Citrate salt, pharmaceutical compositions, and methods of making and using the same
11767330 · 2023-09-26
Assignee
Inventors
Cpc classification
A61K31/4545
HUMAN NECESSITIES
C07D401/04
CHEMISTRY; METALLURGY
C07D519/00
CHEMISTRY; METALLURGY
International classification
C07D519/00
CHEMISTRY; METALLURGY
A61K31/4545
HUMAN NECESSITIES
Abstract
The present disclosure features citrate salts and pharmaceutical compositions useful for the treatment of BAF complex-related disorders. Also disclosed are methods for preparing compounds.
Claims
1. A citrate salt of the compound of Formula I: ##STR00252##
2. A pharmaceutical composition comprising the citrate salt of claim 1.
3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is liquid.
4. The pharmaceutical composition of claim 2, further comprising a buffer.
5. The pharmaceutical composition of claim 4, wherein the buffer is a citrate buffer.
6. The pharmaceutical composition of claim 2, wherein pH of the pharmaceutical composition is 3.5 to 5.5.
7. The pharmaceutical composition of claim 2, further comprising a cyclodextrin-based solubilizer.
8. The pharmaceutical composition of claim 7, wherein the cyclodextrin-based solubilizer is sulfobutylether-β-cyclodextrin.
9. The pharmaceutical composition of claim 2, further comprising saline.
10. The pharmaceutical composition of claim 9, wherein the saline is an isotonic saline.
11. A method of treating a subject having synovial sarcoma, the method comprising administering to the subject an effective amount of the citrate salt of claim 1.
12. A method treating a viral infection related to BAF47 in a subject in need thereof, the method comprising administering to the subject an effective amount of the citrate salt of claim 1.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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DETAILED DESCRIPTION
(55) The present disclosure features compositions and methods useful for the treatment of BAF-related disorders (e.g., cancer and infection) as well for the preparation of compounds. The disclosure further features compositions and methods useful for inhibition of the level and/or activity of BRD9, e.g., for the treatment of disorders such as cancer (e.g., sarcoma) and infection (e.g., viral infection), e.g., in a subject in need thereof.
(56) The invention provides a citrate salt of the compound of Formula I:
(57) ##STR00190##
(58) The invention also provides a citrate salt of the compound of Formula Ia:
(59) ##STR00191##
(60) The citrate salts may be included in a pharmaceutical composition (e.g., a liquid pharmaceutical composition). The pharmaceutical composition may include a buffer (e.g., a buffer sufficient to produce pH of 3.5 to 5.5, 3.5 to 5.0, 4.0 to 5.5, or 4.0 to 5.0, e.g., upon dissolution in water at pH 7, e.g., for solid pharmaceutical compositions). Suitable buffers are known in the art. A non-limiting example of the buffer is a citrate buffer, which is typically prepared from water, citric acid, and sodium citrate or sodium hydroxide. A liquid pharmaceutical composition may have pH of 3.5 to 5.5, 3.5 to 5.0, 4.0 to 5.5, or 4.0 to 5.0. The pharmaceutical composition may also include a cyclodextrin-based solubilizer (e.g., sulfobutylether-β-cyclodextrin). The pharmaceutical composition may also include saline (e.g., isotonic saline). When the pharmaceutical composition is a solid, the pharmaceutical composition may produce pH 3.5 to 5.5, 3.5 to 5.0, 4.0 to 5.5, or 4.0 to 5.0 upon dissolution in water at pH 7.
(61) The invention also provides crystalline forms of the free-base solid form of the compound of Formula I:
(62) ##STR00192##
(63) The first of the crystalline forms is characterized by a powder x-ray diffraction pattern having peaks at 10.4±0.2 2θ and 26.9±0.2 2θ. In some embodiments, the x-ray diffraction pattern further includes peaks at 17.7±0.2 2θ and at 17.9±0.2 2θ. In some embodiments, the x-ray diffraction pattern further includes a peak at 20.8±0.2 2θ. In some embodiments, the x-ray diffraction pattern further includes a peak at 13.8±0.2 2θ. The second of the crystalline forms is characterized by a powder x-ray diffraction pattern having peaks at 13.4±0.2 2θ and 17.4±0.2 2θ. In some embodiments, the x-ray diffraction pattern further includes a peak at 26.3±0.2 2θ. In some embodiments, the x-ray diffraction pattern further includes a peak at 21.0±0.2 2θ. In some embodiments, the x-ray diffraction pattern further includes peaks at 13.6±0.2 2θ and 18.1±0.2 2θ. The crystalline form described herein may be provided in a pharmaceutical composition (e.g., a pharmaceutical composition described herein).
(64) The invention further provides pharmaceutical compositions containing the free-base solid form of the compound of Formula I:
(65) ##STR00193##
(66) The invention further provides pharmaceutical compositions containing the free-base solid form of the compound of Formula Ia:
(67) ##STR00194##
(68) The free-base solid form may be a lyophilizate. The pharmaceutical composition may contain a citrate buffer (e.g., citric acid and sodium citrate) in an amount sufficient produce a solution having pH of 3.5 to 5.5, 3.5 to 5.0, 4.0 to 5.5, or 4.0 to 5.0 upon dissolution in water at pH 7.
(69) The invention provides a pharmaceutical composition comprising an aqueous solvent and the compound of Formula I:
(70) ##STR00195##
(71) The invention also provides a pharmaceutical composition comprising an aqueous solvent and the compound of Formula Ia:
(72) ##STR00196##
(73) The pharmaceutical composition may have pH of 3.5 to 5.5, 3.5 to 5.0, 4.0 to 5.5, or 4.0 to 5.0. The pharmaceutical composition may also include a cyclodextrin-based solubilizer (e.g., sulfobutylether-β-cyclodextrin). The pharmaceutical composition may also include saline (e.g., isotonic saline). The pharmaceutical composition may include a buffer (e.g., a citrate buffer).
(74) The compounds of Formula I or Formula Ia may be administered to a subject according to a non-limiting method involving combining the compound of Formula I or Formula Ia in solid form with an aqueous solvent (e.g., a liquid composition containing water or saline (e.g., isotonic saline) and a buffer (e.g., a citrate buffer)) to produce an aqueous solution, and administering the aqueous solution to the subject intravenously.
(75) Preparation of Compounds
(76) Compounds disclosed herein may be prepared using methods disclosed herein. The methods disclosed herein may be advantageous by providing superior stereochemical purity of the end product, e.g., the compound of Formula I or the compound of Formula A. For example, the syntheses described herein can reduce the number of reactive steps involving stereochemically enriched piperidinedione moiety, thus reducing exposure of the stereochemical center therein to conditions capable of inducing racemization. For example, the reaction may produce a product with less than 15% erosion in the enantiomeric excess (e.g., less than 10% erosion in the enantiomeric excess). Additionally or alternatively, the use of reducing agents, e.g., borohydride agents, such as NHC—BH.sub.3 adduct or NaBH.sub.3CN, may also provide the desired product with a superior stereoretention. Additionally or alternatively, purification steps may be included in the preparation methods disclosed herein may. Advantageously, these purification steps may improve stereochemical enrichment of the desired product (e.g., compound of Formula I or Formula A) without requiring the use of other stereochemically enriched agents (other than the starting material) or chromatographic separation techniques. Thus, the compound of Formula I or Formula A may be obtained with less than 5% erosion in the enantiomeric excess (e.g., less than 4%, less than 3%, or less than 2% erosion in the enantiomeric excess).
(77) The invention provides a method of preparing an enantioenriched compound of Formula I:
(78) ##STR00197##
(79) The method includes the step of reacting an enantioenriched compound of Formula Ib or a salt thereof and a compound of Formula Ic or a salt thereof under reductive amination reaction conditions, where the compound of Formula Ib is of the following structure:
(80) ##STR00198##
and the compound of formula of Formula Ic is of the following structure:
(81) ##STR00199##
where PG is an O-protecting group.
(82) In some embodiments, the reductive amination reaction conditions include the use of a borohydride agent as a reducing agent. In some embodiments, the borohydride agent is an NHC—BH.sub.3 adduct, NaBH.sub.3CN, or NaBH(OAc).sub.3. In some embodiments, the borohydride agent is the NHC—BH.sub.3 adduct. In some embodiments, the borohydride agent is an adduct of N,N′-dimethylimidazolylidene and BH.sub.3. In some embodiments, the reductive amination reaction conditions include the use of a Brønsted acid. In some embodiments, the Brønsted acid is acetic acid.
(83) In some embodiments, the method includes the step of purifying the compound of Formula I.
(84) In some embodiments, the step of purifying the compound of Formula I includes:
(85) (i) an aqueous work-up to produce an aqueous layer and an organic layer,
(86) (ii) separating the organic layer away from the aqueous layer,
(87) (iii) concentrating the organic layer to produce a residue,
(88) (iv) forming a slurry of the residue with dichloromethane/acetonitrile to produce a liquid phase and a solid phase,
(89) (v) isolating the liquid phase, and
(90) (vi) concentrating the liquid phase to produce the compound of Formula I in purified form.
(91) In some embodiments, the method includes the step of further purifying the compound of Formula I, the step of further purifying the compound of Formula I including:
(92) dissolving the compound of Formula I from step (vi) in dichloromethane/acetonitrile to produce a solution,
(93) adding water to the solution to form a wet cake slurry, and
(94) separating the solid away from the wet cake slurry to produce a purified compound of Formula I, and optionally subjecting one or more times the purified compound of Formula I to the steps of dissolving, adding water, and separating the solid to increase the purity of the compound of Formula I.
(95) In some embodiments, the purified compound of Formula I is subjected to the steps of dissolving, adding water, and separating the solid once.
