Hz, 1H), 1.67–1.63 (m, 4H), 1.51 (s, 3H), 1.48–1.44 (m, 2H), 1.17–1.08
(m, 1H), 1.05 (dd, J = 11.2, 3.5 Hz, 1H), 0.98–0.94 (m, 6H) ppm. Anal. HPLC: 100%,
Retention time: 7.56 min (method A).
EXAMPLE 17
This Example demonstrates a method for making compound (11-10R/S), in
Table 1. This example refers to the compound numbering in
(1S,2S,4R,6R,8S,9S,11S,12S,13R)–8–[2–(4–Fluorophenoxy)acetyl]–11–
y–9,13–dimethyl–6–propyl–5,7–dioxapentacyclo[10.8.0.0²,⁹.0⁴,⁸.0¹³,¹⁸]icosa–14,17–
dien–16–one (11-10R/S).
Steroids /S were prepared ing to the method set forth in Example
Compound 11-10R/S was obtained as a white solid (14 mg, 54% yield). ESI
m/z: 525.2 (M + H)+. 1H NMR (400 MHz, MeODd4) δ 7.47 (d, J = 10.1 Hz, 1H), 7.02 (t, J =
8.7 Hz, 2H), 6.94–6.90 (m, 2H), 6.27 (dd, J = 10.1, 1.8 Hz, 1H), 6.03 (s, 1H), 5.06 (d, J = 18.1
Hz, 1H), 4.90–4.88 (m, 1H), 4.82 (d, J = 18.1 Hz, 1H), 4.69 (t, J = 4.4 Hz, 1H), 4.46 (d, J = 2.8
Hz, 1H), 2.71–2.63 (m, 1H), 2.42–2.38 (m, 1H), 2.30–2.11 (m, 2H), 2.05–2.01 (m, 1H), 1.89–
1.84 (m, 1H), 1.77–1.63 (m, 5H), .41 (m, 5H), 1.18–1.02 (m, 2H), 0.97–0.93 (m, 6H)
ppm. Anal. HPLC: 100%, ion time: 9.94 min (method A).
EXAMPLE 18
This Example demonstrates a method for making compound 11-11R/S in Table
1. This example refers to the compound numbering in
N–(4–{2–[(1S,2S,4R,6R,8S,9S,11S,12S,13R)–11–Hydroxy–9,13–dimethyl–16–
oxo–6–propyl–5,7–dioxapentacyclo[10.8.0.0²,⁹.0⁴,⁸.0¹³,¹⁸]icosa–14,17–dien–8–yl]–2–
oxoethoxy}phenyl)acetamide (11-11R/S)
Steroids 11-11R/S were prepared ing to the method set forth in Example
nds 11-11R/S were obtained as a white solid (25 mg, 46% yield). ESI
m/z: 564.3 (M + H)+. 1H NMR (500 MHz, MeODd4) δ 7.49–7.45 (m, 3H), 6.89 (d, J = 9.0 Hz,
2H), 6.28 (d, J = 10.2 Hz, 1H), 6.04 (s, 1H), 5.09 (d, J = 18.1 Hz, 1H), 4.91–4.89 (m, 1H), 4.83
(d, J = 18.1 Hz, 1H), 4.70 (t, J = 4.3 Hz, 1H), 4.47 (d, J = 3 Hz, 1H), 2.72–2.65 (m, 1H), 2.43–
2.39 (m, 1H), 2.30–2.22 (m, 1H), 2.18–2.12 (n, 4H), 2.06–2.03 (m, 1H), 1.90–1.86 (m, 1H),
1.77–1.65 (m, 5H), 1.48 (m, 5H), 1.18–1.09 (m, 1H), 1.07–1.04 (m, 1H), 0.99–0.95 (m, 6H)
ppm. Anal. HPLC: 100%, Retention time: 7.33 min (method B).
EXAMPLE 19
This Example demonstrates a method for making compounds 11-12R/S in
Table 1. This example refers to the compound numbering in
(1S,2S,4R,8S,9S,11S,12R,13S,19S)–8–[2–(4–Aminophenoxy)acetyl]–12,19–
difluoro–11–hydroxy–9,13–dimethyl–6–propyl–5,7–
dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa–14,17–dien–16–one (11-12R/S)
Step 1: nd (9B) was prepared according to the General procedure A
in Example 9. To a solution of (6S,9R)2F–budesonide (80 mg, 0.17 mmol) in DCM (1 mL)
were added dropwise triethylamine (34 mg, 0.34 mmol) and methanesulfonyl chloride (30 mg,
0.26 mmol) at 0 oC. The mixture was stirred at this temperature for half an hour until
)2F–Budesonide was consumed, which was monitored by TLC. The reaction mixture
was then diluted with DCM (100 mL) and quenched with sat. aq. um chloride
(30 mL). The organic solution was washed with sat. aq. um chloride and brine, dried
over sodium sulfate and concentrated in vacuo. The crude product was purified by flash
chromatography (0-2% MeOH in DCM) to give the corresponding mesylate product (9B).
Step 2: Compound 9B was dissolved in acetone (0.5 mL). To the solution were
added 4–aminophenol (10–9, 37 mg, 0.34 mmol) and cesium carbonate (0.11 g, 0.34 mmol).
The reaction mixture was refluxed for 1.5 hours or until (9B) was totally consumed according
to TLC and LCMS. The mixture was then d with ethyl acetate and filtered. The filtrate
was concentrated in vacuo and the residue was purified by prep–HPLC (method B) to give
compounds 11-12R/S (6.0 mg, 6.3% yield from (6S,9R)2F–Budesonide) as a white solid. ESI
m/z: 558 (M + H)+. 1H NMR (500 MHz, ) δ 7.34 (d, J = 10.0 Hz, 1H), 6.78–6.71 (m,
4H), 6.37–6.33 (m, 2H), 5.63–5.49 (m, 1H), 5.10–4.99 (m, 1H), 4.77–4.63 (m, 2H), 4.33 (d, J
= 9.1 Hz, 1H), 2.74–2.57 (m, 1H), 2.39–2.13 (m, 3H), 1.98–1.31 (m, 12H), 1.03–0.93 (m, 6H)
ppm. Anal. HPLC: purity 97.4%, Retention time: 7.55 min (method B).
(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)[2-(4-Aminophenoxy)acetyl]-12,19-
difluorohydroxy-9,13-dimethylpropyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,18]icosa-
14,17-dienone R)
Compound 9BR was prepared ing to the General procedure A in
Example 9. A on of compound 9BR (0.90 g, 1.7 mmol) with 4-aminophenol (0.20 g,
1.8 mmol) and cesium carbonate (1.1 g, 3.4 mmol) in acetonitrile (20 mL) provided (11-12R)
(0.20 g, 54% yield) as yellow oil after purification by silica gel column chromatography (50-
80% ethyl acetate in petroleum ether). ESI m/z: 558 (M/+H)+. 1H NMR (500 MHz, DMSOd6)
δ 7.26 (d, J = 10.5 Hz, 1H), 6.64 (d, J = 5.0 Hz, 2H), 6.50 (d, J = 5.0 Hz, 2H), 6.30 (dd, J = 10
Hz, 2 Hz, 1H), 6.11 (s, 1H), 5.72-5.65 (m, 0.5H), 5.62-5.55 (m, 0.5H), 5.52-5.48 (m, 1H), 5.0
(s, 0.5H), 4.95 (s, 0.5H), 4.80-4.78 (m, 1H), 4.75-4.65 (m, 1H), 4.24-4.16 (m, 1H), 2.70-2.52
(m, 1H), 2.30-2.21 (m, 1H), 2.11-2.00 (m, 2H), 1.77 (d, J z, 1H), .54 (m, 4H),
1.49 (s, 3H), 1.36 (q, J = 7.5 Hz, 3H), 1.23 (s, 1H), 0.87 (d, J = 7.5 Hz, 3H), 0.83 (s, 3H) ppm.
Anal. HPLC: 100%, Retention time: 8.44 min (method B).
EXAMPLE 20
This Example demonstrates a method for making compound 11-13R in Table 1.
This example refers to the compound numbering in
(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)[2-(3-Aminophenoxy)acetyl]-12,19-
difluorohydroxy-9,13-dimethylpropyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-
14,17-dienone (11-13R).
Steroid 11-13R was ed according to the method set forth in Example 19.
Compound (11-13R) was obtained as a light orange solid (9.0 mg, 44% yield)
after purification by prep-HPLC (method A). ESI m/z: 558 (M + H)+. 1H NMR (500 MHz,
MeODd4) δ 7.35 (dd, J = 10.1, 1.3 Hz, 1H), 7.29 (t, J = 8.1 Hz, 1H), 6.76-6.70 (m, 3H), 6.40-
6.29 (m, 2H), 5.66-5.48 (m, 1H), 5.14 (d, J = 18.1 Hz, 1H), 4.93-4.91 (m, 1H), 4.90-4.87 (m,
1H), 4.77 (t, J = 4.3 Hz, 1H), 4.35 (d, J = 9.3 Hz, 1H), 2.76-2.62 (m, 1H), 2.41-2.18 (m, 3H),
1.83-1.56 (m, 9H), 1.50 (dt, J = 15.4, 7.6 Hz, 2H), 0.99-0.96 (m, 6H) ppm. Anal. HPLC: 100%,
Retention time: 7.77 min (method A).
EXAMPLE 21
This Example demonstrates a method for making compounds 11-14R/S in
Table 1. This example refers to the compound numbering in
(1S,2S,4R,8S,9S,11S,12R,13S,19S)[2-(4-Aminofluorophenoxy)acetyl]-
12,19-difluorohydroxy-9,13-dimethylpropyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,18]
icosa-14,17-dienone (11-14R/S).
To a on of (9B) (0.20 g, 0.37 mmol) in DMSO (3 mL) were added 4-
aminofluorophenol (10-14, 0.25 g, 2.0 mmol) and potassium hydroxide (0.11 g, 2.0 mmol)
at RT. The resulting e was d at 60 oC for an hour under nitrogen protection until the
reaction was completed, which was monitored by TLC and LCMS. After cooled to RT and
ed through membrane, the reaction on was directly purified by prep-HPLC (method
A) to give compound 11-14R/S (40 mg, 19% yield) as an ite solid. ESI m/z: 576 (M +
H)+. 1H NMR (500 MHz, MeODd4) δ 7.40-7.31 (m, 1H), 7.20 (td, J = 9.1, 1.9 Hz, 1H), 6.91-
6.84 (m, 1H), 6.80-6.76 (m, 1H), 6.40-6.30 (m, 2H), 5.57 (ddd, J = 48.6, 9.7, 6.8 Hz, 1H), 5.15
(d, J = 18.1 Hz, 1H), 4.90-4.79 (m, 2H), 4.75 (t, J = 4.3 Hz, 1H), 4.41-4.28 (m, 1H), 2.78-2.57
(m, 1H), 2.40-2.12 (m, 3H), .39 (m, 11H), 1.07-0.92 (m, 6H) ppm. Anal. HPLC: 100%,
Retention time: 8.10 min (method A).
EXAMPLE 22
This Example demonstrates a method for making compounds 11-15R/S in
Table 1. This e refers to the compound numbering in
tert-Butyl N-[(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-Difluoro
hydroxy-9,13-dimethyloxopropyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-
-yl]oxoethoxy}phenyl)methyl]carbamate (N-Boc15R/S).
Step 1: To a solution of 4-(aminomethyl)phenol (1.2 g, 10 mmol) in methanol
(70 mL) and water (5 mL) was added Boc2O (2.4 g, 11 mmol) dropwise by e at RT. The
resulting mixture was stirred at RT for an hour until 4-(aminomethyl)phenol was totally
consumed, which was monitored by LCMS and TLC. The volatiles were removed in vacuo
and the residue was ved in ethyl acetate (150 mL). The solution was washed with sat. aq.
citric acid (50 mL x 2) and brine, dried over sodium sulfate and concentrated in vacuo to give
N-Bocaminomethylphenol (2.1 g, 94% yield) as brown oil. ESI m/z: 246 (M + Na)+. 1H
NMR (500 MHz, CDCl3) δ 7.12 (d, J = 7.8 Hz, 2H), 6.82-6.71 (m, 2H), 4.84 (s, 1H), 4.23 (d, J
= 5.3 Hz, 2H), 1.46 (s, 9H) ppm.
Step 2: Compound (N-Boc15R/S) was prepared according to the method
set forth in Example 19.
,4R,8S,9S,11S,12R,13S,19S){2-[4-(Aminomethyl)phenoxy]acetyl}-
12,19-difluorohydroxy-9,13-dimethylpropyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]
icosa-14,17-dienone (11-15R/S)
To a on of (N-Boc15R/S) (30 mg, 45 µmol) in DCM (2 mL) was
added dropwise TFA (0.4 mL) by syringe at 0 oC. The resulting mixture was stirred at RT for
an hour until Boc was y removed, which was monitored by LCMS. The volatiles were
removed in vacuo and the residue was purified by prep-HPLC (method A) to give compound
(11-15R/S) (15 mg, 49% yield) as a white solid. ESI m/z: 572 (M + H)+. 1H NMR (500 MHz,
MeODd4) δ 7.45-7.32 (m, 3H), 7.01-6.96 (m, 2H), 6.41-6.30 (m, 2H), 5.57 (ddd, J = 18.2, 10.4,
7.3 Hz, 1H), 5.21 (dd, J = 19.7 Hz, 1H), 4.93-4.91 (m, 1H), 4.85 (d, J = 18.0 Hz, 1H), 4.77 (t, J
= 4.3 Hz, 1H), 4.37-4.32 (m, 1H), 4.07 (s, 2H), 2.75-2.58 (m, 1H), 2.40-2.15 (m, 3H), 1.86-
1.40 (m, 11H), 1.08-0.92 (m, 6H) ppm. Anal. HPLC: 100%, Retention time: 7.47 min
(method A).
EXAMPLE 23
This Example demonstrates a method for making compounds 11-16R/S in
Table 1. This example refers to the compound numbering in
(1S,2S,4R,6R,8S,9S,11S,12S,13R)Hydroxy[2-(4-
hydroxyphenoxy)acetyl]-9,13-dimethylpropyl-5,7-
dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-dienone (11-16R/S)
Compounds 11-16R/S were ed according to the method set forth in
Example 12.
Compounds 11-16R/S (20 mg, 38% yield) were obtained as a tan solid after
purification by PLC (method A). ESI m/z: 523.2 (M + H)+. 1H NMR (500 MHz,
MeODd4) δ 7.47 (d, J = 10.1 Hz, 1H), 6.82-6.77 (m, 2H), 6.75-6.70 (m, 2H), 6.28 (dd, J = 10.1,
1.8 Hz, 1H), 6.04 (s, 1H), 5.00 (d, J = 18.1 Hz, 1H), 4.91-4.89 (m, 1H), 4.75 (d, J = 18.1 Hz,
1H), 4.67 (t, J = 4.5 Hz, 1H), 4.46 (d, J = 3.1 Hz, 1H), 2.68 (td, J = 13.6, 5.8 Hz, 1H), 2.40 (dd,
J = 13.5, 2.8 Hz, 1H), 2.31-2.21 (m, 1H), 2.17-2.13 (m, 1H), 2.02 (dd, J = 13.7, 3.3 Hz, 1H),
1.86 (dd, J = 13.7, 2.6 Hz, 1H), 1.80-1.58 (m, 5H), 1.53-1.40 (m, 5H), 1.18-0.93 (m, 8H) ppm.
Anal. HPLC: 100%, Retention time: 8.92 min (method A).
EXAMPLE 24
This Example demonstrates a method for making compounds 11-17R/S in
Table 1. This example refers to the compound numbering in
(1S,2S,4R,8S,9S,11S,12R,13S,19S){2-[(6-Aminopyridinyl)oxy]acetyl}-
difluorohydroxy-9,13-dimethylpropyl-5,7-dioxapentacyclo[10.8.0.02,9.04,8.013,18]
icosa-14,17-dienone (11-17R/S)
Compounds 11-17R/S were prepared according to the method set forth in
Example 19.
Compounds /) (50 mg, 24% yield) were obtained as a white solid after
purification by flash tography (10-50% ethyl acetate in petroleum ether). ESI: 559 (M +
H)+. 1H NMR (500 MHz, DMSOd6) δ 7.35-7.31 (m, 2H), 6.31 (d, J = 11.5 Hz, 1H), 6.13 (s,
1H), 6.03 (d, J = 8.0 Hz, 1H), 5.98 (d, J = 7.5 Hz, 1H), 5.84-5.82 (m, 1H), 5.68-5.56 (m, 3H),
.25-4.72 (m, 4H), 4.29 (br s, 1H), 2.66-2.57 (m, 1H), .05 (m, 4H), 1.63-1.58 (m, 4H),
1.50-1.30 (m, 6H), 0.95-0.87 (m, 6H) ppm. Anal. HPLC: 100%, Retention time: 8.65 min
(method A).
EXAMPLE 25
This Example demonstrates a method for making nd 11-19 in Table 1.
This example refers to the compound numbering in
(1S,2S,4R,8S,9S,11S,12R,13S,19S)(2-Azidoacetyl)-12,19-difluoro
hydroxy-9,13-dimethylpropyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-dien-
16-one (11-19)
Step 1: A suspension of compound 9B (1.0 g, 1.8 mmol), sodium azide (1.2 g,
18 mmol) in acetone (15 mL) was stirred at 50 oC overnight, when the reaction was ted
according to LCMS. After cooled, the reaction mixture was poured into cold water (80 mL).
The s mixture was extracted with ethyl acetate (50 mL x 3). The combined organic
solution was washed by brine (30 mL), dried over sodium sulfate and concentrated in vacuo to
afford crude compound azido precursor of (11-19R/S) (0.90 g, > 99% yield) as a yellow solid,
which was used for the next step without further purification. ESI m/z: 492 (M + H)+.