(96) In an aspect, the invention provides a method of preparing an enantioenriched compound of Formula A:
(97) ##STR00200##
where
(98) * designates an enantioenriched stereogenic center; and
(99) each of R.sup.A1, R.sup.A2, R.sup.A3, and R.sup.A4 is, independently, H, A-L-, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, optionally substituted —O—C.sub.3-C.sub.6 carbocyclyl, hydroxyl, thiol, or optionally substituted amino; or R.sup.A1 and R.sup.A2, R.sup.A2 and R.sup.A3, or R.sup.A3 and R.sup.A4, together with the carbon atoms to which each is attached, combine to form
(100) ##STR00201##
is optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heteroaryl, or C.sub.2-C.sub.9 heterocyclyl, any of which is optionally substituted with A-L-, where one of R.sup.A1, R.sup.A2, R.sup.A3, and R.sup.A4 is A-L-, or
(101) ##STR00202##
is substituted with A-L-;
(102) L is a linker; and
(103) A is a BRD9 binding moiety.
(104) The method includes the step of reacting an enantioenriched compound of Formula A1 or a salt thereof and a compound of Formula A2 or a salt thereof under reductive amination reaction conditions, where the compound of Formula A1 is of the following structure:
(105) ##STR00203##
and the compound of formula of Formula A2 is of the following structure:
(106) ##STR00204##
where PG is an O-protecting group.
(107) In some embodiments, the reductive amination reaction conditions include the use of a borohydride agent as a reducing agent.
(108) In some embodiments, the borohydride agent is an NHC—BH.sub.3 adduct, NaBH.sub.3CN, or NaBH(OAc).sub.3. In some embodiments, the borohydride agent is the NHC—BH.sub.3 adduct. In some embodiments, the borohydride agent is an adduct of N,N′-dimethylimidazolylidene and BH.sub.3.
(109) In some embodiments, the reductive amination reaction conditions include the use of a Brønsted acid. In some embodiments, the Brønsted acid is acetic acid.
(110) In some embodiments, the method includes the step of purifying the compound of Formula A.
(111) In some embodiments, the step of purifying the compound of Formula A includes:
(112) (i) an aqueous work-up to produce an aqueous layer and an organic layer,
(113) (ii) separating the organic layer away from the aqueous layer,
(114) (iii) concentrating the organic layer to produce a residue,
(115) (iv) forming a slurry of the residue with dichloromethane/acetonitrile to produce a liquid phase and a solid phase,
(116) (v) isolating the liquid phase, and
(117) (vi) concentrating the liquid phase to produce the compound of Formula Ain purified form.
(118) In some embodiments, the method includes the step of further purifying the compound of Formula A, the step of further purifying the compound of Formula A including:
(119) dissolving the compound of Formula A from step (vi) in dichloromethane/acetonitrile to produce a solution,
(120) adding water to the solution to form a wet cake slurry, and
(121) separating the solid away from the wet cake slurry to produce a purified compound of Formula A, and optionally subjecting one or more times the purified compound of Formula A to the steps of dissolving, adding water, and separating the solid to increase the purity of the compound of Formula A.
(122) In some embodiments, the purified compound of Formula A is subjected to the steps of dissolving, adding water, and separating the solid once.
(123) In some embodiments, PG is C.sub.1-6 alkyl. In some embodiments, PG is methyl.
(124) In the compounds of Formula A, the linkers and BRD9 binding moieties can be those described, e.g., in US 20220098190, US 20220048906, US 20210230190, US 20210009568, US 20190247509, US 20180044335, WO 2020051235, WO 2020160192, WO 2020160193, WO 2020160198, WO 2021055295, and WO 2021178920, the disclosures of which are incorporated herein by reference in their entirety.
(125) The BRD binding moiety may be, e.g., a group of Formula III:
(126) ##STR00205##
(127) where
(128) R.sup.1 is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl;
(129) Z.sup.1 is CR.sup.2 or N;
(130) R.sup.2 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, or optionally substituted C.sub.2-C.sub.9 heteroaryl;
(131) ##STR00206##
(132) X.sup.1 is CR.sup.X1 or N;
(133) X.sup.2 is O or S;
(134) R.sup.X1 is H or optionally substituted C.sub.1-C.sub.6 alkyl;
(135) R.sup.3″ is H,
(136) ##STR00207##
cyano, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.1-C.sub.6 alkoxy, optionally substituted amino, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.3-C.sub.10 heterocyclyl, or optionally substituted C.sub.2-C.sub.9 heteroaryl;
(137) R.sup.3′ is absent, optionally substituted C.sub.1-C.sub.6 alkylene, optionally substituted C.sub.2-C.sub.9 heteroarylene, or optionally substituted C.sub.1-C.sub.6 heteroalkylene;
(138) G″ is
(139) ##STR00208##
optionally substituted C.sub.3-C.sub.10 carbocyclyl, C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, or optionally substituted C.sub.2-C.sub.9 heteroaryl;
(140) G′ is optionally substituted C.sub.3-C.sub.10 carbocyclylene, C.sub.2-C.sub.9 heterocyclylene, optionally substituted C.sub.6-C.sub.10 arylene, or optionally substituted C.sub.2-C.sub.9 heteroarylene; and
(141) A.sup.1 is a bond between A and the linker,
(142) where G″ is
(143) ##STR00209##
or R.sup.3″ is
(144) ##STR00210##
or a pharmaceutically acceptable salt thereof.
(145) The BRD9 binding moiety may be, e.g., a group of Formula IV:
(146) ##STR00211##
(147) where
(148) R.sup.7 is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl;
(149) R.sup.8 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, or optionally substituted C.sub.6-C.sub.10 aryl;
(150) s is 0, 1, 2, 3, or 4;
(151) each R.sup.9 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino;
(152) X.sup.1 is N or CR.sup.10a;
(153) X.sup.2 is N or CR.sup.10b;
(154) X.sup.3 is N or CR.sup.10c;
(155) X.sup.4 is N or CR.sup.10d; and
(156) each of R.sup.10a, R.sup.10b, R.sup.10c, and R.sup.10d is, independently, H, halogen, hydroxy, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino,
(157) or a pharmaceutically acceptable salt thereof.
(158) The BRD9 binding moiety may be, e.g., a group of Formula V:
(159) ##STR00212##
(160) where
(161) each R.sup.11 and R.sup.16 is, independently, H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl;
(162) t is 0, 1, 2, 3, or 4;
(163) each R.sup.12 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino;
(164) u is 0, 1, 2, 3, or 4;
(165) each R.sup.13 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino;
(166) each R.sup.14 and R.sup.15 is, independently, selected form the group consisting of H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl;
(167) A.sup.1 is a bond between A and the linker; and
(168) G is optionally substituted C.sub.1-C.sub.6 alkylene, optionally substituted C.sub.6-C.sub.10 arylene, or optionally substituted C.sub.3-C.sub.6 carbocyclylene;
(169) or a pharmaceutically acceptable salt thereof.
(170) The BRD9 binding moiety may be, e.g., a group of Formula VI:
(171) ##STR00213##
(172) where
(173) Y.sup.2 is CR.sup.17 or N;
(174) R.sup.18 is a bond to the linker, optionally substituted C.sub.6-C.sub.10 aryl, or C.sub.2-C.sub.9 heteroaryl;
(175) R.sup.19 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl;
(176) R.sup.20 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl;
(177) each R.sup.17, R.sup.21, and R.sup.22 is, independently, H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino;
(178) R.sup.23 is H or —NR.sup.24R.sup.25; and
(179) each of R.sup.24 and R.sup.25 is, independently, H, a bond to the linker, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl, or R.sup.24 and R.sup.25 combine to form optionally substituted C.sub.2-C.sub.9 heterocyclyl,
(180) where one of R.sup.18, R.sup.24, or R.sup.25 is a bond to the linker,
(181) or a pharmaceutically acceptable salt thereof.
(182) The BRD9 binding moiety may be, e.g., a group of Formula VII:
(183) ##STR00214##
(184) where
(185) each R.sup.26a, R.sup.26b, and R.sup.26c is, independently, H, a bond to the linker, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino;
(186) each R.sup.27a and R.sup.27b is, independently, H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl;
(187) R.sup.19 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl;
(188) R.sup.20 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl;
(189) each R.sup.17, R.sup.21, and R.sup.22 is, independently, H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino; and
(190) each of R.sup.24 and R.sup.25 is, independently, H, a bond to the linker, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl, or R.sup.24 and R.sup.25 combine to form optionally substituted C.sub.2-C.sub.9 heterocyclyl,
(191) where one of R.sup.26a, R.sup.26b, R.sup.26c, R.sup.24 or R.sup.25 is a bond to the linker,
(192) or a pharmaceutically acceptable salt thereof.
(193) The BRD9 binding moiety may be, e.g., a group of Formula VIII:
(194) ##STR00215##
(195) where
(196) v is 0, 1, 2, 3, or 4;
(197) each R.sup.28 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino;
(198) R.sup.29 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl;
(199) R.sup.31 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl; and
(200) each R.sup.30, R.sup.32, and R.sup.33 is, independently, H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl,
(201) or a pharmaceutically acceptable salt thereof.
(202) The BRD9 binding moiety may be, e.g., a group of Formula IX:
(203) ##STR00216##
(204) where
(205) ##STR00217##
is
(206) ##STR00218##
(207) Z.sup.4 is N or CR.sup.38;
(208) Z.sup.5 is N or CR.sup.39;
(209) R.sup.34 is H, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, or optionally substituted C.sub.3-C.sub.10 carbocyclyl;
(210) R.sup.35 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.6 carbocyclyl, or optionally substituted C.sub.6-C.sub.10 aryl;
(211) R.sup.37 is H, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.1-C.sub.6 heteroalkyl;
(212) R.sup.38 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl;
(213) R.sup.39 is H, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, or optionally substituted C.sub.6-C.sub.10 aryl;
(214) w is 0, 1, 2, 3, or 4; and
(215) each R.sup.36 is, independently, halogen, optionally substituted C.sub.1-C.sub.6 alkyl, optionally substituted C.sub.1-C.sub.6 heteroalkyl, optionally substituted C.sub.3-C.sub.10 carbocyclyl, optionally substituted C.sub.2-C.sub.9 heterocyclyl, optionally substituted C.sub.6-C.sub.10 aryl, optionally substituted C.sub.2-C.sub.9 heteroaryl, optionally substituted C.sub.2-C.sub.6 alkenyl, optionally substituted C.sub.2-C.sub.6 heteroalkenyl, hydroxy, thiol, or optionally substituted amino,
(216) or a pharmaceutically acceptable salt thereof.
(217) The BRD9 binding moiety may be, e.g., a group of the following structure:
(218) ##STR00219## ##STR00220##
Linkers
A linker may be, e.g., as found in the BRD9 inhibitor of Formula II:
A-L-B Formula II,
where
A is a BRD9 binding moiety;
B is a degradation moiety; and
L has the structure of Formula II:
A.sup.1-(E.sup.1)-(F.sup.1)—(C.sup.3).sub.m-(E.sup.3).sub.n—(F.sup.2).sub.o1—(F.sup.3).sub.o2-(E.sup.2).sub.p-A.sup.2, Formula IIA
(219) where
(220) A.sup.1 is a bond between the linker and A;
(221) A.sup.2 is a bond between B and the linker;
(222) each of m, n, o1, o2, and p is, independently, 0 or 1;
(223) each of E.sup.1 and E.sup.2 is, independently, O, S, NR.sup.N, optionally substituted C.sub.1-10 alkylene, optionally substituted C.sub.2-10 alkenylene, optionally substituted C.sub.2-10 alkynylene, optionally substituted C.sub.2-C.sub.10 polyethylene glycol, or optionally substituted C.sub.1-10 heteroalkylene;
(224) E.sup.3 is optionally substituted C.sub.1-C.sub.6 alkylene, optionally substituted C.sub.1-C.sub.6 heteroalkylene, O, S, or NR.sup.N;
(225) each R.sup.N is, independently, H, optionally substituted C.sub.1-4 alkyl, optionally substituted C.sub.2-4 alkenyl, optionally substituted C.sub.2-4 alkynyl, optionally substituted C.sub.2-6 heterocyclyl, optionally substituted C.sub.6-12 aryl, or optionally substituted C.sub.1-7 heteroalkyl;
(226) C.sup.3 is carbonyl, thiocarbonyl, sulphonyl, or phosphoryl; and
(227) each of F.sup.1, F.sup.2, and F.sup.3 is, independently, optionally substituted C.sub.3-C.sub.10 carbocyclylene, optionally substituted C.sub.2-10 heterocyclylene, optionally substituted C.sub.6-C.sub.10 arylene, or optionally substituted C.sub.2-C.sub.9 heteroarylene,
(228) or a pharmaceutically acceptable salt thereof.
(229) In some embodiments, the linker has the structure of Formula IIA-a:
A.sup.1-(E.sup.1)-(F.sup.1)—(C.sup.3).sub.m-(E.sup.2).sub.p-A.sup.2. Formula IIA-a
(230) In some embodiments, the linker has the structure of Formula IIA-b:
A.sup.1-(E.sup.1)-(F.sup.1)-(E.sup.2).sub.p-A.sup.2. Formula IIA-b
(231) In some embodiments, the linker has the structure of Formula IIA-c:
A.sup.1-(E.sup.1)-(F.sup.1)-A.sup.2. Formula IIA-c
(232) In some embodiments, the linker has the structure of Formula IIA-d:
A.sup.1-(E.sup.1)-(F.sup.1)—(C.sup.3).sub.m—(F.sup.2).sub.o1-A.sup.2. Formula IIA-d
(233) In some embodiments, the linker has the structure of Formula IIA-e:
A.sup.1-(E.sup.1)-(F.sup.1)-(E.sup.3).sub.n—(F.sup.2).sub.o1-(E.sup.2).sub.p-A.sup.2. Formula IIA-e
(234) In some embodiments, the linker has the structure of Formula IIA-f:
A.sup.1-(E.sup.1)-(F.sup.1)—(C.sup.3).sub.m-(E.sup.3).sub.n—(F.sup.2).sub.o1-(E.sup.2).sub.p-A.sup.2. Formula IIA-f
(235) In some embodiments, the linker has the structure of Formula IIA-g:
A.sup.1-(E.sup.1)-(F.sup.1)-(E.sup.3).sub.n—(F.sup.2).sub.o1-A, Formula IIA-g
Pharmaceutical Uses
(236) The compounds described herein are useful in the methods of the invention and, while not bound by theory, are believed to exert their desirable effects through their ability to modulate the level, status, and/or activity of a BAF complex, e.g., by inhibiting the activity or level of the BRD9 protein in a cell within the BAF complex in a mammal.
(237) An aspect of the present invention relates to methods of treating disorders related to BRD9 such as cancer in a subject in need thereof. In some embodiments, the compound is administered in an amount and for a time effective to result in one of (or more, e.g., two or more, three or more, four or more of): (a) reduced tumor size, (b) reduced rate of tumor growth, (c) increased tumor cell death (d) reduced tumor progression, (e) reduced number of metastases, (f) reduced rate of metastasis, (g) decreased tumor recurrence (h) increased survival of subject, and (i) increased progression free survival of a subject.
(238) Treating cancer can result in a reduction in size or volume of a tumor. For example, after treatment, tumor size is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) relative to its size prior to treatment. Size of a tumor may be measured by any reproducible means of measurement. For example, the size of a tumor may be measured as a diameter of the tumor.
(239) Treating cancer may further result in a decrease in number of tumors. For example, after treatment, tumor number is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater) relative to number prior to treatment. Number of tumors may be measured by any reproducible means of measurement, e.g., the number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification (e.g., 2×, 3×, 4×, 5×, 10×, or 50×).
(240) Treating cancer can result in a decrease in number of metastatic nodules in other tissues or organs distant from the primary tumor site. For example, after treatment, the number of metastatic nodules is reduced by 5% or greater (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) relative to number prior to treatment. The number of metastatic nodules may be measured by any reproducible means of measurement. For example, the number of metastatic nodules may be measured by counting metastatic nodules visible to the naked eye or at a specified magnification (e.g., 2×, 10×, or 50×).
(241) Treating cancer can result in an increase in average survival time of a population of subjects treated according to the present invention in comparison to a population of untreated subjects. For example, the average survival time is increased by more than 30 days (more than 60 days, 90 days, or 120 days). An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with the compound described herein. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with a pharmaceutically acceptable salt of a compound described herein.
(242) Treating cancer can also result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. For example, the mortality rate is decreased by more than 2% (e.g., more than 5%, 10%, or 25%). A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with a pharmaceutically acceptable salt of a compound described herein. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with a pharmaceutically acceptable salt of a compound described herein.
(243) Combination Therapies
(244) A method of the invention can be used alone or in combination with an additional therapeutic agent, e.g., other agents that treat cancer or symptoms associated therewith, or in combination with other types of therapies to treat cancer. In combination treatments, the dosages of one or more of the therapeutic compounds may be reduced from standard dosages when administered alone. For example, doses may be determined empirically from drug combinations and permutations or may be deduced by isobolographic analysis (e.g., Black et al., Neurology 65:S3-S6 (2005)). In this case, dosages of the compounds when combined should provide a therapeutic effect.
(245) In some embodiments, the second therapeutic agent is a chemotherapeutic agent (e.g., a cytotoxic agent or other chemical compound useful in the treatment of cancer). These include alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-Asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressant, adrenocorticosteroids, progestins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone analog. Also included is 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel, and doxetaxel. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredepa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammaII and calicheamicin omegaII (see, e.g., Agnew, Chem. Intl. Ed Engl. 33:183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin, including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®, cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Two or more chemotherapeutic agents can be used in a cocktail to be administered in combination with the first therapeutic agent described herein. Suitable dosing regimens of combination chemotherapies are known in the art and described in, for example, Saltz et al., Proc. Am. Soc. Clin. Oncol. 18:233a (1999), and Douillard et al., Lancet 355(9209):1041-1047 (2000).
(246) In some embodiments, the second therapeutic agent is a DNA damaging agent (e.g., a platinum-based antineoplastic agent, topoisomerase inhibitors, PARP inhibitors, alkylating antineoplastic agents, and ionizing radiation).
(247) Examples of platinum-based antineoplastic agent that may be used as a second therapeutic agent in the compositions and methods of the invention are cisplatin, carboplatin, oxaliplatin, dicycloplatin, eptaplatin, lobaplatin, miriplatin, nedaplatin, triplatin tetranitrate, phenanthrilplatin, picoplatin, and satraplatin. In some embodiments, the second therapeutic agent is cisplatin and the treated cancer is a testicular cancer, ovarian cancer, or a bladder cancer (e.g., advanced bladder cancer). In some embodiments, the second therapeutic agent is carboplatin and the treated cancer is an ovarian cancer, lung cancer, head and neck cancer, brain cancer, or neuroblastoma. In some embodiments, the second therapeutic agent is oxaliplatin and the treated cancer is a colorectal cancer. In some embodiments, the second therapeutic agent is dicycloplatin and the treated cancer is a non-small cell lung cancer or prostate cancer. In some embodiments, the second therapeutic agent is eptaplatin and the treated cancer is a gastric cancer. In some embodiments, the second therapeutic agent is lobaplatin and the treated cancer is a breast cancer. In some embodiments, the second therapeutic agent is miriplatin and the treated cancer is a hepatocellular carcinoma. In some embodiments, the second therapeutic agent is nedaplatin and the treated cancer is a nasopharyngeal carcinoma, esophageal cancer, squamous cell carcinoma, or cervical cancer. In some embodiments, the second therapeutic agent is triplatin tetranitrate and the treated cancer is a lung cancer (e.g., small cell lung cancer) or pancreatic cancer. In some embodiments, the second therapeutic agent is picoplatin and the treated cancer is a lung cancer (e.g., small cell lung cancer), prostate cancer, bladder cancer, or colorectal cancer. In some embodiments, the second therapeutic agent is satraplatin and the treated cancer is a prostate cancer, breast cancer, or lung cancer.