(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)(2-Aminoacetyl)-12,19-difluoro
hydroxy-9,13-dimethylpropyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-dien-
16-one; trifluoroacetic acid salt (11-19R/S)
Step 2: To a solution of the precursor of nds 11-19R/S (0.85 g, 1.7
mmol) in THF (20 mL) was added aq. hydrochloride (1 N, 10 mL). The mixture was stirred at
28-32 oC until it turned clear, to which was then added triphenylphosphine (0.68 g, 2.6 mmol)
at this temperature. The resulting yellow clear on was stirred at 28-32 oC for 18 hours,
when the reaction was completed ing to TLC and LCMS. The mixture was trated
under vacuum and the residue was purified by reversed phase flash chromatography (0-50%
acetonitrile in aq. TFA (0.05%)) to give compounds 11-19R/S (0.56 g, 57% yield, TFA salt) as
an off-white solid. ESI m/z: 466 (M + H)+. 1H NMR (400 MHz, MeODd4) δ 7.33 (d, J = 9.9
Hz, 1H), 6.40-6.29 (m, 2H), 5.69-5.45 (m, 1H), 4.93-4.92 (m, 1H), 4.71 (t, J = 4.3 Hz, 1H),
4.35-4.27 (m, 2H), 3.90-3.84 (m, 1H), 2.81-2.54 (m, 1H), 2.42-2.06 (m, 3H), 1.82-1.32 (m,
11H), 1.09-0.87 (m, 6H) ppm. 19F NMR (376 MHz, MeODd4) δ
-77.01, -166.24, -166.92, 1, -188.83 ppm. Anal. HPLC: 100%, Retention time: 6.86 min
(method A).
(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)(2-Aminoacetyl)-12,19-difluoro
y-9,13-dimethylpropyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-dien-
16-one; trifluoroacetic acid salt (11-19R)
Step 1: Using the same procedure described above, the azido precursor of (11-
19R) (0.12 g, 87% yield) was obtained from compound (9BR) as a white solid after
purification by flash chromatography (0-50% ethyl acetate in petroleum . ESI m/z: 492
(M + H)+. 1H NMR (500 MHz, CDCl3) δ 7.10 (dd, J = 10.2, 1.3 Hz, 1H), 6.44 (s, 1H), 6.38
(dd, J = 10.2, 1.8 Hz, 1H), 5.48-5.31 (m, 1H), 4.92 (d, J = 5.4 Hz, 1H), 4.62 (t, J = 4.4 Hz,
1H), 4.43 (dd, J = 5.6, 2.7 Hz, 1H), 4.22 (d, J = 18.7 Hz, 1H), 3.94 (d, J = 18.7 Hz, 1H), 2.56-
2.39 (m, 2H), 2.32-2.18 (m, 2H), 1.85-1.71 (m, 3H), 1.67-1.54 (m, 7H), 1.46-1.37 (m, 2H),
0.97-0.90 (m, 6H) ppm.
Step 2: Using the same ure described above, compound 11-19R (30 mg,
66% yield) was obtained as a white solid after purification by prep-HPLC d A). ESI
m/z: 466 (M + H)+. 1H NMR (500 MHz, MeODd4) δ 7.34 (d, J = 10.0 Hz, 1H), 6.40-6.30 (m,
2H), .46 (m, 1H), 4.94-4.91 (m, 1H), 4.72 (t, J = 4.3 Hz, 1H), 4.34-4.28 (m, 2H), 3.88
(d, J = 18.8 Hz, 1H), 2.78-2.60 (m, 1H), 2.39-2.34 (m, 1H), 2.33-2.18 (m, 2H), 1.77-1.54 (m,
9H), 1.53-1.40 (m, 2H), .95 (m, 6H) ppm. Anal. HPLC: 100%, Retention time: 6.85 min
(method A).
EXAMPLE 26
This Example demonstrates a method for making compound 11-20R/S in Table
1. This example refers to the nd numbering in
(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-Difluorohydroxy(2-{[(4-
methoxyphenyl)methyl](methyl)amino}acetyl)-9,13-dimethylpropyl-5,7-
dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-dienone; trifluoroacetic acid (11-20R/S)
To a solution of compound 9B (0.54 g, 1.0 mmol) in acetonitrile (10 mL) were
added N-PMB-methylamine (0.30 g, 2.0 mmol) and potassium carbonate (0.28 g, 2.0 mmol) at
RT successively. The reaction mixture was stirred at 70 oC overnight. After cooled, the mixture
was diluted with DCM and filtered. The filtrate was concentrated in vacuo and the residue was
purified by flash chromatography (10-90% ethyl acetate in eum ether) to afford crude
compound (11-20R/S) (0.20 g, 33% yield) as a white solid. The crude product (30 mg) was
further purified by PLC (method A) to afford pure compound (11-20R/S) as a white
solid (12 mg, 13% yield). ESI m/z: 600 (M + H)+. 1H NMR (500 MHz, MeODd4) δ 7.50-7.43
(m, 2H), 7.34 (d, J = 10.1 Hz, 1H), 7.07 (d, J = 8.5 Hz, 2H), 6.39-6.30 (m, 2H), 5.56 (ddd, J =
48.5, 10.7, 6.5 Hz, 1H), 5.24-5.21 (m, 1H), 4.94-4.92 (m, 1H), .53 (m, 1H), 4.38-4.16
(m, 4H), 3.86 (s, 3H), 2.92-2.91 (m, 3H), 2.76-2.56 (m, 1H), 2.39-2.31 (m, 1H), 2.28-2.09 (m,
2H), 1.97 (td, J = 13.2, 7.8 Hz, 1H), 1.78-1.23 (m, 10H), 1.08-0.88 (m, 6H) ppm. Anal. HPLC:
100%, Retention time: 7.81 min (method A).
This Example demonstrates a method for making compounds 11-21R/S in
Table 1. This example refers to the compound numbering in
(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-Difluorohydroxy-9,13-dimethyl-
8-[2-(methylamino)acetyl]propyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-
dienone; oroacetic acid (11-21R/S)
To compounds 11-20R/S (30 mg, 0.053 mmol) in 4 mL-screw-capped vial were
added 1-chloroethyl carbonochloridate (1 drop) and chloroform (0.4 mL). The mixture was
d at 70 oC for 2 hours until the ng material was consumed by TLC. After cooled to
RT, the mixture was added methanol (1.5 mL). The mixture was stirred at 70 oC for 1 h until
the reaction was completed, which was monitored by TLC and LCMS. The volatiles were
removed in vacuo and the residue was purified by prep-HPLC (method A) to afford
compounds 11-21R/S (8.0 mg, 28% yield) as a white solid. ESI m/z: 480 (M + H)+. 1H NMR
(400 MHz, MeODd4) δ 7.34 (d, J = 10.1 Hz, 1H), 6.41-6.26 (m, 2H), 5.56 (ddd, J = 48.7, 10.0,
6.8 Hz, 1H), 5.28 (t, J = 4.9 Hz, 1H), 5.23 (d, J = 7.4 Hz, 1H), .41 (m, 1H), 4.34-4.30
(m, 1H), 4.07-4.00 (m, 1H), 2.82-2.54 (m, 4H), 2.43-2.09 (m, 3H), 1.96 (td, J = 13.6, 7.9 Hz,
1H), 1.81-1.34 (m, 10H), 1.10-0.85 (m, 6H) ppm. 19F NMR (376 MHz, MeODd4) δ -76.96,
-166.28, -166.95, -188.80, -188.83 ppm. Anal. HPLC: 99%, Retention time: 6.97 min
(method A).
EXAMPLE 28
This example demonstrates a method for making compound 14-2 in Table 1.
This example refers to the compound numbering in
(1R,2S,8S,10S,11S,13S,14R,15S,17S)–1,8–difluoro–17–hydroxy–2,13,15–
trimethyl–14–[2–(methylamino)acetyl]–5–oxotetracyclo[8.7.0.0²,⁷.0¹¹,¹⁵]heptadeca–3,6–dien–
14–yl propanoate (14-2)
The synthesis of mesylate flumethasone (12) was reported in Bioorg. Med.
Chem. Lett., 2015, 25, 843, the entire contents of which are herein orated by
reference in their entirety for all purposes.
A solution of 12 (82 mg crude) in methylamine (2M solution in THF, 1.5 mL,
3.000 mmol) in a sealed tube was stirred at RT for overnight, and then heated at 60oC for 3
hours until the reaction was completed. The solution was concentrated in vacuo and the residue
was purified by prep. HPLC (0–80% itrile in water with 10 mM NH4HCO3) to get
compound 14-2 (8 mg, yield 11% for two steps) as a white solid. ESI m/z: 480.2 (M+H). 1H
NMR d6, 400 MHz) δ 7.27–7.25 (d, J=10.4 Hz, 1H), 6.30–6.27 (dd, J=10.4, 2.0 Hz,
1 H), 6.10 (s, 1H), 5.73–5.56 (m, 1H), .32 (m, 2H), 4.62–4.42 (m, 1H), 4.25–4.18 (m,
1H), 4.15 (brs, 1H), 2.87 (s, 2H), 2.70 (s, 1H), 2.60–2.56 (m, 1H), 2.36–1.90 (m, 7H), 1.49–
1.35 (m, 5H), 1.10–0.91 (m, 10H).
EXAMPLE 29
This example demonstrates a method for making compound 15-5 Table 1.
This example refers to the compound numbering in
(1R,2S,8S,10S,11S,13R,14R,15S,17S)–14–[2–(4–Aminophenoxy)acetyl]–1,8–
difluoro–14,17–dihydroxy–2,13,15–trimethyltetracyclo[8.7.0.02,7.011,15]heptadeca–3,6–dien–5–
one (15-5)
Step 1: A mixture of compound (12) (0.16 g, 0.33 mmol), ophenol (10–5,
92 mg, 0.67 mmol) and potassium carbonate (92 mg, 0.67 mmol) in acetone (15 mL) was
refluxed (60°C) for 18 hours. After cooled down to RT, the volatiles were removed in vacuo.
The residue was purified by flash chromatography (0–1% ethyl acetate in petroleum ether) to
yield a nitro–intermediate (0.14 g, yield 79%) as a white solid. ESI m/z: 532 (M + H)+. 1H
NMR (CDCl3, 400 MHz) δ 8.20 (d, J = 9.0 Hz, 2H), 7.10 (d, J = 10.5 Hz, 1H), 6.94 (d, J = 9.0
Hz, 2H), 6.43 (br s, 1H), 6.39–6.37 (m, 1H), 5.45–5.32 (m, 1H), 5.26 (d, J = 18.0 Hz, 1H),
4.85 (d, J = 18.0 Hz, 1H), .40 (m, 1H), 3.21–3.16 (m, 1H), 2.60 (s, 1H), 2.52–2.40 (m,
2H), 2.30–2.20 (m, 2H), 2.06–1.99 (m, 1H), 1.86–1.68 (m, 3H), 1.53–1.48 (m, 2H), 1.09 (s,
3H), 0.99 (d, J = 7.0 Hz, 3H) ppm.
Step 2: To a on of the nitro–intermediate (0.13 g, 0.25 mmol) in a
combined solution of l (20 mL) and water (2 mL) was added iron powder (0.14 g, 2.5
mmol) and then ammonium chloride (0.14 g, 2.5 mmol). After stirring at 80 oC for 2 hours,
the suspension was cooled down to RT and filtered through Celite to remove the inorganic
salts. The filtrate was trated in vacuo and the residue was purified by prep–HPLC
(method B) to yield compound 15-5 (90 mg, yield 70%) as a white solid. ESI m/z: 502 (M +
H)+. 1H NMR (DMSOd6, 500 MHz) δ 7.27 (d, J = 10.0 Hz, 1H), 6.59 (d, J = 8.5 Hz, 2H), 6.49
(d, J = 8.5 Hz, 2H), 6.31–6.28 (m, 1H), 6.11 (s, 1H), 5.77–5.57 (m, 1H), 5.42–5.39 (m, 1H),
.22 (s, 1H), 5.07 (d, J = 18.5 Hz, 1H), 4.63 (s, 1 H), 4.59 (d, J = 18.5 Hz, 1H), .10 (m,
1H), 2.99–2.91 (m, 1H), 2.55–2.43 (m, 3H), 2.25–2.19 (m, 3H), 1.71–1.64 (m, 1H), 1.56–1.43
(m, 5H), 1.15–1.10 (m, 1H), 0.88 (s, 3H), 0.83 (d, J = 6.0 Hz, 3H) ppm.
EXAMPLE 30
This example demonstrates a method for making compound 16-5in Table 1.
This example refers to the compound numbering in
(1R,2S,10S,11S,13R,14R,15S,17S)–14–[2–(4–Aminophenoxy)acetyl]–1–fluoro–
14,17–dihydroxy–2,13,15–trimethyltetracyclo[8.7.0.02,7.011,15]heptadeca–3,6–dien–5–one
(16-5)
The synthesis of mesylate dexamethasone (13) was reported in J. Pharmacol.,
172, 1360 (2015), the entire contents of which are herein incorporated by reference in their
ty for all es.
A mixture of mesylate dexamethasone (13, 94 mg, 0.20 mmol), 4–nitrophenol
(10–5, 42 mg, 0.30 mmol) and ium carbonate (55 mg, 0.40 mmol) in acetone (10 mL)
was refluxed (60 °C) for 3 hours and was then concentrated. The crude product was
concentrated in vacuo, and then directly purified by flash chromatography (0–50% ethyl
acetate in petroleum ether) to yield a nitro–intermediate (0.10 g, yield 97%) as a white solid.
ESI m/z: 514 (M + H)+. 1H NMR (MeODd4, 400 MHz) δ 8.23 (d, J = 9.0 Hz, 2H), 7.43 (d, J =
.5 Hz, 1H), 7.04 (d, J = 9.0 Hz, 2H), 6.31 (dd, J = 10.0 Hz, 2.0 Hz, 1H), 6.11 (br s, 1H), 5.41
(d, J = 18.0 Hz, 1H), 4.96 (d, J = 18.0 Hz, 1H), 4.34–4.30 (m, 1H), 3.13–3.06 (m, 1H), 2.79–
2.72 (m, 1H), 2.57–2.41 (m, 3H), 2.32–2.26 (m, 1H), 1.94–1.90 (m, 1H), 1.82–1.75 (m, 1H),
1.62 (s, 3H), 1.62–1.53 (m, 2H), 1.28–1.23 (m, 1H), 1.07 (s, 3H), 0.92 (d, J = 7.0 Hz, 3 H)
To a solution of the nitro–intermediate (i.e., NO2-analog in 60 mg,
0.12 mmol) in a combined solution of ethanol (3 mL) and water (0.5 mL) were added iron
powder (67 mg, 1.2 mmol) and then ammonium de (64 mg, 1.2 mmol). After stirring at
80oC for 1.5 hours, the suspension was cooled down to RT and filtered through Celite to
remove the inorganic salts. The filtrate was concentrated in vacuo and the residue was purified
by prep–HPLC (method B) to yield nd 16-5 (20 mg, yield 35%) as a white solid. ESI
m/z: 484 (M + H)+. 1H NMR 4, 500 MHz) δ 7.42 (d, J = 10.5 Hz, 1H), 6.78–6.74 (m,
2H), 6.73–6.70 (m, 2H), 6.31 (dd, J = 10.0 Hz, 2.0 Hz, 1H), 6.10 (br s, 1H), 5.08 (d, J = 18.0
Hz, 1H), 4.71 (d, J = 18.0 Hz, 1H), 4.30–4.27 (m, 1H), 3.14–3.09 (m, 1H), 2.78–2.71 (m, 1H),
2.54–2.37 (m, 3H), 2.30–2.24 (m, 1H), 1.94–1.89 (m, 1H), .74 (m, 1H), 1.62 (s, 3H),
1.59–1.52 (m, 2H), 1.26–1.21 (m, 1H), 1.06 (s, 3H), 0.91 (d, J = 7.5 Hz, 3H) ppm.
EXAMPLE 31
This example demonstrates methods for separating stereoisomers of certain
compounds disclosed herein.
SFC (Supercritical fluid tography) technology was used for the
purification of small lar compounds, which are thermally labile, including chiral
compounds. SFC used ritical fluid carbon dioxide as a mobile phase and organic
polymer bonded solid adsorbent as a stationary phase. Based on different partition coefficient
of the epimers in the two phases, the mixed epimers could be ted by adjusting the mobile
phase’s density. The instrument and column conditions are described as follows: Instrument:
SFC–80 (Thar, Waters), Column: AD 20*250mm, 5um (Decial), Column temperature: 35oC,
Mobile phase: CO2/EtOH(1%Methanol Ammonia)= 65/35, Flow rate: 80 g/min, Back
pressure: 100 bar, Detection ngth: 214 nm, Cycle time: 4.5 min, Sample solution: 130
mg dissolved in 30 ml Methanol, ion volume: 1.5 ml). By using a chiral AD–H column,
grams of 22R/S–budesonide were separated to yield 8.9 grams of R–budesonide and 8.9
grams of S–budesonide in a total of 89% recovery yield. Similarly, two epimers of compound
11-5R/S were also separated by SFC. The detail tion conditions were described below in
Table 5.
Table 5: Conditions of chiral separation of Budesonide and nd (11-5) in Table 1.
Compound Budesonide 11-5R/S
Instrument SFC–200 (Thar, Waters) SFC–200 (Thar, Waters)
Column AD–H 20*250mm, 5um SC 20*250mm, 5um
(Dacel)
Column temperature 35 ºC 35 ºC
Mobile phase CO2 / methanol (0.5% CO2 / methanol (0.5%
NH4OH) = 70/30 NH4OH) = 60/40
Flow rate 120 g/min 140 g/min
Back pressure 100 bar 100 bar
Detection wavelength 214 nm 214 nm
Cycle time 4.0 min 5.0 min
Sample solution 20 g dissolved in 130 ml 10 g dissolved in 130 ml
Methanol Methanol
Injection volume 1.0 ml 0.5 ml
The structures of 22R/S–Budesonide were determined stereospecifically by 2D–
NOESY. Compared with reported proton NMR data of 22R/S–Budesonide, the first compound
from the chiral SFC was determined to be the R–epimer, while the second was determined to
be the S epimer. The configuration at C22 influences the magnetic resonances of the
neighboring protons. A double doublet with J16βH–15βH = 5.0 Hz and J16βH–15αH = 2.5 Hz were
observed in the S–spectrum, which resulted from a steric repulsion from the 22–propyl
substituent deshielding the C16 proton in the S–epimer. This effect is not observed in the R–
epimer. The C22 proton in the S–epimer also moved downfield compared to that of the R–
epimer, ting deshielding of the C22 proton in the S–epimer due to a steric repulsion
n the 17β–ketol substituent and the opyl chain in the S–epimer. Similarly, the C22
proton in the R–epimer was shielded by anisotropy effect from the C20–carbonyl group in the
22R–epimer. The detail al shifts are described below in Table 6.
Table 6
s Chemical shifts (ppm) in D–chloroform
R–epimer reported 1st compound from S–epimer reported 2nd compound
chiral SFC from chiral SFC
C–1 7.26 (d, J1,2 = 10.1) 7.26 (d, J1,2 = 10.1) 7.23 (d, J1,2 = 10.1) 7.26 (d, J1,2 =
.0)
C–2 6.27 (dd, J1,2 = 6.28 (dd, J1,2 = 10.1, 6.27 (dd, J1,2 = 10.1, 6.27 (dd, J1,2 =
.1, J2,4 = 1.8) J2,4 = 1.7) J2,4 = 1.8) 10.1, J2,4 = 1.7)
C–4 6.03 (m) 6.03 (s) 6.02 (m) 6.02 (s)
C–11 4.4–4.6 (m) 4.42–4.60 (m) 4.50 (m) 4.50 (br s)
C–16 4.90 (dd, 15βH 4.90 (d, J16βH–15βH = 5.16 (dd, J16βH–15βH = 5.23–5.11 (m)
= 4.2) 4.4) 5.0, 15αH = 2.5)
C–18 0.92 (s) 0.92 (s) 0.99 (s) 0.99 (s)
C–19 1.45 (s) 1.44 (s) 1.45 (s) 1.46 (s)
C–21 4.50 (dd), 4.25 (dd) 4.50 (m), 4.60 (dd), 4.20 (dd) 4.62 (d), 4.21 (d)
(J21H, H’ = –20.2, 4.26 (dd, J21H, H’ = (J21H, H’ = –20.2, J21H– (J21H, H’ = 19.9)
J21H–21OH = 4.8) 20.1, 1OH = 21OH = 4.8)
C–22 4.55 (t, J22,23 = 4.2) 4.55 (t, J22,23 = 4.6) 5.16 (t, J22,23 = 4.6) 5.23–5.11 (m)
C–25 0.92 (t, J24,25 = 6.7) 0.92 (t, J24,25 = 7.3) 0.91 (t, J24,25 = 7.3) 0.91 (t, J24,25 =
EXAMPLE 32
This example demonstrates methods for making linkers and linker–payloads,
generally.
Three generic approaches for making linker–payloads are shown in In
R' is a steroid amine or aniline; R'' is an alkyne containing moiety, such as fragment A
or B, or a maleimide moiety, such as C; R1 is an amino–acid residue; P is a protective group,
such as Fmoc or Boc; n is an integer from 0–11; m is an integer from 2–4; p is an integer from
0–5. ch I forms an amide (23) from a coupling reaction between the steroid amine or
aniline (21, Q = NH or NR) and a dipeptide (22) followed by N–deprotection. The amine (23)
was then coupled with an acid or its active ester (24), such as V–5, V–7, V in , VI–8
and VI in , and VII in , to generate the linker–payloads (25). Approach II
forms an amide (28) from a coupling reaction between an acid or its active ester (26) and VC–
pAB (27) followed by N–deprotection. Compound 28 was then converted to its PNP
derivative that further reacted with 21 to generate the linker–payload carbamate (29).
ch III forms a carbamate (30) from N–protected–dipeptide–pAB–PNP (19) and the
steroid amine or aniline (21), followed by N–deprotection; the amine moiety in 30 was then
d with an acid or its active ester (26) to generate 29.
EXAMPLE 33
This example demonstrates methods for making linker DIBAC–Suc–NHS (V).
The ing e refers to .
See methods in J. Org. Chem., 2010, 75, 627–636 which are incorporated by
reference herein in their entirety for all es.
Step 1: N–[Tricyclo[9.4.0.03,8]pentadeca–1(11),3,5,7,9,12,14–heptaen–2–
ylidene]hydroxylamine (V–2): A mixture of dibenzosuberenone (V–1) (21 g, 0.10 mol) and
hydroxylamine hydrochloride (9.3 g, 0.14 mol) in a combined solution of absolute ethanol
(100 mL) and pyridine (200 mL) was stirred and refluxed for 15 hours. TLC showed the
starting al was consumed (TLC: 5% methanol in methylene chloride). After cooling to
below 25 oC, the reaction mixture was diluted with methylene de (500 mL) and the
resulting solution was washed with aqueous (aq.) HCl (1N, 3 x 200 mL) and then brine (200
mL). The organic solution was dried over sodium sulfate and concentrated in vacuo to yield
crude V–2 (22 g, 98% crude yield) as a light brown solid. ESI m/z: 222.1 (M + H)+.
Step 2: 2–Azatricyclo[10.4.0.04–9]hexadeca–1(16),4(9),5,7,10,12,14–heptene
(V–3): To a on of the oxime (V–2) (5.5 g, 25 mmol) in dry methylene chloride (herein
also dichloromethane or DCM) (150 mL) at –5 °C was added DIBAL–H (1 M in toluene,
250 mL) dropwise while maintaining the temperature below –5 oC. The on was then
stirred at RT overnight and was subsequently quenched with a solution of sodium fluoride
solid (38 g, 0.90 mol) in water (12 mL) at 0 oC. The slurry was stirred at 0 oC for another 30
minutes and filtered through Celite. The Celite was thoroughly washed with methylene
chloride and the combined organic solution was concentrated in vacuo to yield V–3 (4.6 g,
89% yield) as a yellow solid. ESI m/z: 222.1 (M + H)+.
Step 3: 4–[2–Azatricyclo[10.4.0.04,9]hexadeca–1(16),4(9),5,7,10,12,14–
heptaen–2–yl]–4–oxobutanoic acid (V–5): To a solution of (V–3) (5.0 g, 24 mmol) in
methylene chloride (50 mL) were added DIPEA (3.1 g, 24 mmol) and then succinic ide
(V–4, 2.9 g, 29 mmol). The mixture was then stirred at RT for 4 hours, quenched with aq.
sodium bisulfate (1N, 100 mL), and extracted with ene chloride (3 x 100 mL). The
combined organic solution was washed with water (100 mL) and then brine (100 mL), dried
over sodium sulfate and concentrated in vacuo to afford (V–5) (7.7 g, 95% yield) as a white
solid, which was used without further purification. ESI m/z: 308.2 (M + H)+.
Step 4: 4–{10,11–dibromo–2–azatricyclo[10.4.0.04,9]hexadeca–
1(16),4(9),5,7,12,14–hexaen–2–yl}–4–oxobutanoic acid (V–6): A solution of (V–5) (15 g,
49 mmol) in ene chloride (200 mL) was flushed with nitrogen and cooled to 0 oC. To
the solution was added liquid bromine (23 g, 0.14 mol) dropwise at 0 oC via a syringe. The
reaction was stirred at this temperature for 2 hours and TLC showed the reaction was
completed (TLC: 10% methanol in methylene chloride). The reaction mixture was diluted with
methylene chloride (50 mL) and was allowed to warm to RT. The organic on was washed
with saturated (sat.) aq. sodium sulfite (3 x 50 mL), water (50 mL) and then brine (50 mL),
dried over sodium sulfate and concentrated in vacuo to yield (V–6) (13 g, 99% crude yield) as
an off–white solid. ESI m/z: 467.9 (M + H)+. 1H NMR , 400 MHz): δ 7.71 (d, J = 6.8
Hz, 1H), 7.25–7.01 (m, 6H), 6.94–6.88 (m, 1H), 5.90 (d, J = 9.6 Hz, 1H), 5.84–5.79 (m, 1H),
.25–5.25 (m, 1H), 4.24–4.10 (m, 1H), .80 (m, 1H), 2.68–2.47 (m, 3H) ppm.
Step 5: 4–{2–Azatricyclo[10.4.0.04,9]hexadeca–1(16),4(9),5,7,12,14–hexaen–
–yn–2–yl}–4–oxobutanoic acid (V–7): A solution of (V–6) (5.0 g, 11 mmol) in anhydrous
THF (50 mL) was cooled to –40 oC with a dry–ice/acetonitrile bath and to the solution was
added a solution of potassium tert–butanolate in tetrahydrofuran (1N, 37 mL, 37 mmol)
dropwise under argon atmosphere. The on mixture was stirred at this temperature for half
an hour after the addition. TLC showed that the reaction was ted (TLC: 10% methanol
in methylene chloride). The reaction e was allowed to warm to RT and was quenched
with aq. sodium bisulfate (1N) to pH 1. The mixture was extracted with methylene chloride
(3 x 50 mL). The combined organic solution was washed with water (50 mL) and then brine
(50 mL), dried over sodium sulfate and concentrated in vacuo to yield nd (V–7) (2.7 g,
95% yield) as an off–white solid. ESI m/z: 306.1 (M + H)+. 1H NMR (DMSOd6, 500 MHz):
δ 11.98 (s, 1H), 7.67–7.29 (m, 8H), 5.02 (d, J = 13.5 Hz, 1H), 3.61 (d, J = 14.5 Hz, 1H), 2.61–
2.56 (m, 1H), 2.32–2.27 (m, 1H), 2.21–2.16 (m, 1H), 1.80–1.76 (m, 1H) ppm.
Step 6: 4–{2–Azatricyclo[10.4.0.04,9]hexadeca–1(12),4(9),5,7,13,15–hexaen–
–yn–2–yl}–4–oxobutanoic acid (V): To a solution of acid (V–7) (50 mg, 0.16 mmol) in
methylene chloride (10 mL) were uently added N–hydroxysuccinimide (HOSu, 28 mg,
0.24 mmol) and N–(3–dimethylaminopropyl)–N’–ethylcarbodiimide hydrochloride (EDCI,
47 mg, 0.24 mmol). After stirring at RT ght, the mixture was washed with water and
then brine, dried over sodium sulfate and concentrated in vacuo to yield ediate V, which
was used for next step directly. ESI m/z: 403.0 (M + H)+.
EXAMPLE 34
This example demonstrates methods for making linker DIBAC–Suc–PEG4–
acid/NHS (VI). The following Example refers to .
Step 1: Tert–butyl-1–hydroxy–3,6,9,12–tetraoxapentadecan–15–oate (VI–3):
To a solution of thylene glycol (VI–1, 58 g, 0.30 mol) in dry THF (200 mL) was added
sodium (0.12 g), and the mixture was stirred until the sodium was consumed. To the resulting
solution was then added tert–butyl acrylate (VI–2, 13 g, 0.10 mol) in dry THF (50 mL)
dropwise, and the resulting mixture was stirred at RT overnight. The reaction was quenched
with acetic acid (0.1 mL) first and then water (0.5 mL), and the resulting mixture was d at
RT for half an hour, and subsequently was extracted with ethyl acetate (3 x 200 mL). The
combined organic solution was washed with water (30 mL) and then brine (3 x 100 mL), dried
over sodium sulfate, filtered and concentrated to yield product (VI–3, 26 g, 81% yield) as
colorless oil. ESI m/z: 340 (M + 18)+.
Step 2: tert–Butyl hanesulfonyloxy)–3,6,9,12–tetraoxapentadecan–15–
oate (VI–4): To a solution of (VI–3) (26 g, 81 mmol), triethylamine (12 mL, 89 mmol) in
methylene chloride (150 mL) in an ice–water bath was added a solution of methanesulfonyl
chloride (10 g, 89 mmol) in DCM (50 mL) dropwise. The mixture was d at RT for 14
hours, and was then concentrated in vacuo. The residue was mixed with water (30 mL), and
was then extracted with ethyl acetate (3 x 200 mL). The ed organic layer was washed
with brine (3 x 100 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to yield
the desired product (VI–4) (31 g, 95% yield) as light yellow oil. ESI m/z: 418 (M + 18)+.
Step 3: tert–Butyl 1–azido–3,6,9,12–tetraoxapentadecan–15–oate (VI–5): To a
solution of (VI–4) (27 g, 67 mmol) in DMF (70 mL) was added sodium azide (6.6 g, 0.10
mol), which was then stirred at 80 °C for 4-16 hours. After cooled to RT, the mixture was
diluted with ethyl acetate (3 x 150 mL). The combined solution was washed with water (30
mL) and then brine (3 x 100 mL), dried over sodium sulfate, filtered, and concentrated in
vacuo. The crude product was purified by silica gel column chromatography (petroleum ether /
ethyl acetate (with 1% to 2% methanol) = 4/1) to yield (VI–5) (18 g, 67% yield) as colorless
oil. ESI m/z: 365 (M + 18)+.
Step 4: tert–Butyl 1–amino–3,6,9,12–tetraoxapentadecan–15–oate (VI–6):
To a solution of (VI–5) (1.5 g, 4.3 mmol) in ethyl acetate (20 mL) was added wet Pd/C (10%,
0.15 g) under nitrogen. The mixture was then flushed with hydrogen and stirred at RT under a
en balloon ght. The mixture was then ed through Celite. The Celite was
washed with ethyl acetate (10 mL). The combined filtrate was concentrated in vacuo to yield
crude (VI–6) (1.4 g) as light a yellow oil, which was used on the next step without further
purification. ESI m/z: 322 (M + H)+.
Step 5: 1–Amino–3,6,9,12–tetraoxapentadecan–15–oic acid (VI–7): To a
solution of (VI–6), obtained above (1.4 g) in methylene de (10 mL) was added TFA
(5 mL). The mixture was d at RT for an hour. The volatiles were removed in vacuo to
yield crude product (VI–7) as its TFA salt (1.6 g) as yellow oil, which was used for the next
step without further purification. ESI m/z: 266 (M + H)+.
Step 6: 1–(4–{2–Azatricyclo[10.4.0.04–9]hexadeca–1(12),4(9),5,7,13,15–
hexane–10–yn–2–yl}–4–oxobutanamido)–3,6,9,12–tetraoxapentadecan–15–oic acid :
A mixture of 4–{2–Azatricyclo[10.4.0.04,9]hexadeca–1(12),4(9),5,7,13,15–hexaen–10–yn–2–
yl}–4–oxobutanoic acid (V in , 1.0 g, 2.5 mmol) and (VI–7) (0.91 g, 2.5 mmol) in
DMF (10 mL) was added triethylamine (0.50 g, 5.0 mmol). The mixture was stirred at RT
overnight. The mixture was directly purified by reversed phase flash chromatography (0–100%
acetonitrile in water O3 10 mM)) to yield the (VI–8) (1.0 g, 74% yield in 3 steps from
VI–5) as brown oil. ESI m/z: 553.3 (M + H)+. 1H NMR (MeODd4, 400 MHz): δ 7.65 (d, J =
7.2 Hz, 1H), 7.64–7.58 (m, 1H), 7.49–7.42 (m, 3H), 7.40–7.30 (m, 2H), 7.28–7.22 (m, 1H),
.12 (d, J = 13.6 Hz, 1H), 3.75–3.68 (m, 3H), 3.63–3.50 (m, 12H), 3.50–3.39 (m, 2H), 3.25 (t,
J = 5.6 Hz, 2H), 2.76–2.66 (m, 1H), 2.52 (t, J = 6.0 Hz, 2H), 2.41–2.30 (m, 1H), 2.21–2.14 (m,
1H), 2.03–1.93 (m, 1H) ppm.
Step 7: 2,5–Dioxopyrrolidin–1–yl 1–(4–{2–azatricyclo[10.4.0.04–9]hexadeca–
1(12),4(9),5,7,13,15–hexane–10–yn–2–yl}–4–oxobutanamido)–3,6,9,12–tetraoxapentadecan–
–oate (VI): To a on of (VI–8) (40 mg, 72 µmol) in methylene chloride (10 mL) was
subsequently added HOSu (1–hydroxypyrrolidine–2,5–dione, 12 mg, 0.11 mmol) and EDCI
(21 mg, 0.11 mmol). The e was stirred at RT overnight and was then diluted with
methylene chloride (50 mL). The organic solution was washed with water (50 mL) and then
brine (50 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to generate
ediate (VI), which was used in next step without further purification. ESI m/z: 650 (M +
H)+. 1H NMR (CDCl3, 400 MHz): δ 7.70 (m, 1H), 7.66 (m, 1H), 7.55–7.47 (m, 3H), 7.38–7.24
(m, 4H), 6.33 (br s, 1H), 5.13 (d, J = 13.6 Hz, 1H), 3.83–3.78 (m, 1H), 3.66–3.60 (m, 13H),
3.47–3.35 (m, 2H), 2.99–2.82 (m, 6H), 2.51–2.43 (m, 2H), 2.20–1.89 (m, 4H) ppm.
This example demonstrates s for making 1–((1R,8S,9s)–
Bicyclo[6.1.0]non–4–yn–9–yl)–3–oxo–2,7,10,13,16–pentaoxa–4–azanonadecan–19–oic acid
(BCN–PEG4–Acid, VII). The following Example refers to .