(248) Examples of topoisomerase inhibitors that may be used as a second therapeutic agent in the compositions and methods of the invention are etoposide, teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticine, irinotecan, topotecan, camptothecin, and diflomotecan. In some embodiments, the second therapeutic agent is etoposide and the treated cancer is a lung cancer (e.g., small cell lung cancer) or testicular cancer. In some embodiments, the second therapeutic agent is teniposide and the treated cancer is an acute lymphoblastic leukemia (e.g., childhood acute lymphoblastic leukemia). In some embodiments, the second therapeutic agent is doxorubicin and the treated cancer is an acute lymphoblastic leukemia, acute myeloblastic leukemia, Hodgkin lymphoma, Non-Hodgkin lymphoma, breast cancer, Wilm's tumor, neuroblastoma, soft tissue sarcoma, bone sarcomas, ovarian carcinoma, transitional cell bladder carcinoma, thyroid carcinoma, gastric carcinoma, or bronchogenic carcinoma. In some embodiments, the second therapeutic agent is daunorubicin and the treated cancer is an acute lymphoblastic leukemia or acute myeloid leukemia. In some embodiments, the second therapeutic agent is mitoxantrone and the treated cancer is a prostate cancer or acute nonlymphocytic leukemia. In some embodiments, the second therapeutic agent is amsacrine and the treated cancer is a leukemia (e.g., acute adult leukemia). In some embodiments, the second therapeutic agent is irinotecan and the treated cancer is a colorectal cancer. In some embodiments, the second therapeutic agent is topotecan and the treated cancer is a lung cancer (e.g., small cell lung cancer). In some embodiments, the second therapeutic agent is diflomotecan and the treated cancer is a lung cancer (e.g., small cell lung cancer).
(249) Examples of alkylating antineoplastic agents that may be used as a second therapeutic agent in the compositions and methods of the invention are cyclophosphamide, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, chlorozotocin, fotemustine, nimustine, ranimustine, busulfan, improsulfan, piposulfan, chlornaphazine, cholophosphamide, estramustine, mechlorethamine, mechlorethamine oxide hydrochloride, novembichin, phenesterine, prednimustine, trofosfamide, procarbazine, altretamine, dacarbazine, mitozolomide, and temozolomide. In some embodiments, the second therapeutic agent is cyclophosphamide and the treated cancer is a Non-Hodgkins lymphoma. In some embodiments, the second therapeutic agent is melphalan and the treated cancer is a multiple myeloma, ovarian cancer, or melanoma. In some embodiments, the second therapeutic agent is chlorambucil and the treated cancer is a chronic lymphatic leukemia, malignant lymphoma (e.g., lymphosarcoma, giant follicular lymphoma, or Hodgkin's lymphoma). In some embodiments, the second therapeutic agent is ifosfamide and the treated cancer is a testicular cancer. In some embodiments, the second therapeutic agent is bendamustine and the treated cancer is a chronic lymphocytic leukemia or non-Hodgkin lymphoma. In some embodiments, the second therapeutic agent is carmustine and the treated cancer is a brain cancer (e.g., glioblastoma, brainstem glioma, medulloblastoma, astrocytoma, ependymoma, or a metastatic brain tumor), multiple myeloma, Hodgkin's disease, or Non-Hodgkin's lymphoma. In some embodiments, the second therapeutic agent is lomustine and the treated cancer is a brain cancer or Hodgkin's lymphoma. In some embodiments, the second therapeutic agent is fotemustine and the treated cancer is a melanoma. In some embodiments, the second therapeutic agent is nimustine and the treated cancer is a brain cancer. In some embodiments, the second therapeutic agent is ranimustine and the treated cancer is a chronic myelogenous leukemia or polycythemia vera. In some embodiments, the second therapeutic agent is busulfan and the treated cancer is a chronic myelogenous leukemia. In some embodiments, the second therapeutic agent is improsulfan and the treated cancer is a sarcoma. In some embodiments, the second therapeutic agent is estramustine and the treated cancer is a prostate cancer (e.g., prostate carcinoma). In some embodiments, the second therapeutic agent is mechlorethamine and the treated cancer is a cutaneous T-cell lymphoma. In some embodiments, the second therapeutic agent is trofosfamide and the treated cancer is a sarcoma (e.g., soft tissue sarcoma). In some embodiments, the second therapeutic agent is procarbazine and the treated cancer is a Hodgkin's disease. In some embodiments, the second therapeutic agent is altretamine and the treated cancer is an ovarian cancer. In some embodiments, the second therapeutic agent is dacarbazine and the treated cancer is a melanoma, Hodgkin's lymphoma, or sarcoma. In some embodiments, the second therapeutic agent is temozolomide and the treated cancer is a brain cancer (e.g., astrocytoma or glioblastoma) or lung cancer (e.g., small cell lung cancer).
(250) Examples of PARP inhibitors that may be used as a second therapeutic agent in the compositions and methods of the invention are niraparib, olaparib, rucaparib, talazoparib, veliparib, pamiparib, CK-102, or E7016. Advantageously, the compounds of the invention and a DNA damaging agent may act synergistically to treat cancer. In some embodiments, the second therapeutic agent is niraparib and the treated cancer is an ovarian cancer (e.g., BRCA mutated ovarian cancer), fallopian tube cancer (e.g., BRCA mutated fallopian tube cancer), or primary peritoneal cancer (e.g., BRCA mutated primary peritoneal cancer). In some embodiments, the second therapeutic agent is olaparib and the treated cancer is a lung cancer (e.g., small cell lung cancer), ovarian cancer (e.g., BRCA mutated ovarian cancer), breast cancer (e.g., BRCA mutated breast cancer), fallopian tube cancer (e.g., BRCA mutated fallopian tube cancer), primary peritoneal cancer (e.g., BRCA mutated primary peritoneal cancer), prostate cancer (e.g., castration-resistant prostate cancer), or pancreatic cancer (e.g., pancreatic adenocarcinoma). In some embodiments, the second therapeutic agent is rucaparib and the treated cancer is an ovarian cancer (e.g., BRCA mutated ovarian cancer), fallopian tube cancer (e.g., BRCA mutated fallopian tube cancer), or primary peritoneal cancer (e.g., BRCA mutated primary peritoneal cancer). In some embodiments, the second therapeutic agent is talazoparib and the treated cancer is a breast cancer (e.g., BRCA mutated breast cancer). In some embodiments, the second therapeutic agent is veliparib and the treated cancer is a lung cancer (e.g., non-small cell lung cancer), melanoma, breast cancer, ovarian cancer, prostate cancer, or brain cancer. In some embodiments, the second therapeutic agent is pamiparib and the treated cancer is an ovarian cancer. In some embodiments, the second therapeutic agent is CK-102 and the treated cancer is a lung cancer (e.g., non-small cell lung cancer). In some embodiments, the second therapeutic agent is E7016 and the treated cancer is a melanoma.
(251) Without wishing to be bound by theory, the synergy between the compounds of the invention and DNA damaging agents may be attributed to the necessity of BRD9 for DNA repair; inhibition of BRD9 may sensitize cancer (e.g., cancer cell or cancer tissue) to DNA damaging agents.
(252) In some embodiments, the second therapeutic agent is a JAK inhibitor (e.g., JAK1 inhibitor). Non-limiting examples of JAK inhibitors that may be used as a second therapeutic agent in the compositions and methods of the invention include tofacitinib, ruxolitinib, oclacitinib, baricitinib, peficitinib, fedratinib, upadacitinib, filgotinib, cerdulatinib, gandotinib, lestaurtinib, momelotinib, pacritinib, abrocitinib, solcitinib, itacitinib, or SHR0302. Without wishing to be bound by theory, the synergy between the compounds of the invention and JAK inhibitors may be inhibitor of SAGA complex to their combined effect of downregulating Foxp3+ Treg cells. In some embodiments, the second therapeutic agent is ruxolitinib and the treated cancer is a myeloproliferative neoplasm (e.g., polycythemia or myelofibrosis), ovarian cancer, breast cancer, pancreatic cancer. In some embodiments, the second therapeutic agent is fedratinib and the treated cancer is a myeloproliferative neoplasm (e.g., myelofibrosis). In some embodiments, the second therapeutic agent is cerdulatinib and the treated cancer is a lymphoma (e.g., peripheral T-cell lymphoma). In some embodiments, the second therapeutic agent is gandotinib and the treated cancer is a myeloproliferative neoplasm (e.g., polycythemia or myelofibrosis). In some embodiments, the second therapeutic agent is lestaurtinib and the treated cancer is a myeloproliferative neoplasm (e.g., polycythemia or myelofibrosis), leukemia (e.g., acute myeloid leukemia), pancreatic cancer, prostate cancer, or neuroblastoma. In some embodiments, the second therapeutic agent is momelotinib and the treated cancer is a myeloproliferative neoplasm (e.g., polycythemia or myelofibrosis) or pancreatic cancer (e.g., pancreatic ductal adenocarcinoma). In some embodiments, the second therapeutic agent is momelotinib and the treated cancer is a myeloproliferative neoplasm (e.g., polycythemia or myelofibrosis). In some embodiments, the second therapeutic agent is momelotinib and the treated cancer is a myeloproliferative neoplasm (e.g., polycythemia or myelofibrosis) or pancreatic cancer (e.g., pancreatic ductal adenocarcinoma).