To a on of intermediate VII–1 (0.10 g, 0.33 mmol) in tetrahydrofuran
(THF) (5 mL) were subsequently added diisopropylethylamine (0.17 g, 1.3 mmol),
intermediate (VI–7) (89 mg, 0.33 mmol), and oxybenzotriazole (HOBt, 43 mg, 0.33
mmol). The mixture was stirred at RT overnight. After filtered to remove the insoluble solid
and concentrated in vacuo, the reaction mixture was directly purified by prep–HPLC (method
B) to yield BCN–PEG4–acid (VII) (25 mg, 17% yield) as colorless oil. 1H NMR (CDCl3, 400
MHz): δ 5.07 (br s, 1H), 4.14 (d, J = 7.6 Hz, 2H), 3.77 (t, J = 6.4 Hz, 2H), 3.70–3.55 (m, 14H),
3.40–3.31 (m, 2H), 2.58 (t, J = 6.0 Hz, 2H), .19 (m, 6H), 1.61–1.52 (m, 2H), 1.43–1.32
(m, 1H), 92 (m, 2H) ppm.
EXAMPLE 36
This example demonstrates methods for making {4–[(2S)–2–[(2S)–2–[1–(4–
{2–azatricyclo[10.4.0.04,9]hexadeca–1(12),4(9),5,7,13,15–hexaen–10–yn–2–yl}–4–
oxobutanamido)–3,6,9,12–tetraoxapentadecan–15–amido]–3–methylbutanamido]–5–
(carbamoylamino)pentanamido]phenyl}methyl 4–nitrophenyl carbonate (DIBAC–Suc–PEG4–
VC–pAB–PNP, VIII). The following Example refers to .
1–(4–{2–azatricyclo[10.4.0.04–9]hexadeca–1(12),4(9),5,7,13,15–hexane–10–
yn–2–yl}–4–oxobutanamido)–N–[(1S)–1–{[(1S)–4–(carbamoylamino)–1–{[4–
(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}–2–methylpropyl]–3,6,9,12–
tetraoxapentadecan–15–amide (VIII–3)
Step 1: To a solution of compound (VIII–1) (300 mg, 0.54 mmol) and
compound (VIII–2, 205 mg, 0.54 mmol) in DMF (10 ml) were added HATU (309 mg, 0.81
mmol) and then DIEA (140 mg, 1.08 mmol). The mixture was stirred at RT for 3 hours. After
filtering to remove the insoluble solid and trated in vacuo, the reaction mixture was
directly purified by reverse flash (NH4HCO3 as buffer), and a white solid (VIII–3) (300 mg,
60%) was obtained. ESI m/z: 617(M+1).
{4–[(2S)–2–[(2S)–2–[1–(4–{2–azatricyclo[10.4.0.04–9]hexadeca–
1(12),4(9),5,7,13,15–hexane–10–yn–2–yl}–4–oxobutanamido)–3,6,9,12–tetraoxapentadecan–
–amido]–3–methylbutanamido]–5–(carbamoylamino)pentanamido]phenyl}methyl 4–
nitrophenyl carbonate (VIII)
Step 2: To a solution of (VIII–3) (150 mg, 0.16 mmol) and (VIII–4) (150 mg,
0.49 mmol) in DMF (10 mL) was added DIEA (63 mg, 0.49 mmol). The mixture was d at
RT for 3 hours. After filtered to remove the insoluble solid and concentrated in vacuo, the
reaction mixture was directly purified by reverse flash chromatography (NH4HCO3 as ),
and (VIII) as a yellow solid (50 mg, 28%) was obtained. ESI m/z: 1079 (M+1).
This example demonstrates methods for making Linker–Payload (LP1). The
following Example refers to .
Tert–Butyl )–1–({4–[(1S,2S,4R,6R,8S,9S,11S,12S,13R)–11–hydroxy–8–
(2–hydroxyacetyl)–9,13–dimethyl–16–oxo–5,7–dioxapentacyclo[10.8.002,9.04,8.013,18]icosa–
14,17–dien–6–yl]phenyl}carbamoyl)ethyl]carbamate (31)
Step 1: A mixture of a–OH (0.20 g, 0.42 mmol), DIPEA (0.12 g, 0.84
mmol) and HATU (0.24 g, 0.63 mmol) in DMF (5 mL) was d at 23 oC for 30 minutes.
To the solution was then added compound 7–1R (87 mg, 0.46 mmol). After stirring at
23 oC for another 2 hours, the e was directly purified by prep–HPLC (method B) to yield
compound 31 (0.11 g, 40% yield) as a white solid. ESI m/z: 651 (M + H)+.
(2S)–2–Amino–N–{4–[(1S,2S,4R,6R,8S,9S,11S,12S,13R)–11–hydroxy–8–(2–
hydroxyacetyl)–9,13–dimethyl–16–oxo–5,7–dioxapentacyclo[10.8.002,9.04,8.013,18]icosa–
14,17–dien–6–yl]phenyl}propanamide (32)
Step 2: To a solution of compound 31 (0.10 g, 0.15 mmol) in methylene
chloride (3 mL) was added TFA (0.3 mL) dropwise. The mixture was stirred at 23 oC for an
hour, and the volatiles were removed in vacuo to yield crude (32) (83 mg) as an oil, which was
used next step without further purification. ESI m/z: 551 (M + H)+.
Tert–Butyl N–[(1S)–1–{[(1S)–1–({4–[(1S,2S,4R,6R,8S,9S,11S,12S,13R)–11–
hydroxy–8–(2–hydroxyacetyl)–9,13–dimethyl–16–oxo–5,7–
dioxapentacyclo[10.8.002,9.04,8.013,18]icosa–14,17–dien–6–
yl]phenyl}carbamoyl)ethyl]carbamoyl}–2–methylpropyl]carbamate (33)
Step 3: A mixture of (32) (83 mg, 0.15 mmol), triethylamine (31 mg, 0.31
mmol) and l–NHS (58 mg, 0.19 mmol) in DMF (5 mL) was stirred 23 oC for 4 hours
and the on mixture was directly purified by prep–HPLC (method B) to yield (33) (52 mg,
% yield in 2 steps) as a white solid. ESI m/z: 750 (M + H)+. 1H NMR (500 MHz, )
δ 10.00 (s, 1H), 8.07 (d, J =7.0 Hz, 1H), 7.58 (d, J = 8.5 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.31
(d, J = 10.0 Hz, 1H), 6.72 (d, J = 9.0 Hz, 1H), 6.16 (dd, J = 1.5, 10.0 Hz, 1H), 5.91 (s, 1H),
.38 (s, 1H), 5.08 (t, J = 6.5Hz, 1H), 4.92 (d, J = 5.1 Hz, 1H), 4.78 (d, J =3.0 Hz, 1H), 4.55–
4.46 (m, 1H), 4.42 (t, J = 7.0Hz, 1H), 4.29 (s, 1H), 4.21–4.14 (m, 1H), 3.82 (t, J = 8.5 Hz, 1H),
2.65–2.52 (m, 1H), 2.37–2.25 (m, 1H), 2.18–2.06 (m, 1H), 2.04–1.88 (m, 2H), 1.85–1.57 (m,
5H), 1.40 (s, 3H), 1.37 (s, 9H), 1.29 (d, J = 7.0 Hz, 3H), 1.15–0.98 (m, 2H), 0.96–0.76 (m, 9H)
(2S)–2–Amino–N–[(1S)–1–({4–[(1S,2S,4R,6R,8S,9S,11S,12S,13R)–11–
y–8–(2–hydroxyacetyl)–9,13–dimethyl–16–oxo–5,7–
entacyclo[10.8.002,9.04,8.013,18]icosa–14,17–dien–6–yl]phenyl}carbamoyl)ethyl]–3–
methylbutanamide (34g)
Step 4: To a solution of compound 33 (50 mg, 67 µmol) in methylene chloride
(3 mL) was added TFA (0.3 mL) dropwise, which was then stirred at 23 oC for an hour. The
volatiles were removed in vacuo to yield crude compound 34g (42 mg) as an oil, which was
used the next step without further purification. ESI m/z: 650 (M + H)+.
1–(4–{2–Azatricyclo[10.4.0.04,9]hexadeca–1(12),4(9),5,7,13,15–hexane–10–
yn–2–yl}–4–oxobutanamido)–N–[(1S)–1–{[(1S)–1–({4–[(1S,2S,4R,6R,8S,9S,11S,12S,13R)–
11–hydroxy–8–(2–hydroxyacetyl)–9,13–dimethyl–16–oxo–5,7–
dioxapentacyclo[10.8.002,9.04,8.013,18]icosa–14,17–dien–6–
yl]phenyl}carbamoyl)ethyl]carbamoyl}–2–methylpropyl]–3,6,9,12–tetraoxapentadecan–15–
amide (LP1)
Step 5: A solution of DIBAC–suc–PEG4–OH (VI–8, 41 mg, 74 µmol), DIPEA
(24 mg, 0.19 mmol) and HATU (47 mg, 0.12 mmol) in DMF (5 mL) was stirred at 23 oC for
minutes, and then (34g) (40 mg, 62 µmol) was added. After being stirred at
23 oC for another 2 hours, the reaction mixture was directly purified by prep–HPLC (method
B) to yield LP1 (33 mg, 44% yield in 2 steps) as a white solid. ESI m/z: 1185 (M + H)+. 1H
NMR (500 MHz, DMSOd6) δ 9.97 (s, 1H), 8.18 (d, J = 6.5 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H),
7.75 (t, J = 5.5 Hz, 1H), 7.67 (d, J = 6.5 Hz, 1H), 7.63–7.56 (m, 3H), 7.53–7.41 (m, 3H), 7.42
7.27 (m, 6H), 6.19–6.14 (m, 1H), 5.93 (s, 1H), 5.38 (s, 1H), 5.08 (t, J = 6.5 Hz, 1H), 5.03 (d, J
= 14.0 Hz, 1H), 4.92 (d, J = 5.1 Hz, 1H), 4.78 (d, J = 3.0 Hz, 1H), 4.55–4.46 (m, 1H), 4.42 (t, J
= 7.0 Hz, 1H), 4.29 (s, 1H), .14 (m, 2H), 3.63–3.55 (m, 3H), 3.50–3.40 (m, 12H), 3.32–
3.26 (m, 2H), .05 (m, 2H), 2.65–2.52 (m, 2H), 2.48–2.48 (m, 2H), 2.40–2.25 (m, 3H),
.06 (m, 1H), 2.04–1.88 (m, 3H), 1.85–1.57 (m, 5H), 1.40 (s, 3H), 1.28 (d, J = 7.0 Hz,
3H), 1.15–0.98 (m, 2H), 0.96–0.84 (m, 6H), 0.84–0.80 (d, J = 7.0 Hz, 3H) ppm.
EXAMPLE 38
The example demonstrates a method for making Linker–Payload (LP2). The
following Example refers to FIG 15.
Tert–Butyl N–[(1S)–1–{[(1S)–4–(carbamoylamino)–1–[(4–{2–
[(1R,2S,8S,10S,11S,13R,14R,15S,17S)–1,8–difluoro–14,17–dihydroxy–2,13,15–trimethyl–5–
oxotetracyclo[8.7.0.02,7.011,15]heptadeca–3,6–dien–14–yl]–2–
oxoethoxy}phenyl)carbamoyl]butyl]carbamoyl}–2–methylpropyl]carbamate (34e)
General ure C: To a solution of Boc-Val-Ala-OH or Boc-Val-Cit-OH
(1.0 equiv.) in an organic solvent (such as DCM or DMF) were added a base (such as DIPEA)
(2.0 equiv.) and HATU (1.2 equiv.) at 20-25 oC. The mixture was stirred at
-25 oC for 30 minutes followed with the addition of an aniline (1.1 equiv.). The mixture was
further stirred for 16 hours until the peptide was consumed ing to LCMS. To the
reaction mixture was then added TFA (0.05 mL per 10 mg of peptide). The mixture was stirred
at 20-25 oC for another hour. The volatiles were removed under reduced pressure and the
residue was ly ed by PLC (method B).
Step 1: To a solution of Boc–VC (VC is Val–Cit) (67 mg, 0.18 mmol) in DMF
(3 mL) were added HATU (68 mg, 0.18 mmol) and NMM (30 mg, 0.30 mmol), and the
resulting solution was stirred at 23 oC for 10 minutes. To the reaction mixture was then added
compound 15–5 (75 mg, 0.15 mmol). After stirring at 23 oC overnight, the reaction mixture
was poured into ethyl acetate (80 mL), washed with brine, and then dried over anhydrous
sodium e. The combined organic solution was concentrated in vacuo and the residue was
purified by flash chromatography (0–10% methanol in methylene de) to yield (34e)
(0.12 g, yield 89%) as a white solid. ESI m/z: 858 (M + H)+. 1H NMR (MeODd4, 500 MHz)
δ 7.54–7.47 (m, 2H), 7.36 (d, J = 10.0 Hz, 1H), 6.90–6.87 (m, 2H), 6.34 (dd, J = 10.0, 1.5 Hz,
1H), 6.31 (s, 1H), 5.63–5.50 (m, 1H), 5.20 (d, J = 18.0 Hz, 1H), 4.80 (d, J = 18.0 Hz, 1H),
4.54–4.47 (m, 1H), 4.32–4.30 (m, 1H), 3.92–3.81 (m, 1H), 3.23–3.11 (m, 3H), 2.65–2.52 (m,
1H), 2.43–2.32 (m, 3H), 2.11–1.99 (m, 1H), 1.79–1.58 (m, 9H), 1.46–1.24 (m, 11H), 1.06 (s,
3H), 1.00–0.92 (m, 9H) ppm.
Bicyclo[6.1.0]non–4–yn–9–ylmethyl N–(14–{[(1S)–1–{[(1S)–4–
(carbamoylamino)–1–[(4–{2–[(1R,2S,8S,10S,11S,13R,14R,15S,17S)–1,8–difluoro–14,17–
dihydroxy–2,13,15–trimethyl–5–oxotetracyclo[8.7.0.02,7.011,15]heptadeca–3,6–dien–14–yl]–2–
oxoethoxy}phenyl)carbamoyl]butyl]carbamoyl}–2–methylpropyl]carbamoyl}–3,6,9,12–
tetraoxatetradecan–1–yl)carbamate (LP2)
Step 2: To a solution of intermediate compound 34e (25 mg, 29 µmol) in
methylene chloride (2 mL) was added TFA (1 mL), and the resulting e was d at
23 oC for an hour. The volatiles were removed in vacuo to yield a residue (25 mg, ESI m/z:
758.3 (M + H)+) as brown oil residue.
To a on of BCN–PEG4–acid (VII in , 18 mg, 41 µmol) in DMF (2
mL) were added HATU (15 mg, 41 µmol) and NMM (6.9 mg, 41 µmol), and the resulting
on was stirred at 23 oC for a half hour. To the reaction solution was then added a solution
of the brown oil residue obtained above in DMF (1 mL). After stirring at 23 oC overnight, the
mixture was worked up and purified directly by prep–HPLC (method B) to yield LP2 (15 mg,
37% yield) as a white solid. ESI m/z: 1181.4 (M + H)+. 1H NMR (DMSOd6, 400 MHz)
(rotamer) δ 9.82 and 9.37 (s, 1H), 8.39 (d, J = 8.0 Hz, 0.4H), 8.09 (d, J = 7.2 Hz, 0.6H), 8.00
(d, J = 8.0 Hz, 0.4H), 7.88 (d, J = 8.8 Hz, 0.6H), 7.55 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz,
1H), 7.27 (d, J = 10.0 Hz, 1H), 7.10 (br s, 1H), 6.80 (m, 2H), 6.29 (dd, J = 10.0, 1.0 Hz, 1H),
6.11 (s, 1H), 5.99–5.94 (m, 1H), 5.72–5.56 (m, 1H), 5.43–5.41 (m, 3H), 5.31 (s, 1H), 5.22 (d, J
= 18.0 Hz, 1H), 4.71 (d, J = 18.0 Hz, 1H), 4.37–4.31 (m, 1H), 4.24–4.14 (m, 2H), 4.04 (s, 1H),
4.02 (s, 1H), 3.62–3.56 (m, 2H), 3.50–3.45 (m, 12H), 3.40–3.37 (m, 2H), 3.13–3.08 (m, 2H),
3.00–2.92 (m, 3H), 2.54–2.33 (m, 2H), .08 (m, 8H), 2.09–1.90 (m, 1H), 1.78–1.23 (m,
15H), 1.14–1.09 (m, 1H), 0.89–0.82 (m, 14H) ppm.
EXAMPLE 39
The e demonstrates a method for making Linker–Payload (LP3). The
following Example refers to .
{4–[(2S)–2–[(2S)–2–Amino–3–methylbutanamido]–5–(carbamoylamino)
pentanamido]phenyl} methyl N–(4–{2–[(1R,2S,8S,10S,11S,13R,14R,15S,17S)–1,8–difluoro–
14,17–dihydroxy–2,13,15–trimethyl–5–oxotetracyclo[8.7.0.0²,70¹¹,¹5]heptadeca–3,6–dien–14–
yl]–2–oxoethoxy}phenyl) carbamate (34f)
General procedure D: Step 1: To a solution of payload an aniline (1.0 equiv.)
in DMF were added Fmoc-vcPAB-PNP (1.1 equiv.), HOBt (1.5 equiv.) and DIPEA (2.0
equiv.) at RT. The mixture was stirred at RT (18-30 oC) until the starting material was
consumed ing to LCMS. Step 2: To the reaction mixture was added piperidine (0.03 mL
per 10 mg of payload) and the mixture was stirred at RT (18-30 oC) for an hour until Fmoc was
removed monitored by LCMS. After filtered through membrane, the reaction on was
directly purified by reversed phase flash chromatography or prep-HPLC to generate the vcPAB
carbonate.
When N-Boc-vcPAB-PNP was used to replace Fmoc-vcPAB-PNP in the Step 1
reaction, the N-Boc vcPAB carbonate was obtained from Step 1. After purification, the N-Boc
vcPAB carbonate was redissolved in DCM, and was treated with TFA (TFA concentration <
%) at 0 oC until the Boc was d monitored by LCMS. The reaction mixture was
concentrated to remove the volatiles and the resulting residue was purified by chromatography
or prep-HPLC to generate the vcPAB carbonate.