(253) In some embodiments, the second therapeutic agent is an inhibitor of SAGA complex or a component thereof. A SAGA complex inhibitor may be, e.g., an inhibitory antibody or small molecule inhibitor, of CCDC101, Tada2B, Tada3, Usp22, Tada1, Taf6I, Supt5, Supt20, or a combination thereof. Without wishing to be bound by theory, the synergy between the compounds of the invention and inhibitors of SAGA complex may be attributed to their combined effect of downregulating Foxp3+ Treg cells. In some embodiments, the second therapeutic agent is a therapeutic agent which is a biologic such a cytokine (e.g., interferon or an interleukin (e.g., IL-2)) used in cancer treatment. In some embodiments the biologic is an anti-angiogenic agent, such as an anti-VEGF agent, e.g., bevacizumab (AVASTIN®). In some embodiments the biologic is an immunoglobulin-based biologic, e.g., a monoclonal antibody (e.g., a humanized antibody, a fully human antibody, an Fc fusion protein or a functional fragment thereof) that agonizes a target to stimulate an anti-cancer response, or antagonizes an antigen important for cancer. Such agents include RITUXAN® (rituximab); ZENAPAX® (daclizumab); SIMULECT® (basiliximab); SYNAGIS® (palivizumab); REMICADE® (infliximab); HERCEPTIN® (trastuzumab); MYLOTARG® (gemtuzumab ozogamicin); CAMPATH® (alemtuzumab); ZEVALIN® (ibritumomab tiuxetan); HUMIRA® (adalimumab); XOLAIR® (omalizumab); BEXXAR® (tositumomab-1-131); RAPTIVA® (efalizumab); ERBITUX® (cetuximab); AVASTIN® (bevacizumab); TYSABRI® (natalizumab); ACTEMRA® (tocilizumab); VECTIBIX® (panitumumab); LUCENTIS® (ranibizumab); SOLIRIS® (eculizumab); CIMZIA® (certolizumab pegol); SIMPONI® (golimumab); ILARIS® (canakinumab); STELARA® (ustekinumab); ARZERRA® (ofatumumab); PROLIA® (denosumab); NUMAX® (motavizumab); ABTHRAX® (raxibacumab); BENLYSTA® (belimumab); YERVOY® (ipilimumab); ADCETRIS® (brentuximab vedotin); PERJETA® (pertuzumab); KADCYLA® (ado-trastuzumab emtansine); and GAZYVA® (obinutuzumab). Also included are antibody-drug conjugates.
(254) The second agent may be a therapeutic agent which is a non-drug treatment. For example, the second therapeutic agent is radiation therapy, cryotherapy, hyperthermia, and/or surgical excision of tumor tissue.
(255) The second agent may be a checkpoint inhibitor. In one embodiment, the inhibitor of checkpoint is an inhibitory antibody (e.g., a monospecific antibody such as a monoclonal antibody). The antibody may be, e.g., humanized or fully human. In some embodiments, the inhibitor of checkpoint is a fusion protein, e.g., an Fc-receptor fusion protein. In some embodiments, the inhibitor of checkpoint is an agent, such as an antibody, that interacts with a checkpoint protein. In some embodiments, the inhibitor of checkpoint is an agent, such as an antibody, that interacts with the ligand of a checkpoint protein. In some embodiments, the inhibitor of checkpoint is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of CTLA-4 (e.g., an anti-CTLA4 antibody or fusion a protein such as ipilimumab/YERVOY® or tremelimumab). In some embodiments, the inhibitor of checkpoint is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of PD-1 (e.g., nivolumab/OPDIVO®; pembrolizumab/KEYTRUDA®; pidilizumab/CT-011). In some embodiments, the inhibitor of checkpoint is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of PDL1 (e.g., MPDL3280A/RG7446; MEDI4736; MSB0010718C; BMS 936559). In some embodiments, the inhibitor of checkpoint is an inhibitor (e.g., an inhibitory antibody or Fc fusion or small molecule inhibitor) of PDL2 (e.g., a PDL2/Ig fusion protein such as AMP 224). In some embodiments, the inhibitor of checkpoint is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of B7-H3 (e.g., MGA271), B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, B-7 family ligands, or a combination thereof. In some embodiments, the second therapeutic agent is ipilimumab and the treated cancer is a melanoma, kidney cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), or prostate cancer. In some embodiments, the second therapeutic agent is tremelimumab and the treated cancer is a melanoma, mesothelioma, or lung cancer (e.g., non-small cell lung cancer). In some embodiments, the second therapeutic agent is nivolumab and the treated cancer is a melanoma, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), kidney cancer, Hodgkin lymphoma, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), urothelial carcinoma, hepatocellular carcinoma, or colorectal cancer. In some embodiments, the second therapeutic agent is pembrolizumab and the treated cancer is a melanoma, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), Hodgkin lymphoma, head and neck cancer (e.g., squamous cell carcinoma of the head and neck), primary mediastinal large B-cell lymphoma, urothelial carcinoma, hepatocellular carcinoma, microsatellite instability-high cancer, gastric cancer, esophageal cancer, cervical cancer, Merkel cell carcinoma, kidney carcinoma, or endometrial carcinoma. In some embodiments, the second therapeutic agent is MPDL3280A and the treated cancer is a lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), urothelial carcinoma, hepatocellular carcinoma, or breast cancer. In some embodiments, the second therapeutic agent is MEDI4736 and the treated cancer is a lung cancer (e.g., non-small cell lung cancer or small cell lung cancer) or urothelial carcinoma. In some embodiments, the second therapeutic agent is MSB0010718C and the treated cancer is a urothelial carcinoma. In some embodiments, the second therapeutic agent is MSB0010718C and the treated cancer is a melanoma, lung cancer (e.g., non-small cell lung cancer), colorectal cancer, kidney cancer, ovarian cancer, pancreatic cancer, gastric cancer, and breast cancer.
(256) Advantageously, the compounds of the invention and a checkpoint inhibitor may act synergistically to treat cancer. Without wishing to be bound by theory, the synergy between the compounds of the invention and checkpoint inhibitors may be attributed to the checkpoint inhibitor efficacy enhancement associated with the BRD9 inhibition-induced downregulation of Foxp3+ Treg cells.
(257) In some embodiments, the anti-cancer therapy is a T cell adoptive transfer (ACT) therapy. In some embodiments, the T cell is an activated T cell. The T cell may be modified to express a chimeric antigen receptor (CAR). CAR modified T (CAR-T) cells can be generated by any method known in the art. For example, the CAR-T cells can be generated by introducing a suitable expression vector encoding the CAR to a T cell. Prior to expansion and genetic modification of the T cells, a source of T cells is obtained from a subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art, may be used. In some embodiments, the T cell is an autologous T cell. Whether prior to or after genetic modification of the T cells to express a desirable protein (e.g., a CAR), the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.
(258) In any of the combination embodiments described herein, the first and second therapeutic agents are administered simultaneously or sequentially, in either order. The first therapeutic agent may be administered immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to hours 16, up to 17 hours, up 18 hours, up to 19 hours up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours up to 24 hours or up to 1-7, 1-14, 1-21 or 1-30 days before or after the second therapeutic agent.
(259) Pharmaceutical Compositions
(260) The pharmaceutical compositions described herein are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.
(261) The compounds described herein may be used in the form of the free base, in the form of salts, solvates, and as prodrugs. All forms are within the methods described herein. In accordance with the methods of the invention, the described compounds or salts, solvates, or prodrugs thereof may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compounds described herein may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, intratumoral, or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.
(262) A compound described herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, a compound described herein may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. A compound described herein may also be administered parenterally. Solutions of a compound described herein can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO, and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2012, 22nd ed.) and in The United States Pharmacopeia: The National Formulary (USP 41 NF36), published in 2018. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that may be easily administered via syringe. Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels, and powders. Aerosol formulations typically include a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device, such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form includes an aerosol dispenser, it will contain a propellant, which can be a compressed gas, such as compressed air or an organic propellant, such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier, such as sugar, acacia, tragacanth, gelatin, and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter. A compound described herein may be administered intratumorally, for example, as an intratumoral injection. Intratumoral injection is injection directly into the tumor vasculature and is specifically contemplated for discrete, solid, accessible tumors. Local, regional, or systemic administration also may be appropriate. A compound described herein may advantageously be contacted by administering an injection or multiple injections to the tumor, spaced for example, at approximately, 1 cm intervals. In the case of surgical intervention, the present invention may be used preoperatively, such as to render an inoperable tumor subject to resection. Continuous administration also may be applied where appropriate, for example, by implanting a catheter into a tumor or into tumor vasculature.
(263) The compounds described herein may be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration, and standard pharmaceutical practice.
(264) Dosages
(265) The dosage of the compounds described herein, and/or compositions including a compound described herein, can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. The compounds described herein may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In general, satisfactory results may be obtained when the compounds described herein are administered to a human at a daily dosage of, for example, between 0.05 mg and 3000 mg (measured as the solid form). Dose ranges include, for example, between 10-1000 mg (e.g., 50-800 mg). In some embodiments, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg of the compound is administered.
(266) Alternatively, the dosage amount can be calculated using the body weight of the patient. For example, the dose of a compound, or pharmaceutical composition thereof, administered to a patient may range from 0.1-100 mg/kg.
(267) Kits
(268) The invention also features kits including (a) a pharmaceutical composition including an agent that reduces the level and/or activity of BRD9 in a cell or subject described herein, and (b) a package insert with instructions to perform any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition including an agent that reduces the level and/or activity of BRD9 in a cell or subject described herein, (b) an additional therapeutic agent (e.g., an anti-cancer agent), and (c) a package insert with instructions to perform any of the methods described herein.
EXAMPLES
(269) Throughout the Examples, compound numbers
(270) TABLE-US-00001 TABLE 1 Compounds Com- pound No. Structure D1
Example 1—BRD9 Degrader Depletes BRD9 Protein
(271) The following example demonstrates the depletion of the BRD9 protein in synovial sarcoma cells treated with a BRD9 degrader.
(272) Procedure: Cells were treated with DMSO or the BRD9 degrader, Compound 1 (also known as dBRD9, see Remillard et al, Angew. Chem. Int. Ed. Engl. 56(21):5738-5743 (2017); see structure of compound 1 below), for indicated doses and timepoints.
(273) ##STR00225##
(274) Whole cell extracts were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer's protocols (Bio-Rad). After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was incubated with antibodies against BRD9 (1:1,000, Bethyl laboratory A303-781A), GAPDH (1:5,000, Cell Signaling Technology), and/or MBP (1:1,000, BioRad) overnight at 4° C. Membranes were washed three times for 10 min and incubated with anti-mouse or anti-rabbit antibodies conjugated with either horseradish peroxidase (HRP,
(275) Results: Treatment of SYO1 synovial sarcoma cells with the BRD9 degrader Compound 1 results in dose dependent (
Example 2—Inhibition of Growth of Synovial Cell Lines by BRD9 Inhibitors and BRD9 Degraders
(276) The following example demonstrates that BRD9 degraders and inhibitors selectively inhibit growth of synovial sarcoma cells.
(277) Procedures:
(278) Cells were treated with DMSO or the BRD9 degrader, Compound 1, at indicated concentrations, and proliferation was monitored from day 7 to day 14 by measuring confluency overtime using an IncuCyte live cell analysis system (
(279) Cells were seeded into 12-well plates and treated with DMSO, 1 μM BRD9 inhibitor, Compound 2 (also known as BI-7273, see Martin et al, J Med Chem. 59(10):4462-4475 (2016); see structure of compound 2 below), or 1 μM BRD9 degrader, Compound 1.
(280) ##STR00226##
(281) The number of cells was optimized for each cell line. Growth medium and compounds were refreshed every 3-5 days. SYO1, Yamato, A549, 293T and HS-SY-II cells were fixed and stained at day 11. ASKA cells were fixed and stained at day 23. Staining was done by incubation with crystal violet solution (0.5 g Crystal Violet, 27 ml 37% Formaldehyde, 100 mL 10×PBS, 10 mL Methanol, 863 dH20 to 1 L) for 30 min followed by 3× washes with water and drying the plates for at least 24 h at room temperature. Subsequently plates were scanned on an Odyssey CLx Imaging system (
(282) Cells were seeded into 96-well ultra-low cluster plate (Costar, #7007) in 200 μL complete media and treated at day 2 with DMSO, Staurosporin, or BRD9 degrader, Compound 1, at indicated doses (
(283) Results: As shown in
Example 3—Selective Inhibition of Growth of Synovial Cell Lines by BRD9 Degraders and BRD9 Binders
(284) The following example demonstrates that BRD9 degraders and binders selectively inhibit growth of synovial sarcoma cells.
(285) Procedure: Cells were seeded into 6-well or 12-well plates and were treated daily with a BRD9 degrader (Compound 1), a bromo-domain BRD9 binder (Compound 2), E3 ligase binder (lenalidomide), DMSO, or staurosporin (positive control for cell killing), at indicated concentrations. The number of cells was optimized for each cell line. Growth media was refreshed every 5 days. By day 14, medium was removed, cells were washed with PBS, and stained using 500 μL of 0.005% (w/v) crystal violet solution in 25% (v/v) methanol for at least 1 hour at room temperature. Subsequently plates were scanned on an Odyssey CLx Imaging system.
(286) Results: As shown in
Example 4—Inhibition of Cell Growth in Synovial Sarcoma Cells
(287) The following example shows that BRD9 degraders inhibit cell growth and induce apoptosis in synovial sarcoma cells.
(288) Procedure: SYO1 cells were treated for 8 or 13 days with DMSO, a BRD9 degrader (Compound 1) at 200 nM or 1 μM, or an E3 ligase binder (lenalidomide) at 200 nM. Compounds were refreshed every 5 days. Cell cycle analysis was performed using the Click-iT™ Plus EdU Flow Cytometry Assay (Invitrogen). The apoptosis assay was performed using the Annexin V-FITC Apoptosis Detection Kit (Sigma A9210). Assays were performed according to the manufacturer's protocol.
(289) Results: As shown in
Example 5—Composition for SS18-SSX1-BAF
(290) The following example shows the identification of BRD9 as a component of SS18-SSX containing BAF complexes.
(291) Procedure: A stable 293T cell line expressing HA-SS18SSX1 was generated using lentiviral integration. SS18-SSX1 containing BAF complexes were subject to affinity purification and subsequent mass spectrometry analysis revealed SS18-SSX1 interacting proteins.
(292) Results: As shown in
Example 6—Preparation of 4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxybenzaldehyde (Intermediate H)
(293) ##STR00227##
Step 1: Preparation of 6-chloro-4-methylpyridine-3-carboxamide (Intermediate B)
(294) ##STR00228##
(295) To a stirred mixture of 6-chloro-4-methylpyridine-3-carboxylic acid (20.00 g, 116.564 mmol, 1.00 equiv) and NH.sub.4Cl (62.35 g, 1.17 mol, 10.00 equiv) in dichloromethane (DCM; 400 mL) was added DIEA (22.60 g, 174.846 mmol, 3.00 equiv). After stirring for 5 minutes, HATU (66.48 g, 174.846 mmol, 1.50 equiv) was added in portions. The resulting mixture was stirred for 3 hours at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography, eluted with petroleum ether/ethyl acetate (PE/EtOAc) from 1/1 to 3/2 to afford 6-chloro-4-methylpyridine-3-carboxamide (18.30 g, 61.3%) as a yellow solid. LCMS (ESI) m/z: [M+H].sup.+=171.
Step 2: Preparation of 6-chloro-N-[(1E)-(dimethylamino)methylidene]-4-methylpyridine-3-carboxamide (Intermediate C)
(296) ##STR00229##
(297) To a stirred mixture of 6-chloro-4-methylpyridine-3-carboxamide (18.30 g, 107.268 mmol, 1.00 equiv) and in 2-methyltetrahydrofuran (100 mL) was added DMF-DMA (19.17 g, 160.903 mmol, 1.50 equiv) at 80° C. under nitrogen atmosphere, and stirred for additional 1 hour. Then the mixture was cooled and concentrated to afford 6-chloro-N-[(1E)-(dimethylamino)methylidene]-4-methylpyridine-3-carboxamide (26.3 g, 91.3%) as a yellow crude solid, which was used directly without further purification. LCMS (ESI) m/z: [M+H].sup.+=226.
Step 3: Preparation of 6-chloro-2H-2,7-naphthyridin-1-one (Intermediate D)
(298) ##STR00230##
(299) To a stirred mixture of 6-chloro-N-[(1E)-(dimethylamino)methylidene]-4-methylpyridine-3-carboxamide (26.30 g) in THE (170.00 mL) was added t-BuOK (174.00 mL, 1 mol/L in THF). The resulting solution was stirred at 60° C. under nitrogen atmosphere for 30 minutes. Then the mixture was cooled and concentrated under reduced pressure. The crude solid was washed with saturated NaHCO.sub.3 solution (100 mL) and collected to give 6-chloro-2H-2,7-naphthyridin-1-one (14.1 g, 67.0%) as a pink solid, which was used directly without further purification. LCMS (ESI) m/z: [M+H].sup.+=181.
Step 4: Preparation of 6-chloro-2-methyl-2,7-naphthyridin-1-one (Intermediate E)
(300) ##STR00231##
(301) To a stirred mixture of 6-chloro-2H-2,7-naphthyridin-1-one (14.10 g, 78.077 mmol, 1.00 equiv) in anhydrous THE (280.00 mL) was added NaH (9.37 g, 234.232 mmol, 3.00 equiv, 60%) in portions at 0° C. After 10 minutes, Mel (33.25 g, 234.232 mmol, 3.00 equiv) was added at 0° C., and the mixture was allowed to stir for 10 minutes at 0° C., and then the mixture was allowed to stir for 12 hours at room temperature. The resulting mixture was concentrated under reduced pressure. The crude solid was slurried with water (100 mL), and the solid was filtered and collected to give the 6-chloro-2-methyl-2,7-naphthyridin-1-one (14.6 g, 94.1%) as a yellow solid, which was used directly without further purification. LCMS (ESI) m/z: [M+H].sup.+=195.
Step 5: Preparation of 4-bromo-6-chloro-2-methyl-2,7-naphthyridin-1-one (Intermediate F)
(302) ##STR00232##
(303) To a stirred mixture of 6-chloro-2-methyl-2,7-naphthyridin-1-one (8.00 g, 41.106 mmol, 1.00 equiv) in DMF (160.00 mL) was added NBS (8.78 g, 49.327 mmol, 1.20 equiv), and the resulting mixture was stirred for 2 hours at 90° C. The reaction mixture was cooled and diluted with DCM (150 mL) and washed with water (3×100 mL). The organic layers were dried and concentrated. Then the residue was slurried with EtOAc (20 mL), and the slurry was filtered. The filter cake was washed with EtOAc (20 mL) to give 4-bromo-6-chloro-2-methyl-2,7-naphthyridin-1-one (6.32 g, 55.7%) as a white solid, which was used directly without further purification. LCMS (ESI) m/z: [M+H].sup.+=273.