To a on of Fmoc–vcPAB–PNP (73 mg, 96 µmol) in DMF (1 mL) were
added compound 15–5 (40 mg, 80 µmol), DMAP (20 mg, 0.16 mmol), HOBt (23 mg, 0.16
mmol) and DIPEA (55 mg, 0.40 mmol) successively at RT. The reaction mixture was stirred at
RT for half an hour until (15–5) was totally consumed according to LCMS. (ESI: 565.3 (M +
H)+). To the resulting mixture was then added piperidine (34 mg, 0.40 mmol) at RT. After
ng at RT for further 30 minutes, which was monitored by LCMS, the resulting mixture
was directly purified by reversed phase flash tography (0–30% acetonitrile in water) to
(34f) (50 mg, yield 69%) as a pale yellow solid. ESI: 907 (M + H)+
o[6.1.0]non–4–yn–9–ylmethyl N–(14–{[(1S)–1–{[(1S)–4–
(carbamoylamino)–1–{[4–({[(4–{2–[(1R,2S,8S,10S,11S,13R,14R,15S,17S)–1,8–difluoro–
dihydroxy–2,13,15–trimethyl–5–oxotetracyclo[8.7.0.0²,70¹¹,¹5]heptadeca–3,6–dien–14–
yl]–2–oxoethoxy}phenyl)carbamoyl] thyl)phenyl]carbamoyl}butyl]carbamoyl}–2–
methylpropyl]carbamoyl}–3,6,9,12–tetraoxatetradecan–1–yl)carbamate (LP3)
Step 3: To a solution of BCN–PEG4–acid (60 mg, 67 µmol) in DMF (3.6 mL)
were added HATU (27 mg, 70 µmol) and DIPEA (20 mg, 0.15 mmol) sively at RT. The
reaction mixture was stirred at RT for half an hour followed by the addition of compound (34f)
(50 mg, 60 µmol) portionwise. The reaction mixture was then stirred at RT for 2 hours until
compound 34f was totally consumed ing to LCMS. The reaction mixture was then
directly purified by prep–HPLC (method B) to yield compound LP3 (36 mg, yield 54%) as a
white solid . ESI: 1330 (M + H)+. 1H NMR (400 MHz, DMSOd6) δ 10.02 (s, 1H), 9.56 (s, 1H),
8.14 (d, J = 7.2 Hz, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz,
4H), 7.27 (d, J = 10.4 Hz, 1H), 7.11 (t, J = 4.4 Hz, 1H), 6.78 (d, J = 8.8 Hz, 2H), 6.33–6.26 (m,
1H), 6.10 (s, 1H), 5.98 (t, J = 5.4 Hz, 1H), 5.75–5.52 (m, 1H), 5.42 (s, 3H), 5.30 (s, 1H), 5.20
(d, J = 18.4 Hz, 1H), 5.05 (s, 2H), 4.70 (d, J = 18.4 Hz, 1H), 4.43–4.35 (m, 1H), 4.26–4.15 (m,
2H), 4.02 (d, J = 7.6 Hz, 2H), .55 (m, 2H), 3.49 (s, 11H), 3.38 (t, J = 6.0 Hz, 2H), 3.11
(dd, J = 11.8, 5.9 Hz, 2H), 3.05–2.88 (m, 3H), 2.44–2.31 (m, 2H), 2.28–2.08 (m, 9H), 2.02–
1.90 (m, 1H), 1.76–1.10 (m, 16H), 0.91–0.77 (m, 14H) ppm. HPLC purity: >99%, retention
time: 7.03 min.
The example demonstrates a method for making Linker–Payload (LP4). The
following Example refers to .
(2S)–2–Amino–N–[(1S)–1–[(4–{2–[(1S,2S,4R,8S,9S,11S,12S,13R)–11–
hydroxy–9,13–dimethyl–16–oxo–6–propyl–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–
14,17–dien–8–yl]–2–oxoethoxy}phenyl)carbamoyl]ethyl]–3–methylbutanamide (34a)
General procedure E: To a on of Fmoc-Val-Ala-OH (1.2 equiv.) in DMF
(0.2 mL per 10 mg of peptide) were added DIPEA (3.0 equiv.) and HATU (1.4 equiv.) at 20-
oC. The e was d at 20-25 oC for 5 minutes followed with the addition of aniline
(1.0 equiv.). The mixture was stirred for additional 2 hours until the peptide was totally
consumed, according to LCMS. To the reaction mixture was then added piperidine (5.0
). The mixture was stirred at 20-25 oC for 2 hour. After filtering through membrane, the
reaction solution was directly purified by reversed phase flash chromatography (0-100%
acetonitrile in aq. um bicarbonate (10 mM)) or prep-HPLC (method B). Compound
(34a) was ed following this General procedure.
Alternatively compound (34a) was obtained according to l Procedure
C. To a solution of Boc–Val–Ala–OH (0.29 g, 1.0 mmol) in methylene chloride (5 mL) were
added DIPEA (0.26 g, 2.0 mmol) and HATU (0.46 g, 1.2 mmol), and the mixture was stirred at
23 oC for 30 minutes and to the reaction mixture was then added compound (11–5) (0.57 g, 1.1
mmol). After stirring at 23 oC for additional 16 hours, to the reaction mixture was added TFA
(1.5 mL) and the resulting mixture was stirred at 23 oC for another hour. The volatiles were
removed under reduced pressure and the residue was directly purified by prep–HPLC (method
B) to yield 34a (0.17 g, 25% yield in 2 steps) as a white solid. ESI m/z: 692 (M + H)+. 1H
NMR (500 MHz, DMSOd6) δ 10.00 (s, 1H), 8.47 (d, J = 6.5 Hz, 1H), 7.57–7.47 (m, 2H), 7.33
(d, J = 10 Hz, 1H), 6.87–6.82 (m, 2H), 6.18 (d, J = 10 Hz, 1H), 5.93 (s, 3H), 5.25–5.11 (m,
1H), 5.09 (d, J = 6.5 Hz, 1H), 4.92–4.65 (m, 3H), 4.55–4.40 (m, 1H), 4.40–4.30 (m, 1H), 2.32–
2.22 (m, 1H), 2.18–1.80 (m, 5H), 1.65–1.45 (m, 5H), 1.45–1.25 (m, 9H), 1.25–0.98 (m, 2H),
0.96–0.76 (m, 13H) ppm.
Bicyclo[6.1.0]non–4–yn–9–ylmethyl N–(14–{[(1S)–1–{[(1S)–1–[(4–{2–
[(1S,2S,4R,8S,9S,11S,12S,13R)–11–hydroxy–9,13–dimethyl–16–oxo–6–propyl–5,7–
dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–8–yl]–2–
oxoethoxy}phenyl)carbamoyl]ethyl]carbamoyl}–2–methylpropyl]carbamoyl}–3,6,9,12–
tetraoxatetradecan–1–yl)carbamate (LP4)
General procedure F: To a on of an BCN–PEG4–acid or its NHS-ester in
DMF were added HATU (1 eq.) and DIPEA (2.5 eq.). The mixture was stirred at 25 oC for 30
minutes followed by the addition of a solution of an amine. After stirring at 25 oC for 2 hours
monitored by LC-MS, the starting materials were consumed and the mixture was purified
directly by prep–HPLC to yield the desired amide.
To a solution of G4–acid (IX, 70 mg, 0.16 mmol) in DMF (8 mL) were
added HATU (66 mg, 0.17 mmol) and DIPEA (56 mg, 0.43 mmol) successively. The mixture
was stirred at 25 oC for 30 minutes followed by the addition of a on of 34a (0.10 g, 0.15
mmol). After stirring at 25 oC for 2 hours, the mixture was purified directly by prep–HPLC
(method B) to yield LP4 (25 mg, 16% yield) as a white solid. ESI m/z =1116 (M + H)+.
Using chiral compound 11–5R as the starting material, chiral (R)–LP4 was
ed as a white solid (24 mg, 31% yield) ing to General procedure F. ESI m/z:
1115 (M + H)+. 1H NMR (500 MHz, DMSOd6) (rotamers) δ 9.78 (s, 0.5H), 9.69 (s, 0.5H), 8.40
(d, J = 7.5 Hz, 0.5H), 8.15 (d, J = 7.0 Hz, 0.5H), 8.01 (d, J = 8.0 Hz, 0.5H), 7.89 (d, J = 9.0 Hz,
0.5H), 7.57 (d, J = 9.0 Hz, 1H), 7.51 (d, J = 9.0 Hz, 1H), 7.32 (d, J = 10.1 Hz, 1H), 7.09 (s,
1H), 6.85 (d, J = 9.1 Hz, 2H), 6.18 (d, J = 11.4 Hz, 1H), 5.93 (s, 1H), 5.10 (d, J = 18.5 Hz,
1H), 4.86–4.67 (m, 4H), 4.45–4.36 (m, 1H), 4.33 (s, 1H), 4.20 (t, J = 7.5 Hz, 0.5H), 4.10 (t, J =
7.8 Hz, 0.5H), 4.03 (d, J = 8.0 Hz, 2H), 3.59 (d, J = 6.6 Hz, 2H), 3.49–3.45 (m, 11H), 3.39 (s,
2H), 3.30 (s, 2H), 3.11 (dd, J = 11.4, 5.9 Hz, 2H), 2.47–2.43 (m, 1H), 2.38–2.12 (m, 8H),
2.03–1.83 (s, 5H), 1.62–1.51 (m, 6H), 1.42–1.24 (m, 10H), 1.02–0.94 (m, 2H), .82 (m,
14H) ppm. Anal. HPLC: 100%, Retention time: 9.49 min (method A).
EXAMPLE 41
The example demonstrates a method for making Linker–Payload (LP5). The
following Example refers to .
(2S)–2–[(2S)–2–Amino–3–methylbutanamido]–5–(carbamoylamino)–N–(4–{2–
[(1S,2S,4R,8S,9S,11S,12S,13R)–11–hydroxy–9,13–dimethyl–16–oxo–6–propyl–5,7–
dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–8–yl]–2–
oxoethoxy}phenyl)pentanamide (34c)
Compound 34c was ed following the l ure C. A mixture
of Boc–vc (0.26 g, 0.50 mmol), DIPEA (0.19 g, 0.60 mmol) and HATU (0.23 g, 0.60 mmol) in
DMF (10 mL) was stirred at 23 oC for 30 minutes and to the mixture was then added 11–5
(0.28 g, 0.55 mmol). After stirring at 23 oC for 16 hours, the reaction mixture was directly
purified by reversed phase flash chromatography (0–50% acetonitrile in water) to yield a crude
(ESI m/z 878 (M + H)+), which was dissolved in methylene chloride (8 mL) and treated with
TFA (3 mL). The resulting mixture was stirred at 23 oC for one hour. The volatiles were
removed under reduced pressure and the residue was directly purified by prep–HPLC (method
B) to yield compound 34c (0.12 g, 31% yield in 2 steps) as a white solid. ESI m/z: 778 (M +
H)+. 1H NMR (500 MHz, ) δ 9.97 (d, J = 12.0 Hz, 1H), 8.10 (m, 1H), 7.51 (d, J = 6.5
Hz, 2H), 7.32 (dd, J = 10.1, 2.5 Hz, 1H), 6.83 (dd, J = 15.9, 9.0 Hz, 2H), 6.17 (d, J = 10.0 Hz,
1H), 5.97 (t, J = 5.0 Hz ,1H), 5.93 (s, 1H), 5.40 (s, 2H), 5.22 (t, J = 4.8 Hz, 1H), 5.12 (d, J =
6.0 Hz, 1H), 5.09 (d, J = 6.5 Hz, 1H), 4.83–4.67 (m, 3H), 4.47–4.37 (m, 1H), 4.35 – 4.29 (m,
1H), 3.05 –2.90 (m, 3H), 2.57–2.51(m, 1H), 2.30 (d, J = 12.0 Hz, 1H), 2.13–1.74 (m, 7H),
1.70– 1.46 (m, 7H), 1.45–1.29 (m, 7H), 1.17–0.93 (m, 2H), 0.91– 0.82 (m, 9H), 0.77 (dd, J =
6.7, 2.7 Hz, 3H) ppm.
Bicyclo[6.1.0]non–4–yn–9–ylmethyl N–(14–{[(1S)–1–{[(1S)–4–
moylamino)–1–[(4–{2–[(1S,2S,4R,8S,9S,11S,12S,13R)–11–hydroxy–9,13–dimethyl–16–
oxo–6–propyl–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–8–yl]–2–
oxoethoxy}phenyl)carbamoyl]butyl]carbamoyl}–2–methylpropyl]carbamoyl}–3,6,9,12–
tetraoxatetradecan–1–yl)carbamate (LP5)
LP5 was obtained following the General procedure F. A solution of BCN-
PEG4-acid (IX in , 0.28 g) in methylene chloride (6 mL) was added to a mixture of
HATU (59 mg, 0.15 mmol) and DIPEA (50 mg, 0.39 mmol) in DMF (5 mL). The reaction
e was stirred at 25 oC for 30 minutes and to it was added compound 34c (0.10 g, 0.13
mmol) in one portion. The resulting mixture was stirred at 25 oC overnight and was directly
purified by prep–HPLC (method B) to yield LP5 (35 mg, 23% yield) as a pale yellow solid.
ESI m/z =1202 (M + H)+. 1H NMR (400 MHz, MeODd4) δ 7.61–7.43 (m, 3H), 6.87 (t, J = 8.6
Hz, 2H), 6.26 (d, J = 10.0 Hz, 1H), 6.02 (s, 1H), 5.29–5.02 (m, 2H), 4.84–4.65 (m, 2H), 4.51–
4.44 (s, 2H), 4.22–4.05 (m, 3H), 3.80–3.68 (m, 2H), 3.67–3.45 (m, 14H), 3.22–3.08 (m, 2H),
2.72–2.50 (m, 3H), 2.45–2.33 (m, 1H), 2.30–2.02 (m, 10H), 1.99–1.82 (m, 2H), .32 (m,
17H), 1.26–0.85 (m, 17H) ppm.
EXAMPLE 42
The e demonstrates a method for making Linker–Payload (LP6). The
following Example refers to .
{4–[(2S)–2–[(2S)–2–Amino–3–methylbutanamido]–5–
(carbamoylamino)pentanamido]phenyl}methyl N–(4–{2–[(1S,2S,4R,8S,9S,11S,12S,13R)–11–
hydroxy–9,13–dimethyl–16–oxo–6–propyl–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–
dien–8–yl]–2–oxoethoxy}phenyl)carbamate (34d)
Compound 34d was prepared according to General procedure D.
Step 1: To a solution of compound (11-5) from Table 1 (66 mg, 0.10 mmol) in
DMF (3.5 mL) were added sively Boc–vcPAB–PNP (64 mg, 0.12 mmol), HOBt (14 mg,
0.10 mmol) and DIPEA (13.0 mg, 0.10 mmol). The reaction mixture was stirred at 13 oC
overnight and was purified directly by prep–HPLC (method B) to yield intermediate Boc–34d
(61 mg, yield 58%) as a white solid. ESI m/z: 1027.3 (M + H)+. 1H NMR (MeODd4, 400 MHz)
δ 7.60 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 10.4 Hz, 1H), .33 (m, 4H), 6.87–6.83 (m, 2H),
6.26 (dt, J = 10.0, 2.0 Hz, 1H), 6.02 (s, 1H), 5.26–5.03 (m, 4.2H), 4.82–4.67 (m, 1.8H), 4.54–
4.51 (m, 1H), 4.48–4.43 (m, 1H), 3.91 (d, J = 6.4 Hz, 1H), .18 (m, 1H), 3.14–3.08 (m,
1H), 2.70–2.63 (m, 1H), 2.40–2.37 (m, 1H), 2.26–2.00 (m, 4H), 1.94–1.72 (m, 4H), 1.68–1.35
(m, 20H), 1.22–0.92 (m, 14H) ppm.
Step 2: To a solution of Boc–34d (59 mg, 58 µmol) in DCM (2 mL) and
MeOH (1 mL) was added dropwise HCl in e (4 N, 1.5 mL) at 0 oC. The mixture was
then stirred at RT (14 oC) for 4 hours. The volatiles were removed in vacuo to yield 34d (60
mg, crude) as brown oil, which was used directly for the next step. ESI m/z: 927 (M + H)+.
Bicyclo[6.1.0]non–4–yn–9–ylmethyl N–(14–{[(1S)–1–{[(1S)–4–
(carbamoylamino)–1–{[4–({[(4–{2–[(1S,2S,4R,8S,9S,11S,12S,13R)–11–hydroxy–9,13–
dimethyl–16–oxo–6–propyl–5,7–dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa–14,17–dien–8–
yl]–2–oxoethoxy}phenyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}–2–
methylpropyl]carbamoyl}–3,6,9,12–tetraoxatetradecan–1–yl)carbamate (LP6)
LP6 was obtained as a white solid (24 mg, 31% yield) ing the General
procedure F. ESI m/z: 1350.5 (M + H)+. 1H NMR (DMSOd6, 400 MHz) δ 10.02 (s, 1H), 9.58
(s, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.88 (d, J =8.4 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.36–7.30
(m, 5H), 7.11 (t, J =4.8 Hz, 1H), 6.84–6.78 (m, 2H), 6.19–6.16 (m, 1H), 5.98 (t, J =5.2 Hz,
1H), 5.93 (s, 1H), 5.42 (s, 2H), 5.23–5.06 (m, 4H), 4.80–4.67 (m, 3H), 4.39–4.31 (m, 2H), 4.23
(t, J =7.2 Hz, 1H), 4.02 (d, J =8.0 Hz, 2H), 3.64–3.55 (m, 2H), 3.49 (m, 12H), 3.42–3.27 (m,
3H), 3.13–2.89 (m, 4H), 2.41–2.12 (m, 9H), 2.03–1.95 (m, 2H), 1.91–1.82 (m, 2H), 1.75–1.68
(m, 1H), 1.61–1.20 (m, 16H), 1.15–0.95 (m, 2H), 0.92–0.81 (m, 15H) ppm. Anal. HPLC:
69%+31%=100%, Retention time: 8.86 min and 8.92 min d B).