Step 6: Preparation of 6-(azetidin-1-yl)-4-bromo-2-methyl-2,7-naphthyridin-1-one (Intermediate G)
(304) ##STR00233##
(305) To a solution of 4-bromo-6-chloro-2-methyl-2,7-naphthyridin-1-one (5.00 g, 18.281 mmol, 1.00 equiv) and azetidine hydrochloride (3.2 g, 54.843 mmol, 3 equiv) in DMSO (50.00 mL) was added K.sub.2CO.sub.3 (12.6 g, 91.404 mmol, 5 equiv). The resulting solution was stirred at 130° C. for 2 hours. The resulting mixture was cooled and diluted with water (100 mL), and then extracted with EtOAc (3×100 mL). The combined organic layers were washed with saturated NaCl solution (3×50 mL), dried over anhydrous Na.sub.2SO.sub.4, and concentrated under reduced pressure to afford 6-(azetidin-1-yl)-4-bromo-2-methyl-2,7-naphthyridin-1-one (3.7 g, 68.8%) as a grey solid, which was used directly without further purification. LCMS (ESI) m/z: [M+H].sup.+=294.
Step 7: Preparation of 4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxybenzaldehyde (Intermediate H)
(306) ##STR00234##
(307) To a solution of 6-(azetidin-1-yl)-4-bromo-2-methyl-2,7-naphthyridin-1-one (1.42 g, 4.827 mmol, 1.00 equiv) and 4-formyl-3,5-dimethoxyphenylboronic acid (1.52 g, 7.241 mmol, 1.5 equiv) in dioxane (16.00 mL) and H.sub.2O (4.00 mL) was added Pd(dppf)Cl.sub.2 (353.2 mg, 0.483 mmol, 0.1 equiv) and Cs.sub.2CO.sub.3 (3.15 g, 9.655 mmol, 2 equiv), and the resulting solution was stirred at 70° C. for 2 hours. The resulting mixture was cooled and concentrated under reduced pressure. The residue was slurried with water (30 mL) and filtered, and the filter cake was collected. This solid was further slurried with MeOH (30 mL) and filtered. The solid was collected to afford 4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxybenzaldehyde (1.42 g, 77.5%) as a grey solid. LCMS (ESI) m/z: [M+H].sup.+=380.
Example 7—Preparation of 3-[1-oxo-6-[7-(piperidin-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-2-yl]-3H-isoindol-2-yl]piperidine-2,6-dione (Intermediate P)
(308) ##STR00235##
Step 1: Preparation of methyl 5-bromo-2-(bromomethyl)benzoate (Intermediate J)
(309) ##STR00236##
(310) To a stirred mixture of methyl 5-bromo-2-methylbenzoate (50.00 g, 218.271 mmol, 1.00 equiv) in CCl.sub.4 (500.00 mL) was added NBS (38.85 g, 218.271 mmol, 1.00 equiv) and BPO (5.59 g, 21.827 mmol, 0.10 equiv). After stirring for overnight at 80° C., the mixture was purified by silica gel column chromatography, eluted with PE/EtOAc (50:1) to afford methyl 5-bromo-2-(bromomethyl)benzoate (67 g, 74.75%) as a yellow oil. LCMS (ESI) m/z: [M+H].sup.+=307.
Step 2: Preparation of tert-butyl 4-(6-bromo-1-oxo-3H-isoindol-2-yl)-4-carbamoylbutanoate (Intermediate K)
(311) ##STR00237##
(312) To a stirred mixture of methyl 5-bromo-2-(bromomethyl)benzoate (67.00 g, 217.554 mmol, 1.00 equiv) and tert-butyl (4S)-4-amino-4-carbamoylbutanoate hydrochloride (62.32 g, 261.070 mmol, 1.20 equiv) in DMF (100.00 mL) was added DIEA (112.47 g, 870.217 mmol, 4 equiv). After stirring for overnight at 80° C., the mixture was concentrated under reduced pressure. The residue was added water (200 mL) and stirred for 1 h at room temperature. The resulting mixture was filtered, the filter cake was added water (100 mL) and stirred. The precipitated solids were collected by filtration and washed with water (3×30 mL). This resulted in tert-butyl 4-(6-bromo-1-oxo-3H-isoindol-2-yl)-4-carbamoylbutanoate (55 g, 60.46%) as an off-white solid. LCMS (ESI) m/z: [M+H].sup.+=397.
Step 3: Preparation of tert-butyl 2-[2-[4-(tert-butoxy)-1-carbamoyl-4-oxobutyl]-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonane-7-carboxylate (Intermediate L)
(313) ##STR00238##
(314) To a stirred solution of tert-butyl 4-(6-bromo-1-oxo-3H-isoindol-2-yl)-4-carbamoylbutanoate (10.00 g, 25.172 mmol, 1.00 equiv) and tert-butyl 2,7-diazaspiro[3.5]nonane-7-carboxylate hydrochloride (8.60 g, 32.723 mmol, 1.30 equiv) in dioxane (200.00 mL) was added Cs.sub.2CO.sub.3 (24.60 g, 75.516 mmol, 3.00 equiv) and RuPhos Palladacycle Gen. 3 (2.11 g, 2.517 mmol, 0.10 equiv). After stirring for overnight at 100° C. under nitrogen atmosphere, the resulting mixture was filtered while hot, and the filter cake was washed with 1,4-dioxane (3×50 mL). The filtrate was concentrated under reduced pressure to afford tert-butyl 2-[2-[4-(tert-butoxy)-1-carbamoyl-4-oxobutyl]-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro [3.5]nonane-7-carboxylate (21 g, crude) as a black solid. LCMS (ESI) m/z: [M+H].sup.+=543.
Step 4: Preparation of tert-butyl 2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonane-7-carboxylate (Intermediate M)
(315) ##STR00239##
(316) To a stirred mixture of tert-butyl 2-[2-[(1S)-4-(tert-butoxy)-1-carbamoyl-4-oxobutyl]-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonane-7-carboxylate (13.68 g, 25.208 mmol, 1.00 equiv) in THE (100.00 mL) was added t-BuOK in THE (25.00 mL, 25.000 mmol, 0.99 equiv). The resulting mixture was stirred for 2 hours at room temperature. The mixture was acidified to pH 6 with 1 M HCl (aq.) and then neutralized to pH 7 with saturated NaHCO.sub.3 (aq.). The resulting mixture was extracted with EtOAc (3×200 mL). The combined organic layers were concentrated under reduced pressure. This resulted in tert-butyl 2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonane-7-carboxylate (7.8 g, 59.43%) as a yellow solid. LCMS (ESI) m/z: [M+H].sup.+=469.
Step 5: Preparation of 3-(6-[2,7-diazaspiro[3.5]nonan-2-yl]-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione (Intermediate N)
(317) ##STR00240##
(318) To a stirred mixture of tert-butyl 2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonane-7-carboxylate (7.80 g, 16.647 mmol, 1.00 equiv) in DCM (10.00 mL) was added trifluoroacetic acid (TFA; 5.00 mL). After stirring for 2 hours at room temperature, the resulting mixture was concentrated under vacuum. This resulted in 3-(6-[2,7-diazaspiro[3.5]nonan-2-yl]-1-oxo-3H-isoindol-2-yl)piperidine-2,6-dione (6 g, 92.93%) as a light yellow solid. LCMS (ESI) m/z: [M+H].sup.+=369.
Step 6: Preparation of tert-butyl 4-([2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonan-7-yl]methyl)piperidine-1-carboxylate (Intermediate O)
(319) ##STR00241##
(320) To a stirred solution of 3-(6-[2,7-diazaspiro[3.5]nonan-2-yl]-1-oxo-3H-isoindol-2-yl) piperidine-2,6-dione (4.00 g, 8.685 mmol, 1.00 equiv, 80%) and tert-butyl 4-formylpiperidine-1-carboxylate (1.48 g, 6.939 mmol, 0.80 equiv) in DMF (20.00 mL) was added NaBH(OAc).sub.3 (3.68 g, 17.363 mmol, 2.00 equiv) at room temperature. The resulting mixture was stirred for 2 hours at room temperature. The reaction was quenched with water at room temperature. The resulting mixture was purified by reverse flash chromatography with the following conditions (column, C18 silica gel; mobile phase, CH.sub.3CN in water (0.1% FA), 0 to 100% gradient in 40 minutes; detector, UV 254 nm). This resulted in tert-butyl 4-([2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro [3.5]nonan-7-yl]methyl)piperidine-1-carboxylate (2.8 g, 51.29%) as a dark yellow solid. LCMS (ESI) m/z: [M+H].sup.+=566.
Step 7: Preparation of 3-[1-oxo-6-[7-(piperidin-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-2-yl]-3H-isoindol-2-yl]piperidine-2,6-dione (Intermediate P)
(321) ##STR00242##
(322) To a stirred mixture of tert-butyl 4-([2-[2-(2,6-dioxopiperidin-3-yl)-3-oxo-1H-isoindol-5-yl]-2,7-diazaspiro[3.5]nonan-7-yl]methyl)piperidine-1-carboxylate (2.80 g, 4.949 mmol, 1.00 equiv) in DCM (5.00 mL) was added TFA (2.00 mL). The mixture was stirred for 2 hours at room temperature. The resulting mixture was concentrated under reduced pressure to afford 3-[1-oxo-6-[7-(piperidin-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-2-yl]-3H-isoindol-2-yl]piperidine-2,6-dione (3.9 g, crude) as a yellow solid. LCMS (ESI) m/z: [M+H].sup.+=466.