EXAMPLE 43
The example demonstrates a method for making –Payload LP7. The
following e refers to .
(Bicyclo[6.1.0]nonynylmethyl N-(14-{[(1S){[(1S)[(4-{2-[(1S,2S,4R,
6R,8S,9S,11S,12S,13R)hydroxy-9,13-dimethyloxopropyl-5,7-dioxapentacyclo
[10.8.0.02,9.04,8.013,18]icosa-14,17-dienyl]
oxoethoxy}phenyl)carbamoyl]ethyl]carbamoyl}methylpropyl]carbamoyl}-3,6,9,12-
tetraoxatetradecanyl)carbamate (LP7)
LP7 (24 mg, 31% yield in 3 steps from 34a) was obtained as a white solid
according to General procedure F. ESI m/z: 1115 (M + H)+. 1H NMR (500 MHz, )
(rotamers) δ 9.78 (s, 0.5H), 9.69 (s, 0.5H), 8.40 (d, J = 7.5 Hz, 0.5H), 8.15 (d, J = 7.0 Hz,
0.5H), 8.01 (d, J = 8.0 Hz, 0.5H), 7.89 (d, J = 9.0 Hz, 0.5H), 7.57 (d, J = 9.0 Hz, 1H), 7.51 (d,
J = 9.0 Hz, 1H), 7.32 (d, J = 10.1 Hz, 1H), 7.09 (s, 1H), 6.85 (d, J = 9.1 Hz, 2H), 6.18 (d, J =
11.4 Hz, 1H), 5.93 (s, 1H), 5.10 (d, J = 18.5 Hz, 1H), 4.86-4.67 (m, 4H), 4.45-4.36 (m, 1H),
4.33 (s, 1H), 4.20 (t, J = 7.5 Hz, 0.5H), 4.10 (t, J = 7.8 Hz, 0.5H), 4.03 (d, J = 8.0 Hz, 2H),
3.59 (d, J = 6.6 Hz, 2H), 3.49-3.45 (m, 11H), 3.39 (s, 2H), 3.30 (s, 2H), 3.11 (dd, J = 11.4, 5.9
Hz, 2H), 2.47-2.43 (m, 1H), 2.38-2.12 (m, 8H), 2.03-1.83 (s, 5H), 1.62-1.51 (m, 6H), 1.42-1.24
(m, 10H), 1.02-0.94 (m, 2H), 0.90-0.82 (m, 14H) ppm. Anal. HPLC: 100%, Retention time:
9.47 min (method A).
EXAMPLE 44
The example also demonstrates a method for making –Payload (LP7).
The following Example refers to . The following reaction conditions were used:
Step 1
Amine mg
Acid HATU DIPEA MS
mg DMF Temp. Time %
mg (µmol) mg mg m/z
(µmol) (mL) (oC) (hr) Yield
(µmol) (µmol)
26 30 VI- 48 40 17 30 1227.6
1 25 16
b (43) 8 (87) (105) (132) 56% (M+H)+
To a solution of acid (VI-8) (1.0-2.5 equiv.) in DMF (or DCM/DMF) were
added DIPEA (1.5-10 equiv.) and HATU (2.5-4.0 equiv.) at room temperature successively.
The resulting mixture was stirred at this temperature for 0.5-1 hour before the amine (26b) (1.0
equiv.) was added. The reaction mixture was d at room temperature for 2-16 hours until
the amine was y consumed, as monitored by LCMS. The reaction mixture was filtered
through a membrane and the filtrate was concentrated then separated by prep-HPLC (method
B) to give the example compound LP7 (20-69% yield) as a white solid.
1-(4-{2-azatricyclo[10.4.0.04,9]hexadeca-1(12),4(9),5,7,13,15-hexaenynyl}
anamido)-N-[(1S){[(1S)[(4-{2-[(1S,2S,4R,6R,8S,9S,11S,12S,13R)hydroxy-
9,13-dimethyloxopropyl-5,7-dioxapentacyclo[10.8.0.0²,⁹.0⁴,⁸.0¹³,¹⁸]icosa-14,17-dien
yl]oxoethoxy}phenyl)carbamoyl]ethyl]carbamoyl}methylpropyl]-3,6,9,12-
tetraoxapentadecanamide (LP7)
ESI m/z: 1227.6 (M + H)+.
1H NMR (500 MHz, DMSO
d6) (rotamers) δ 9.79 (s, 0.5H), 9.70 (s, 0.5H), 8.41 (d, J = 7.5 Hz,
0.5H), 8.17 (d, J = 7.0 Hz, 0.5H), 8.02 (d, J = 8.0 Hz, 0.5H), 7.89 (d, J = 8.6 Hz, 0.5H), 7.77 (t,
J = 4.8 Hz, 1H), 7.68 (d, J = 7.3 Hz, 1H), 7.62 (d, J = 7.3 Hz, 1H), 7.58 (d, J = 9.0 Hz, 1H),
7.53-7.43 (m, 4H), 7.40-7.28 (m, 4H), 6.88-6.82 (m, 2H), 6.18 (d, J = 9.1 Hz, 1H), 5.93 (s,
1H), 5.10 (d, J = 18.4 Hz, 1H), 5.03 (d, J = 14.0 Hz, 1H), 4.83-4.67 (m, 4H), 4.45-4.29 (m,
2H), .17 (m, 0.5H), 4.11 (t, J = 7.7 Hz, 0.5H), 3.64-3.40 (m, 15H), 3.31-3.26 (m, 2H),
3.13-3.03 (m, 2H), 2.65-2.52 (m, 2H), 2.47-1.26 (m, 24H), 1.06-0.93 (m, 2H), 0.90-0.80 (m,
12H) ppm.
Anal. HPLC: 99%, Retention time: 8.55 min (method B).
Solubility: <0.1 mg/mL water; 0.06 mg/mL 20% DMSO in water; 0.07 mg/mL
% DMSO in water.
This example demonstrates a method for making –Payload (LP15). The
following Example refers to FIGs. 27-28. Note that in , compound 11b is identical to
compound 11-5 in
Step 1: Making Compound (13b), with reference to .
To a solution of acid Fmoc-Val-Ala-OH (12b) in DMF were added HATU (1.0-
2.8 equiv.) and TEA (2.0-5.0 equiv.) at 25 oC. After the mixture was d at 25 oC for 30
minutes, a solution of amine (11b, i.e., payload, 1.0 equiv.) in DMF (1 mL) was added by
syringe. The resulting e was stirred at 25 oC for 2-24 hours until the amine was mostly
consumed according to LCMS. To the mixture was then added piperidine or diethylamine
(excess), and the mixture was stirred at 25 oC for 1-16 hours until Fmoc was totally removed,
as monitored by LCMS. The reaction mixture was filtered through a ne and the filtrate
was concentrated and directly purified by prep-HPLC (method B) or reversed phase flash
chromatography to give compound 13b % yield) as a white solid. Specifically, the
following conditions were used:
Step 1 Step 2
Amine Acid HAT DIPE DM Et2 Ti Purif %
mg mg Tim m/z
U mg A mg F NH me icati Yield
(M+1)+
(mmoL) (mmoL) e(hr
(mmo (mm (m (mL (hr on* mg
l) ol) L) ) )
85 69 TEA 832.2
11 12 69 17,
(0.0 (0.0 18 3 2 0.5 16 RP (M/2 +
b b (0.18) 43%
76) 88) (0.18) H)+
Step 2: Making Compound (17a) , with reference to .
To a solution of compound 13b in DMF were added HATU .8 equiv.) and
DIPEA or TEA (2.0-5.0 equiv.) at 25 oC. After the mixture was stirred at 25 oC for 30 minutes,
a solution of Fmoc-Lys-(PEG)4-COT (13c, 1.0 equiv.) in DMF (1 mL) was added by syringe.
The resulting e was stirred at 25 oC for 2-24 hours until the amine (13b) was mostly
consumed according to LCMS. To the e was then added piperidine or diethylamine
(excess), and the mixture was stirred at 25 oC for 1-16 hours until Fmoc was totally removed,
as monitored by LCMS. The reaction mixture was filtered through a membrane and the filtrate
was concentrated and directly purified by prep-HPLC (method B) or reversed phase flash
chromatography to give compound (17a) (23-64% yield) as a white solid.
Step 3: Making Compound (27b) , with reference to .
To a solution of alkyne (17a) (1.0 ) in DMF or DMSO was added αcyclodextrin-azide
(16a) (See Synth. Commun., 2002, 32(21), 3367-3372; J. Am. Chem. Soc.,
2012, 134(46), 19108-19117; J. Med. Chem., 1997, 40(17), 2755-2761; J. Am. Chem. Soc.,
1993, the entire contents of each of these publications is herein orated by reference in
their entirety for all purposes, 115(12), 5035-5040) (1.5-3.0 equiv.). The resulting mixture was
then stirred at 20-30 oC for 16 hours to 3 days until the nd 16a was mostly consumed
and the desired intermediate mass was detected, as monitored by LCMS. After filtration, the
resulting mixture was directly purified by prep-HPLC (or used directly) to give compound 27b
(25-58% yield) as a white solid (with triazole regioisomers). ically, the following
conditions were used.
Alkyne
16a Solvent Temp. Time
mg cation Yield m/z
mg (mmol) (mL) (oC) (hr)
(mmol)
50 DMSO 887.9
60 (0.06) 25 48 RP-B 46 mg, 58%
(0.030) (2) (M/3 + H)+.
Step 4: Making Compound (LP15) , with reference to .
The following reaction conditions were used:
Amine mg
Acid HATU DIPEA MS
mg DMF Temp. Time %
mg (µmol) mg mg m/z
(µmol) (mL) (oC) (hr) Yield
(µmol) (µmol)
27 13 VI- 20 4.0 6.0 1259.1
(39) 2 25 2
b (6.0) 8 (36) (31) 36% )+
To a solution of acid (VI-8) (1.0-2.5 equiv.) in DMF (or DCM/DMF) were
added DIPEA (1.5-10 equiv.) and HATU (2.5-4.0 equiv.) at room ature successively.
The resulting e was stirred at this temperature for 0.5-1 hour before the amine (27b) (1.0
) was added. The reaction mixture was stirred at room temperature for 2-16 hours until
the amine (27b) was totally consumed, as monitored by LCMS. The reaction mixture was
filtered h a membrane, the filtrate was concentrated, and then separated by prep-HPLC
(method B) to give the example nd (20-69% yield) as a white solid.
1-(4-{2-Azatricyclo[10.4.0.04,9]hexadeca-1(12),4(9),5,7,13,15-hexaenynyl}
oxobutanamido)-N-[(1R){2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-
,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-
dodecaoxaheptacyclo[26.2.2.2³,⁶.2⁸,¹¹.2¹³,¹⁶.2¹⁸,²¹.2²³,²⁶]dotetracontanyl]methyl}-
1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazolyl)oxy]acetamido}{[(1S){[(1S)-
1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12S,13R)hydroxy-9,13-dimethyloxopropyl-5,7-
dioxapentacyclo[10.8.0.0²,⁹.0⁴,⁸.0¹³,¹⁸]icosa-14,17-dienyl]
oxoethoxy}phenyl)carbamoyl]ethyl]carbamoyl}methylpropyl]carbamoyl}pentyl]-
3,6,9,12-tetraoxapentadecanamide (LP15)
O OH
O O O
H H H
N O O N N
N O O N N
H H
O O O
HN O O
O O
H O OHHO
O O
N N OH OH
N O
O HO
OH O
HO HO OH
HO OH
O OHO O
ESI m/z: 1259.1 (M/2 + H)+.
1H NMR (500 MHz, DMSO
d6) ers) δ 9.84 (s, 1H), 8.34 (s, 0.5H), 8.15 (d, J = 7.3 Hz,
1H), 8.04 (d, J = 6.6 Hz, 1H), 7.90-7.84 (m, 1H), 7.81-7.74 (m, 1.5H), 7.72-7.56 (m, 4H), 7.56-
7.27 (m, 11H), 6.89-6.79 (m, 2H), 6.17 (d, J = 10.0 Hz, 1H), 5.93 (s, 1H), 5.64-5.44 (m, 12H),
.24-5.00 (m, 5H), 4.86-4.51 (m, 16H), 4.40-4.16 (m, 5H), 4.05-3.96 (m, 1H), 3.86-3.73 (m,
10H), 3.67-2.88 (m, 35H), 2.80-2.69 (m, 1H), 2.62-2.55 (m, 1H), 2.41-2.20 (m, 6H), 2.10-1.71
(m, 10H), 1.66-1.07 (m, 26H), 1.05-0.79 (m, 17H) ppm.
Anal. HPLC: 97%, Retention time: 6.62 and 6.67 min (method B). Retention times are from
two triazole-regioisomers.
EXAMPLE 46
This example demonstrates a method for making Linker–Payload (LP16). The
following Example refers to FIGS. 27-28. The method for making LP16 was the same as the
method for making LP15, in Example 45 herein, except that a ent payload was used, as
shown in FIGs. 27-28. The following reaction conditions were used:
Step 1
Amine Acid purifi mg
HATU DIPEA Tem MS
mg mg DMF Time catio %
mg mg p. m/z
(µmol) (µmol) (mL) (hr) n Yield
(µmol) (µmol) (oC)
VI 10 6.0 18 1259.1
7 8.0 (21) 1 15-20 16 B
(15) -8 (18) (47) 47% (M/2+H)+
To a solution of acid VI-8 (1.0-2.5 equiv.) in DMF (or DCM/DMF) were added
DIPEA 0 equiv.) and HATU (2.5-4.0 equiv.) at room temperature successively. The
ing mixture was stirred at this temperature for 0.5-1 hour before the amine (27b) (1.0
equiv.) was added. The reaction mixture was stirred at room temperature for 2-16 hours until
the amine (27b) was totally consumed, as monitored by LCMS. The reaction mixture was
filtered through a membrane and the filtrate was concentrated and then separated by prep-
HPLC (method B) to give the example nd (20-69% yield) as a white solid.
1-(4-{2-Azatricyclo[10.4.0.04,9]hexadeca-1(12),4(9),5,7,13,15-hexaenynyl}
oxobutanamido)-N-[(1R){2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-
,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-
dodecaoxaheptacyclo[26.2.2.2³,⁶.2⁸,¹¹.2¹³,¹⁶.2¹⁸,²¹.2²³,²⁶]dotetracontanyl]methyl}-
1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazolyl)oxy]acetamido}{[(1S){[(1S)-
1-[(4-{2-[(1S,2S,4R,6R,8S,9S,11S,12S,13R)hydroxy-9,13-dimethyloxopropyl-
,7-dioxapentacyclo[10.8.0.0²,⁹.0⁴,⁸.0¹³,¹⁸]icosa-14,17-dienyl]
oxoethoxy}phenyl)carbamoyl]ethyl]carbamoyl}methylpropyl]carbamoyl}pentyl]-
3,6,9,12-tetraoxapentadecanamide (LP16)
ESI m/z: 839.5 (M/3 + H)+, 1259.1 (M/2 + H)+ (60%).
1H NMR (500 MHz, DMSO
d6) (with triazole regioisomer) δ 9.77-9.42 (m, 1H), 8.27-8.20 (m,
0.5H), 8.17-8.01 (m, 2H), 7.86-7.74 (m, 2.5H), 7.70-7.60 (m, 4H), 7.57-7.43 (m, 7H), 7.39-
7.28 (m, 6H), 6.88-6.81 (m, 2H), 6.21-6.14 (m, 1H), 5.93 (s, 1H), 5.61-5.42 (m, 10H), 5.16-
4.97 (m, 4H), .48 (m, 17H), 4.40-4.28 (m, 4H), 4.16-4.10 (m, 1H), 4.04-3.94 (m, 1H),
3.83-3.74 (m, 7H), 3.65-3.56 (m, 9H), 3.48-3.21 (m, 23H), 3.15-3.06 (m, 4H), 2.97-2.89 (m,
1H), 2.81-2.69 (m, 1H), 2.61-2.53 (m, 2H), 2.40-2.20 (m, 6H), .06 (m, 2H), 2.03-1.95
(m, 4H), 1.91-1.70 (m, 5H), 1.64-1.52 (m, 9H), .25 (m, 14H), 1.13-0.81 (m, 19H) ppm.
Anal. HPLC: 98%, ion time: 6.61(59%) and 6.73 (39%) min (method B). Retention
times are from two triazole-regioisomers.
Solubility: 0.1 mg/mL 10% DMSO in water.
EXAMPLE 47
The example trates a method for making Linker–Payload (LP8). The
following Example refers to .
(2S)[(2S)Aminomethylbutanamido](carbamoylamino)-N-(4-{2-
[(1S,2S,4R,8S,9S,11S,12S,13R)hydroxy-9,13-dimethyloxopropyl-5,7-dioxapenta
cyclo[10.8.0.02,9.04,8.013,18]icosa-14,17-dienyl]oxoethoxy}phenyl)pentanamide (34h)
Compound (34h) as a white solid was prepared according to General
procedure C after purification by prep-HPLC (method B). ESI m/z: 778 (M + H)+. 1H NMR
(500 MHz, DMSOd6) δ 9.97 (d, J = 12.0 Hz, 1H), 8.10 (m, 1H), 7.51 (d, J = 6.5 Hz, 2H), 7.32
(dd, J = 10.1, 2.5 Hz, 1H), 6.83 (dd, J = 15.9, 9.0 Hz, 2H), 6.17 (d, J = 10.0 Hz, 1H), 5.97 (t, J
= 5.0 Hz ,1H), 5.93 (s, 1H), 5.40 (s, 2H), 5.22 (t, J = 4.8 Hz, 1H), 5.12 (d, J = 6.0 Hz, 1H),
.09 (d, J = 6.5 Hz, 1H), 4.83-4.67 (m, 3H), 4.47-4.37 (m, 1H), 4.35-4.29 (m, 1H), .90
(m, 3H), 2.57-2.51(m, 1H), 2.30 (d, J = 12.0 Hz, 1H), 2.13-1.74 (m, 7H), 1.70- 1.46 (m, 7H),
1.45-1.29 (m, 7H), 1.17-0.93 (m, 2H), 0.91- 0.82 (m, 9H), 0.77 (dd, J = 6.7, 2.7 Hz, 3H) ppm.
1-(4-{2-Azatricyclo[10.4.0.04,9]hexadeca-1(12),4(9),5,7,13,15-hexaenyn
yl}oxobutanamido)-N-[(1S){[(1S)[(4-{2-[(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-
difluorohydroxy-9,13-dimethyloxopropyl-5,7-
dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8] icosa-14,17-dienyl]
oxoethoxy}phenyl)carbamoyl]ethyl]carbamoyl}methylpropyl]-3,6,9,12-tetraoxapentadecan-
-amide (LP8)
Compound LP8 (25 mg, 20% yield) was obtained as a white solid according to
General procedure F. ESI m/z: 1263 (M/+H)+. 1H NMR (400 MHz, ) δ 9.79 (s,
0.7H), 9.69 (s, 0.3H),8.41 (d, J = 8.0 Hz, 0.3H), 8.16 (d, J = 8.0 Hz, 0.7H), 8.01 (d, J = 7.6 Hz,
0.3H), 7.89 (d, J = 7.6 Hz, 0.7H), 7.77 (t, J = 5.2 Hz, 1H), 7.70-7.66 (m, 1H), 7.64-7.60 (m,
1H), .54 (m, 1H), 7.54-7.44 (m, 4H), 7.40-7.24 (m, 4H), 6.90-6.82 (m, 2H), 6.30 (dd, J =
Hz, 1.2 Hz, 1H), 6.11 (s, 1H), 5.72-5.55 (m, 1H), 5.52-5.48 (m, 1H), 5.16-5.08 (m, 1H),
.06-5.00 (m, 1H), 4.88-4.80 (m, 1H), 4.80-4.76 (m, 1H), 4.74 (t, J = 4.0 Hz, 1H), 4.42-4.33
(m, 1H), .06 (m, 2H), 3.64-3.54 (m, 3H), 3.50-3.40 (m, 12H), 3.12-3.02 (m, 2H), 2.70-
2.55 (m, 2H), 2.40-2.20 (m, 4H), 2.12-1.90 (m, 4H), 1.86-1.70 (m, 2H), .54 (m, 4H),
1.49 (s, 4H), 1.46-1.34 (m, 3H), 1.29 (d, J = 6.8 Hz, 3H), 0.90-0.80 (m, 13H) ppm. Anal.
HPLC: 100%, Retention time: 8.26 min (method B).
EXAMPLE 48
The example demonstrates a method for making Linker–Payload (LP9). The
following Example refers to .
S)[(2S)[1-(4-{2-Azatricyclo[10.4.0.04,9]hexadeca-1(12),4(9),5,7,13,
-hexaenynyl}oxobutanamido)-3,6,9,12-tetraoxapentadecanamido]methyl
butanamido](carbamoylamino)pentanamido]phenyl}methyl N-(4-{2-[(1S,2S,4R,8S,9S,11S,
12R,13S,19S)-12,19-difluorohydroxy-9,13-dimethyloxopropyl-5,7-dioxapenta
cyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-dienyl]oxoethoxy}phenyl)carbamate (LP9)
Compound (34i) as a white solid was prepared according to General procedure
Compound LP9 (20 mg, 22% yield) was obtained according to General
procedure F. ESI m/z: 1499 (M + H)+. 1H NMR (400 MHz, DMSOd6) δ 10.02 (s, 1H), 9.59 (s,
1H),8.14 (d, J = 7.6 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.80-7.75 (m, 1H), 7.70-7.66 (m, 1H),
7.65-7.60 (m, 3H), 7.53-7.45 (m, 3H), .28 (m, 7H), 6.84 (d, J = 9.2 Hz, 2H), 6.30 (dd, J
= 10.4 Hz, J = 1.6 Hz, 1H), 6.11 (s, 1H), 6.10-6.0 (m, 1H), 5.72-5.55 (m,1H), 5.52 (s, 1H),
.43 (s, 2H), 5.16-5.05 (m, 4H), .70 (m, 3H), 4.43-4.33 (m, 1H), 4.25-4.20 (m, 2H),
3.65-3.55 (m, 3H), 3.50-3.40 (m, 12H), 3.30-3.25 (m, 2H), 3.12-2.90 (m, 4H), 2.70-2.55 (m,
2H), 2.48-2.43 (m, 1H), 2.40-2.35 (m, 1H), 2.30-2.20 (m, 2H), 2.15-1.95 (m, 4H), 1.86-1.75
(m, 2H), 1.64-1.54 (m, 5H), 1.49 (s, 4H), 1.46-1.34 (m, 4H), 1.23 (s, 2H), .80 (m, 12H)
ppm. Anal. HPLC: 100%, Retention time: 7.83 min (method B).
EXAMPLE 49
The example demonstrates a method for making Linker–Payload (LP10). The
ing Example refers to .
(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)(2-Aminoacetyl)-12,19-difluoro
hydroxy-9,13-dimethylpropyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-dien-
16-one (34j)
Compound 34j (80 mg, 64% yield) was obtained from compound 1-19
according to the General procedure D. ESI m/z: 871 (M + H)+.
{4-[(2S)[(2S)[1-(4-{2-Azatricyclo[10.4.0.04,9]hexadeca-1(12),4(9),5,7,13, 15-hexaen-
-ynyl}oxobutanamido)-3,6,9,12-tetraoxapentadecanamido]methyl
butanamido](carbamoylamino)pentanamido]phenyl}methyl 2-
[(1S,2S,4R,8S,9S,11S, 12R,13S,19S)-12,19-difluorohydroxy-9,13-dimethyloxo
propyl-5,7-dioxapenta cyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-dienyl]
oxoethoxy}phenyl)carbamate (LP10)
Following the l procedure F, compound (LP10) (20 mg, 22% yield)
was obtained from the reaction of 34j (43 mg, 50 µmol) with DIBAC-suc-PEG4-NHS ester
(VI), after purification by prep-HPLC (method B). ESI m/z: 1406 (M+H)+. 1H NMR (DMSOd6,
500 MHz) δ 9.99 (s, 1H), 8.11 (d, J = 7.5 Hz, 1H), 7.88 (d, J = 8.5 Hz, 1H), 7.80-7.75 (m, 1H),
7.70-7.66 (m, 1H), 7.65-7.60 (m, 3H), 7.53-7.33 (m, 6H), 7.33-7.28 (m, 3H), 6.30 (dd, J = 10.0
Hz and 1.5 Hz, 1H), 6.11 (s, 1H), 6.10-6.00 (m, 1H), 5.72-5.55 (m, 2H), 5.41 (s, 2H), 5.05-
.01 (m, 1H), 4.97 (s, 2H), 4.80-4.72 (m, 1H), 4.60-4.58 (m, 1H), .33 (m, 1H), .10
(m, 3H), 3.88-3.80 (m, 1H), 3.65-3.55 (m, 3H), 3.50-3.40 (m, 12H), 3.30-3.25 (m, 2H), 3.12-
2.90 (m, 4H), 2.70-2.55 (m, 2H), 2.48-2.35 (m, 2H), 2.30-2.20 (m, 2H), 2.15-1.95 (m, 4H),
1.86-1.65 (m, 3H), 1.64-1.54 (m, 5H), 1.49 (s, 4H), 1.46-1.34 (m, 5H), .80 (m, 12H)
ppm. Anal. HPLC: 100%, Retention time: 7.40 min (method B).
EXAMPLE 50
The example demonstrates a method for making Linker–Payload LP11. The
following Example refers to .
{4-[(2S)[(2S)[1-(4-{2-Azatricyclo[10.4.0.04,9]hexadeca-1(12),4(9),5,7,13,15-hexaen-
-ynyl}oxobutanamido)-3,6,9,12-tetraoxapentadecanamido]
methylbutanamido](carbamoylamino)pentanamido]phenyl}methyl N-[(4-{2-
[(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-12,19-difluorohydroxy-9,13-dimethyloxo-
6-propyl-5,7-dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-dienyl]
oxoethoxy}phenyl)methyl]carbamate (LP11)
Compound 34k (80 mg, 64% yield) was obtained from (11-19) according to the
l procedure D.
Following the General procedure C, compound (LP11) (18 mg, 31% yield) as
a white solid was obtained from the reaction of compound (34k). ESI m/z: 756.5 (M/2 + H)+.
1H NMR (500 MHz, DMSO
d6) δ 10.02 (s, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz,
1H), 7.76(t, J = 5.5 Hz, 1H), 7.72(t, J = 5.5 Hz, 1H), 7.70-7.66 (m, 1H), 7.65-7.60 (m, 3H),
7.53-7.45 (m, 3H), 7.40-7.31 (m, 2H), 7.31-7.25 (m, 4H), 7.20-7.15 (m, 2H), 6.86-6.80 (m,
2H), 6.30 (dd, J = 10.4Hz, 1.6 Hz, 1H), 6.11 (s, 1H), 6.10-6.00 (m, 1H), .55 (m, 1H),
.52 (s, 1H), 5.43 (s, 2H), 5.16-5.10 (m, 1H), 5.06-5.00 (m, 1H), 5.00-4.93 (m, 2H), 4.90-4.76
(m, 2H), 4.75 (t, J = 4.0 Hz, 1H), 4.43-4.33 (m, 1H), 4.25-4.20 (m, 2H), 4.12 (d, J = 6.0 Hz,
2H), .55 (m, 3H), 3.50-3.40 (m, 12H), .25 (m, 2H), 3.12-2.90 (m, 4H), .55
(m, 2H), 2.48-2.43 (m, 1H), 2.40-2.35 (m, 1H), 2.30-2.20 (m, 2H), 2.15-1.95 (m, 4H), 1.86-
1.70 (m, 3H), 1.64-1.54 (m, 5H), 1.49 (s, 4H), 1.46-1.34 (m, 4H), 0.90-0.80 (m, 12H) ppm.
Anal. HPLC: 99%, Retention time: 7.89 min (method B).
EXAMPLE 51
The example demonstrates a method for making Linker–Payload LP12. The
following Example refers to .
[(2R,3R,4S,5R,6S)–3,4,5–Tris(acetyloxy)–6–[4–formyl–3–(prop–2–yn–1–
yloxy)phenoxy]oxan–2–yl]methyl acetate (45)
Step 1: The sis of [(2R,3R,4S,5R,6S)–3,4,5–Tris(acetyloxy)–6–(4–
formyl–3–hydroxyphenoxy)oxan–2–yl]methyl acetate (43) was reported in Carbohydrate
Research, 1986, 146, 241–249, the entire contents of which are herein incorporated by
reference in its entirety. To a solution of intermediate compound 43 (2.8 g, 6.0 mmol) in
acetone (40 mL) was simultaneously added potassium carbonate (1.7 g, 12 mmol) and 3–
bromoprop–1–yne (44, 3.5 g, 30 mmol), and the resulting mixture was refluxed overnight. The
mixture was then concentrated in vacuo and the e was purified by flash chromatography
(0–33% ethyl acetate in petroleum ether) to yield compound 45 (1.9 g, yield 63%) as a brown
solid. ESI m/z: 507 (M + H)+. 1H NMR (MeODd4, 500 MHz) δ 10.26 (s, 1H), 7.78 (d, J = 8.5
Hz, 1H), 6.87 (d, J = 2.0 Hz, 1H), 6.77 (dd, J = 8.5, 2.0 Hz, 1H), 5.51 (d, J = 8.0 Hz, 1H), 5.41
(t, J = 9.5 Hz, 1H), 4.93 (t, J = 2.5 Hz, 2H), 5.23–5.19 (m, 1H), 5.14 (t, J = 9.5 Hz, 1H), 4.34–
4.30 (m, 1H), 4.22–4.15 (m, 2H), 3.11 (t, J = 2.0 Hz, 1H), 2.05–1.99 (m, 12H) ppm.
[(2R,3R,4S,5R,6S)–3,4,5–Tris(acetyloxy)–6–[4–(hydroxymethyl)–3–(prop–2–yn–1–
yloxy)phenoxy]oxan–2–yl]methyl acetate (46)
Step 2: To a solution of compound 45 (0.83 g, 1.6 mmol) in isopropanol (50
mL) was added sodium borohydride (31 mg, 0.82 mmol). The mixture was stirred at 23oC for 2
hours and was then concentrated in vacuo. The residue was diluted with ethyl acetate and
washed with brine. The organic on was dried over sodium sulfate and concentrated to
afford nd 46 (0.70 g, yield 84%) as brown oil. ESI m/z: 526.1 (M + H2O)+. 1H NMR
(MeODd4, 500 MHz) δ 7.32 (d, J = 8.0 Hz, 1H), 6.78 (d, J = 2.0 Hz, 1H), 6.67 (dd, J = 8.0, 2.0
Hz, 1H), 5.40 (t, J = 9.0 Hz, 1H), 5.33 (dd, J = 7.5 Hz, 1H), 5.20–5.11 (m, 2H), 4.78 (t, J = 2.5
Hz, 2H), 4.59 (s, 2H), 4.32 (d, J = 12.5, 5.0 Hz, 1H), 4.21 (dd, J = 12.5, 2.5 Hz, 1H), 4.12–4.08
(m, 1H), 3.02 (t, J = 2.0 Hz, 1H), 2.07–2.0
[(2R,3R,4S,5R,6S)–3,4,5–Tris(acetyloxy)–6–(4–{[(4–nitrophenoxycarbonyl)
oxy]methyl}–3–(prop–2–yn–1–yloxy)phenoxy)oxan–2–yl]methyl acetate (48)
Step 3: To a solution of nd 46 (0.40 g, 0.79 mmol) in methylene
de (30 mL) were added 4–nitrophenyl carbonochloridate (47, 0.24 g, 1.2 mmol), 4–
dimethylaminopyridine (0.19 g, 1.6 mmol) and diisopropylethylamine (0.20 g, 1.6 mmol). The
mixture was stirred at 23 oC overnight and diluted with methylene chloride (50 mL). The
organic solution was washed with saturated aqueous ammonium de solution (50 mL) and
then brine (50 mL), dried over sodium sulfate and trated. The residue was purified by
flash chromatography (0–33% ethyl e in petroleum ether) to afford compound 48 (0.30 g,
yield 57%) as an off–white solid. ESI m/z: 691.0 (M + H2O)+. 1H NMR (CDCl3, 500 MHz) δ
8.27 (d, J = 9.0 Hz, 2H), 7.38 (d, J = 9.0 Hz, 2H), 7.35 (d, J = 8.5 Hz, 1H), 6.75 (d, J = 2.5 Hz,
1H), 6.64 (dd, J = 9.0, 2.5 Hz, 1H), 5.33–5.26 (m, 4H), 5.21–5.17 (m, 1H), 4.76 (t, J = 2.0 Hz,
2H), 4.28 (dd, J = 12.5, 5.0 Hz, 1H), 4.20 (dd, J = 12.5, 2.5 Hz, 1H), 3.89–3.88 (m, 1H), 2.56
(t, J = 7.0 Hz, 1H), 2.08–2.04 (m, 12H) ppm.
[(2R,3R,4S,5R,6S)–3,4,5–Tris(acetyloxy)–6–[4–({[(4–{2–[(1S,2S,4R,8S,9S,11S,12S,13R)–
11–hydroxy–9,13–dimethyl–16–oxo–6–propyl–5,7–
dioxapentacyclo[10.8.002,9.04,8.013,18]icosa–14,17–dien–8–yl]–2–
oxoethoxy}phenyl)carbamoyl]oxy}methyl)–3–(prop–2–yn–1–yloxy)phenoxy]oxan–2–
yl]methyl acetate (49)
Step 4: To a on of compound 48 (0.15 g, 0.22 mmol) in DMF (5 mL)
were added 11–5 (0.14 g, 0.26 mmol), HOBt (59 mg, 0.44 mmol) and ropylethylamine
(57 mg, 0.44 mmol) successively. The mixture was stirred at 23oC overnight and was then
purified by prep–HPLC d B) to yield compound 49 (0.14 g, 62% yield) as a white solid.
ESI m/z: 1056.3 (M + H)+. 1H NMR 4, 400 MHz) δ 7.46 (d, J = 10.4 Hz, 1H), 7.35–
7.26 (m, 3H), 6.87–6.80 (m, 3H), 6.67 (dd, J = 8.0, 2.4 Hz, 1H), 6.26 (dt, J = 10.0, 2.4 Hz, 1H),
6.03 (br s, 1H), 5.42–5.34 (m, 2.5H), 5.26–5.03 (m, 5.5H), 4.88–4.64 (m, 4H), 4.46–4.43 (m,
1H), 4.34–4.30 (m, 1H), 4.21–4.18 (m, 1H), 4.12–4.08 (m, 1H), 3.03 (t, J = 2.0 Hz, 1H), 2.71–
2.62 (m, 1H), 2.41–2.38 (m, 1H), 2.28–2.15 (m, 2H), 2.06–2.04 (m, 12H), 1.90–1.39 (m, 12H),
.89 (m, 8H) ppm.
[2–(Prop–2–yn–1–yloxy)–4–{[(2S,3R,4S,5S,6R)–3,4,5–trihydroxy–6–
(hydroxymethyl)oxan–2–yl]oxy}phenyl]methyl N–(4–{2–[(1S,2S,4S,8R,9S,11S,12S,13R)–
11–hydroxy–4,9,13–trimethyl–16–oxo–6–propyl–7–oxapentacyclo[10.8.
002,9.04,8.013,18]icosa–14,17–dien–8–yl]–2–oxoethoxy}phenyl)carbamate (LP12)
Step 5: To a solution of compound 49 (35 mg, 33 µmol) in methanol (3 mL)
was added another solution of LiOH in H2O (14 mg, 0.33 mmol) in water (1 mL). The mixture
was stirred at 23 oC for 1.5 hours and was quenched with HOAc (20 mg). The mixture was
concentrated in vacuo and the residue was purified by prep–HPLC (method B) to yield linkerpayload
LP12 (26 mg, 88% yield) as a white solid. ESI m/z: 888 (M + H)+. 1H NMR
(MeODd4, 400 MHz) δ 7.46 (d, J = 10.0 Hz, 1H), 7.35–7.30 (m, 3H), 6.91 (d, J = 2.0 Hz, 1H),
6.87–6.83 (m, 2H), 6.74 (dd, J = 8.0, 2.0 Hz, 1H), 6.27 (dt, J = 10.0, 2.0 Hz, 1H), 6.03 (s, 1H),
.25 (t, J = 4.8 Hz, 0.5H), 5.19 (d, J = 7.2 Hz, 0.5H), .03 (m, 3H), 4.94–4.91 (m, 1H),
4.82–4.75 (m, 3H), 4.71–4.67 (m, 1H), 4.46–4.43 (m, 1H), 3.91 (dd, J = 12.0, 2.0 Hz, 1H),
3.70 (dd, J = 12.0, 5.2 Hz, 1H), 3.48–3.36 (m, 4H), 2.99 (t, J = 2.4 Hz, 1H), 2.71–2.62 (m, 1H),
2.40–2.37 (m, 1H), 2.26–2.12 (m, 2H), 2.07–2.00 (m, 1H), 1.88–1.61 (m, 5H), 1.56–1.35 (m,
6H), 1.20–0.92 (m, 8H) ppm.
EXAMPLE 52
The example demonstrates a method for making Linker–Payload LP13. The
following Example refers to .
2–[(1S,2S,4S,8S,9S,11S,12S,13R)–11–hydroxy–9,13–dimethyl–16–oxo–6–propyl–5,7–
dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa–14,17–dien–8–yl]–2–oxoethyl N–(2–{[({4–
[(2S)–5–(carbamoylamino)–2–[(2S)–2–[6–(2,5–dioxo–2,5–dihydro–1H–pyrrol–1–
yl)hexanamido]–3–methylbutanamido]pentanamido]phenyl}methoxy)carbonyl]
l)amino}ethyl)–N–methylcarbamate (LP13)
To a on of nide–DME carbonate (20 mg, 0.037 mmol) in DMF (1
ml) was subsequently added MC–VC–PAB–PNP (22 mg, 0.03 mmol), DIPEA (12 mg, 0.09
mmol), and HOBt (6 mg, 0.05mmol). This mixture was stirred at RT for 12 hours, then prep–
HPLC was performed to get two epimers: Epimer 1: 3.3 mg (yield 10%) and Epimer 2: 4.1
mg (yield 12%).
Epimer 1: ESI m/z: 1143.4 (M+1). 1H NMR (400 MHz, MeOD) δ 7.63–7.62
(m, 2H), 7.50–7.49 (m, 1H), 7.37–7.35 (m, 2H), 6.81 (s, 1H), 6.29–6.27 (m, 1H), 6.03 (brs,
1H), 5.37–5.08 (m, 5H), 4.83–4.79 (m, 3H), 4.53–4.46 (m, 2H), 4.18–4.15 (m, 1H), 3.69–3.37
(m, 6H), 3.25–3.13 (m, 3H), 3.12–2.96 (m, 5H), 2.90–2.86 (m, 2H), 2.68–2.64 (m, 1H), 2.41–
2.40 (m, 1H), 2.31–2.28 (m, 2H), 2.26–1.95 (m, 7H), 1.93–1.77 (m, 11H), 1.51 (s, 3H), 1.42–
1.30 (m, 10H), 1.25–0.89 (m, 15H)
Epimer 2: ESI m/z: 1143.4 (M+1). 1H NMR (400 MHz, MeOD) δ 7.62–7.34
(m, 5H), 6.81 (brs, 1H), 6.29–6.23 (m, 1H), 6.05–6.00 (m, 1H), .17 (m, 4H), 4.92–4.79
(m, 2H), 3.75–3.37 (m, 7H), 3.03–2.86 (m, 5H), 2.72–2.63 (m, 1H), 2.41–2.28 (m, 3H), 2.23–
2.04 (m, 7H), 1.91–1.32 (m, 31H), 1.19–0.90 (m, 14H).
EXAMPLE 53
The e demonstrates a method for making –Payload LP14. The
following Example refers to .
N-[(1S){[(1S)[(4-{2-[(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-12,19-
Difluorohydroxy-9,13-dimethyloxopropyl-5,7-
dioxapentacyclo[10.8.0.0²,9.04,8.0¹³,¹8]icosa-14,17-dienyl]
oxoethoxy}phenyl)carbamoyl]ethyl]carbamoyl}methylpropyl]{2-[4-(2,5-dioxo-2,5-
dihydro-1H-pyrrolyl)phenyl]acetamido}-3,6,9,12-tetraoxapentadecanamide (LP14)
Compound 34h-2 (0.18 g, 74% yield in 2 steps) was obtained according to the
General procedure F. ESI m/z: 728 (M + H)+.
Compound LP14 (20 mg, 14% yield in 3 steps from 34h) was obtained as a
white solid. ESI: 1189 (M + H)+. 1H NMR (500 MHz, DMSOd6) δ 9.81-9.67 (m, 1H), 8.43-
8.13 (m, 2H), 8.03-7.84 (m, 1H), 7.61-7.47 (m, 2H), 7.35 (d, J = 8.4 Hz, 2H), 7.29-7.21 (m,
3H), 7.17 (s, 2H), 6.88-6.81 (m, 2H), 6.33-6.28 (dd, J = 10.1, 1.8 Hz, 1H), 6.11 (s, 1H), 5.71-
.56 (m, 1H), 5.51 (s, 1H), 5.12 (d, J = 18.5 Hz, 1H), 4.84 (d, J = 18.5 Hz, 1H), 4.79-4.76 (m,
1H), 4.74 (t, J = 4.3 Hz, 2H), .33 (m, 1H), 4.25-4.17 (m, 2H), 3.63-3.55 (m, 2H), 3.52-
3.44 (m, 14H), 3.42 (t, J = 5.8 Hz, 2H), 3.21 (q, J = 5.7 Hz, 1H), 2.69-2.55 (m, 1H), 2.47-2.41
(m, 1H), 2.41-2.34 (m, 1H), 2.29-2.23 (m, 1H), 2.14-2.02 (m, 2H), 1.99-1.90 (m, 1H), 1.82 (d,
J = 13.0 Hz, 1H), 1.65-1.53 (m, 4H), 1.49 (s, 3H), 1.47-1.41 (m, 1H), 1.40-1.33 (m, 2H), 1.29
(d, J = 7.1 Hz, 3H), 0.90-0.80 (m, 12H) ppm. Anal. HPLC: 100%, Retention time: 8.45 min
(method A).
Table 7 below summarizes n physical properties of 16.
Table 7: Physical Properties of Certain Linker–Payloads
Purity MS
LP No. MF MW Highest m/z RT
(%) m/z (100%)
(min)
LP1 C66H81N5O15 1184.4 98 593 (M/2+H) 6.53 (B)
(M+H, 20%)
1181.4
LP2 C61H86F2N6O15 1181.4 100 1181.4 (M+H) 7.83 (B)
(M+H)
1330.4
LP3 C70H94F2N6O17 1329.5 100 1330.4 (M+H) 7.03 (B)
(M+H)
1115 8.17 (A)
LP4 C61H86N4O15 1115.4 100 1115 [M+H]
[M+H] 8.24 (B)
1201 7.34 (A)
LP5 C64H92N6O16 1201.5 100 1201 [M+H]
[M+H] 7.44 (B)
1350.5
LP6 N7O18 1350.6 100 1350.5 (M+H) 8.87 (A)
(M+H)
LP7 C61H86N4O15 1226.5 100 1227.8 (M+H) 1227.8 (M+H) 9.47 (A)
1262.4
LP8 C69H85F2N5O15 1262.4 100 1262.4 (M+H) 8.26 (B)
(M+H)
LP9 C80H98F2N8O18 1497.7 100 749.5 (M/2+H) 1497.7 7.99 (B)
(M+H)
1405.7 (M+H)
LP10 C74H94F2N8O17 1405.6 100 703.5 (M/2+H) 7.40 (B)
756.5
LP11 C81H100F2N8O18 1511.7 99.3 756.5 (M/2+H) 7.89 (B)
(M/2+H)
566.2(M- 889.1 8.02 (A)
LP12 C48H57NO15 888.0 100
glucose-PAB) (M+H, 25%) 8.08 (B)
1350.5
LP13 C72H99N7O18 1350.6 100 1350.5 (M+H) 8.87 (A)
(M+H)
1188.5
LP14 F2N5O16 1188.31 100 594.8 ) 7.99 (B)
(M+H, 40%)
1259.1 6.62 and
LP15 C121H170N10O47 2516.71 97 1259 (M/2 +H)
(M/2 + H)+. 6.67 (B)
6.61(59%)
1259.1
LP16 C121H170N10O47 2516.71 98 1258 (M/2+H) and 6.73
(M/2+H,60%)
(39%) (B)
This example demonstrates a method for site–specific conjugation, generally, of
a payload to an antibody or antigen–binding fragment thereof. This example refers to .
In one example, site–specific conjugates were ed via Microbial
lutaminase (MTG EC 2.3.2.13, Zedira, Darmstadt, Germany) n “MTG–based”)
two–step conjugation of an N297Q or N297D d antibody. In the first step, the mutated
antibody was functionalized with azido-PEG3-amine via MTG based enzymatic reaction. See,
e.g., International PCT Patent Application No. PCT/US17/19537, filed February 24, 2017,
entitled OPTIMIZED TRANSGLUTAMINASE SITE-SPECIFIC ANTIBODY
CONJUGATION, incorporated herein by reference in its entirety for all purposes. In the
second step, an alkyne-functionalized linker–payload was attached to the azido–functionalized
antibody via [2+3] 1, 3–dipolar cycloaddition reaction (see, e.g., , which depicts a
DIBAC-functionalized –payload conjugated with an azido–functionalized antibody
derived via [2+3] cyclization). This s provided site–specific and stoichiometric
conjugates in about 50–80% isolated yield.
EXAMPLE 55
This Example demonstrates specific procedures for site-specific conjugation of
an alkyne-linker-payload to antibody.
This e refers to the compounds depicted in .
In this example, the site–specific ates were produced in two steps. The
first step is Microbial transglutaminase (MTG)-based enzymatic attachment of a small
molecule, such as azide–PEG3–amine (supra), to the antibody having a Q-tag (references for
the Qtag) (hereinafter “MTG–based” conjugation). The second step employed the attachment
of a linker–payload to the azido–functionalized antibody via a [2+3] ddition, for
example, the 1,3–dipolar cycloaddition between the azides and the cyclooctynes (aka copper–
free click chemistry). See, , J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang,
P. V.; Miller, I. A.; Lo, A.; i, J. A.; Bertozzi, C. R. PNAS 2007, 104 (43), 16793–7, the
entire contents of which are herein incorporated by nce in its entirety for all purposes.
Shown in is an example of a linker–payload having a DIBAC moiety conjugated with
an azido–functionalized antibody via a [2+3] cycloaddition. This s provided the site–
specific and iometric conjugates in about 50–80% isolated yield.
ADC conjugation via [2+3] click reaction.
Step 1: Preparation of an azido–functionalized antibody.
Aglycosylated human antibody IgG (IgG1, IgG4, etc.) or a human IgG1 isotype
with N297Q mutation, in PBS (pH 6.5–8.0) was mixed with ≥200 molar lents of azido–
dPEG3–amine (MW= 218.26 g/mol). The resulting solution was mixed with MTG (EC
13 from Zedira, Darmstadt, Germany, or Modernist Pantry [L# 210115A]
- ACTIVA TI contains Maltodextrin from Ajinomoto, Japan) (25 U/mL; 5U MTG per mg of
antibody) resulting in a final concentration of the antibody at 0.5–5 mg/mL, and the on
was then incubated at 37 °C for 4–24 h while gently shaking. The on was monitored by
ESI–MS. Upon reaction completion, the excess amine and MTG were removed by SEC or
protein A column chromatography, to generate the azido–functionalized antibody. This
product was characterized by SDS–PAGE and ESI–MS. The dPEG3–amine added to
two sites of the antibody resulting in a 204 Da increase for the 2DAR antibody–PEG3–azide
conjugate.
In a specific experimental, the N-terminal Q tag antibody (24 mg) in 7 mL
potassium-free PBS buffer (pH 7.3) was incubated with > 200 molar equivalent of the azido-
PEG3-amine (MW 218.26) in the presence of MTG (0.350 mL, 35 U, mTGase, Zedira,
Darmstadt, y). The reaction was incubated at 37 °C overnight while gently mixing.
Excess azido-PEG3-amine and mTGase were removed by size exclusion chromatography
(SEC, Superdex 200 PG, GE Healthcare).
Step 2: Preparation of site–specific conjugates of a drug to an antibody using
click chemistry reactions.
The site–specific antibody drug conjugates with a human IgG (IgG1, IgG4, etc.)
in Table 10 were prepared by a [2+3] click reaction between azido–functionalized dies
and an alkyne containing linker–payload. The detailed ation procedure follows. A sitespecific
antibody conjugate with linker–payload (LP) was prepared by incubating mAb–
PEG3–N3 (1–3 mg / mL) in an aqueous medium (e.g., PBS, PBS containing 5% ol,
HBS) with ≥6 molar equivalents of an LP ved in a suitable organic solvent, such as
DMSO, DMF or DMA (i.e., the reaction mixture contains 5-20% organic solvent, v/v) at 24 oC
to 37 oC for over 6 h. The progress of the reaction was monitored by ESI–MS and the absence
of mAb–PEG3–N3 indicated the completion of the conjugation. The excess amount of the LP
and organic solvent were removed by SEC via elution with PBS, or via n A column
chromatography via n with acidic buffer followed by neutralization with Tris (pH8.0).
In a specific example, the azido-functionalized antibody (1 mg) in 0.800 mL
PBSg (PBS, 5% ol, pH 7.4) was treated with six molar lents of DIBAC-PEG4-DLys
(COT-∝-CD)-VC-PABC-payload (conc. 10 mg/mL in DMSO) for 6 -12 hours at room
temperature and the excess linker payload (LP) was removed by size exclusion
chromatography (SEC, Superdex 200 HR, GE Healthcare).
The final product was concentrated by ultra centrifugation and characterized by
UV, SEC, SDS-PAGE and ESI-MS.
EXAMPLE 56
This example demonstrates a method for making an azido–functionalized
dy drug conjugate.
Aglycosylated antibody with a human IgG1 isotype in BupHTM (pH 7.6–7.8)
was mixed with ≥200 molar equivalents of azido–dPEG3–amine (MW. 218.26 g/mol). The
resulting on was mixed with transglutaminase (25 U/mL; 5U MTG per mg of antibody,
Zedira, Darmstadt, Germany) resulting in a final concentration of the antibody at 0.5–3
mg/mL, and the solution was then incubated at 37 °C for 4–24 hours while gently shaking. The
reaction was monitored by SDS–PAGE or ESI–MS. Upon the completion, the excess amine
and MTG were removed by Size Exclusion Chromatography (see ) to generate the
functionalized antibody. This product was analyzed on SDS–PAGE (see ) and
ESI–MS (see ). The azido–dPEG3–amine added to two sites – Q295 and Q297– of the
antibody resulting in an 804 Da increase for the 4DAR aglycosylated antibody–PEG3–azide
conjugate. The conjugation sites were identified and confirmed at EEQLinkerYQLinkerSTYR for
the 4DAR azido–functionalized antibody via peptide sequence g of trypsin digested
heavy chains.
This e demonstrates a method for making a site–specific conjugations of
a drug to an antibody using click chemistry reactions.
The site–specific aglycosylated antibody drug conjugates with an human IgG1
ning an N297Q mutation in Table 8 described below were prepared by a [2+3] click
reaction between azido–functionalized antibodies with an alkyne containing linker–payload.
As shown in Table 8, Anti Her2–PEG3–N3 was conjugated to compounds LP1, LP2, LP3,
LP4, LP5, LP6, LP7, LP8, LP9, LP10, and LP11. As shown in Table 8, Anti PRLR– PEG3–
N3 was ated to LP1 LP2, LP3, LP4, LP5, LP6, LP7, LP8, LP9, LP10, LP11, LP15,
and LP16. As shown in Table 8, Anti–IL2Rg–PEG3–N3 was conjugated to LP4 and LP7. As
shown in Table 8, Anti–Fel d 1–PEG3–N3 was conjugated to LP4.
For the ation, an azido–functionalized aglycosylated human IgG1
antibody EG3–N3) and linker–payload (LP) conjugate was prepared by incubating
mAb–PEG3–N3 (1–3 mg / mL) in an aqueous medium (e.g., PBS, PBS containing 5%
glycerol, HBS) with ≥6 molar equivalent of an LP dissolved in a suitable organic solvent, such
as DMSO, DMF or DMA (reaction mixture contains 10 – 20% organic solvent, v/v) at 24oC to
37oC for over 6 hours. The ss of the reaction was monitored by ESI–MS. The reaction
was monitored by ESI–MS, and the absence of mAb–PEG3–N3 indicated the completion of the
conjugation. The excess amount of the LP and organic t were removed by SEC eluting
with PBS. The purified conjugates were analyzed by SEC, SDS–PAGE, and ESI–MS. Shown
in Table 8 is a list of non–toxic steroid antibody conjugates (ncADCs) from the corresponding
LPs, their molecular weights and ESI–DAR values. In Table 8, Ab refers to an antibody, Ab–
N3 refers to the azide functionalized antibody, and ncADC refers to a non–cytotoxic antibody
drug conjugate.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US62/419,365 | 2016-11-08 | ||
US62/508,317 | 2017-05-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
NZ793831A true NZ793831A (en) | 2022-11-25 |
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