Example 8—Preparation of 3-[6-(7-[[1-([4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxyphenyl]methyl)piperidin-4-yl]methyl]-2,7-diazaspiro[3.5]nonan-2-yl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione TFA Salt (Compound D1 TFA Salt)
(323) ##STR00243##
(324) A solution of 3-[1-oxo-6-[7-(piperidin-4-ylmethyl)-2,7-diazaspiro[3.5]nonan-2-yl]-3H-isoindol-2-yl]piperidine-2,6-dione (4.5 g, 10.52 mmol, 1.00 equiv) and 4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxybenzaldehyde (4.0 g, 10.52 mmol, 1.00 equiv) and titanium(IV) isopropoxide (3.0 g, 10.52 mmol, 1.00 equiv) in DMSO (100 mL) was stirred at room temperature for 3 hours. Then NaBH(OAc).sub.3 (8.92 g, 42.08 mmol, 4.00 equiv) was added in batches to the above resulting solution, and the resulting mixture was stirred at room temperature overnight. The reaction was quenched by the addition of water (30 mL), and then the solution was filtered. The filter cake was wash by water and acetonitrile. Then the filtrate was concentrated in vacuo. The crude product was purified by reverse phase flash chromatography with the following conditions (Column: AQ C18 Column, 50×250 mm 10 μm; Mobile Phase A: Water (TFA 0.1%), Mobile Phase B: ACN; Flow rate: 100 mL/minute; Gradient: 5 B to 25 B in 35 minutes; 254/220 nm). Pure fractions were evaporated to dryness to afford 3-[6-(7-[[1-([4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxyphenyl]methyl)piperidin-4-yl]methyl]-2,7-diazaspiro[3.5]nonan-2-yl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione TFA salt (3.2 g, 32.3%) as a white solid. .sup.1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 9.01 (s, 1H), 7.59 (s, 1H), 7.36 (d, J=8.0 Hz, 1H), 6.72 (s, 2H), 6.68 (d, J=8.1 Hz, 2H), 6.20 (s, 1H), 5.07 (dd, J=13.3, 5.1 Hz, 1H), 4.35-4.13 (m, 2H), 4.06-3.95 (m, 4H), 3.80 (s, 6H), 3.57 (s, 4H), 3.47 (s, 5H), 2.97-2.75 (m, 3H), 2.70-2.55 (m, 1H), 2.44-2.16 (m, 7H), 2.13-1.88 (m, 5H), 1.80-1.67 (m, 4H), 1.61 (d, J=12.4 Hz, 2H), 1.53-1.33 (m, 1H), 1.13-0.94 (m, 2H). LCMS (ESI) m/z: [M+H].sup.+=829.55.
(325) Enantiomers of compound D1 were separated by supercritical fluid chromatography on chiral support to produce compound S-D1 and compound R-D1.
(326) Compound D1 is of the following structure:
(327) ##STR00244##
(328) Compound S-D1 is of the following structure:
(329) ##STR00245##
(330) Compound R-D1 is of the following structure:
(331) ##STR00246##
Example 9—Preparation of Compound D2
(332) In analogy to the procedures described in the examples above, compound D2 was prepared using the appropriate starting materials.
(333) Compound D2: .sup.1H NMR (300 MHz, DMSO-d6) δ 10.98 (s, 1H), 9.02 (d, J=0.7 Hz, 1H), 7.63 (d, J=2.3 Hz, 1H), 7.41 (d, J=8.8 Hz, 1H), 6.88 (s, 2H), 6.70 (h, J=2.4 Hz, 2H), 6.24 (d, J=6.0 Hz, 1H), 5.08 (dd, J=13.2, 5.1 Hz, 1H), 4.41-4.14 (m, 4H), 4.04 (t, J=7.4 Hz, 4H), 3.91 (s, 1H), 3.70 (d, J=22.5 Hz, 4H), 3.50 (s, 3H), 3.45 (s, 1H), 3.22 (s, 1H), 3.14-2.82 (m, 6H), 2.60 (d, J=16.2 Hz, 1H), 2.55 (s, 2H), 2.44-2.28 (m, 3H), 2.18-2.05 (m, 3H), 1.97 (t, J=13.9 Hz, 5H), 1.51 (q, J=12.2, 11.1 Hz, 2H). LCMS (ESI) m/z: [M+H].sup.+=835.45.
(334) Compound D2 is of the following structure:
(335) ##STR00247##
Example 10—SYO1 BRD9 NanoLuc Degradation Assay
(336) This example demonstrates the ability of the compounds of the disclosure to degrade a Nanoluciferase-BRD9 fusion protein in a cell-based degradation assay.
(337) Procedure: A stable SYO-1 cell line expressing 3×FLAG-NLuc-BRD9 was generated. On day 0 cells were seeded in 30 μL media into each well of 384-well cell culture plates. The seeding density was 8000 cells/well. On day 1, cells were treated with 30 nL DMSO or 30 nL of 3-fold serially DMSO-diluted compounds (10 points in duplicates with 1 μM as final top dose). Subsequently plates were incubated for 6 hours in a standard tissue culture incubator and equilibrated at room temperature for 15 minutes. Nanoluciferase activity was measured by adding 15 μL of freshly prepared Nano-Glo Luciferase Assay Reagent (Promega N1130), shaking the plates for 10 minutes and reading the bioluminescence using an EnVision reader.
(338) Results: The Inhibition % was calculated using the following formula: % Inhibition=100×(Lum.sub.HC−Lum.sub.Sample)/(Lum.sub.HC−Lum.sub.LC). DMSO treated cells are employed as High Control (HC) and 1 μM of a known BRD9 degrader standard treated cells are employed as Low Control (LC). The data was fit to a four parameter, non-linear curve fit to calculate IC.sub.50 (μM) values as shown in Table 2. As shown by the results in Table 2, a number of compounds of the present disclosure exhibit an IC.sub.50 value of <1 μM for the degradation of BRD9, indicating their use as compounds for reducing the levels and/or activity of BRD9 and their potential for treating BRD9-related disorders.
(339) TABLE-US-00002 TABLE 2 SYO1 BRD9-NanoLuc Degradation SYO1 BRD9-NanoLuc Compound No. degradation IC.sub.50 (nM) D1 0.13 D2 0.18
Example 11—Degradation of BRD9 Inhibits the Growth of Synovial Sarcoma Tumor In Vivo
(340) Method: NOD SCID mice (Beijing Anikeeper Biotech, Beijing) were inoculated subcutaneously on the right flank with the single cell suspension of SYO-1 human biphasic synovial sarcoma tumor cells (5×106) in 100 μL Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). The mice were randomized into either control group [10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol (PEG400) and 50% water], or treatment group D1 when the mean tumor size reached about 117 mm.sup.3. Mice were dosed daily through intraperitoneal (i.p.) route over the course of 3 weeks. All dose volumes were adjusted by body weights in terms of mg/kg.
(341) Results: As shown in
Example 12—Compound D1 Causes Degradation of BRD9 in Synovial Sarcoma Tumor In Vivo
(342) Method: Mice were treated with D1, 1 mg/kg, i.p. for 4 weeks. Mice were then euthanized, and tumors were collected at 8 hours, 72 hours, and 168 hours post last dose. Tumors were lysed with 1×RIPA lysis buffer (Boston BioProducts, BP-115D) with protease and phosphatase protein inhibitor (Roche Applied Science #04906837001 & 05892791001). Equal amounts of lysate (30 μg) were loaded in in 4-12% Bis-Tris Midi Protein Gels in 1×MOPS buffer; samples ran at 120 V for 120 minutes. Protein was transferred to membrane with TransBlot at 250 mA for 150 minutes, and then membranes were blocked with Odyssey blocking buffer for 1 hour at room temperature. Membranes were hybridized overnight in cold room with primary antibodies. Images acquired using Li—COR imaging system (Li—COR Biotechnology, Lincoln, Nebr.).
(343) Table 3 shows detection antibody information.
(344) TABLE-US-00003 TABLE 3 Antibody Vendor Cat# Species Dilution BRD9 Bethyl, (Montgomery, TX) A303-781A Rabbit 1:1000 GAPDH CST, (Danvers, MA) 97166 Mouse 1:2000
(345) Results: As shown in
Example 13—the Effect of Compounds S-D1 and R-D1 on Synovial Sarcoma Cells Method
(346) Synovial sarcoma cells were plated in 6-well plate at 500-100k cells/well and treated with serial concentrations of BRD9 degrader (10 nM top concentration, diluted 1:3) the next day for two time points at 37° C. Cells were then harvested, washed with cold PBS, and frozen in cell pellets at −80° C. Lysates were prepared by resuspending thawed pellets in 1×RIPA Lysis and Extraction buffer (Thermo Fisher, Cat #89900) with 1× Halt™ Protease and Phosphatase Inhibitor Cocktail, EDTA-free (Thermo Fisher, Cat #78441) and 1:1000 dilution Pierce™ Universal Nuclease for Cell Lysis 25 ku (Thermo Fisher, Cat #88700). Lysates were incubated on ice for 10 minutes and then centrifuged in 4° C. at maximum speed (15,000 rpm) for 10 minutes. Samples were then analyzed for total protein using BCA protein quantification assay and diluted to 1 μg/μL with lysis buffer and 1× NuPAGE™ LDS Sample Buffer (4×) (Thermo Fisher, Cat #NP0007) and 1×DTT from 30× stock (Cell Signaling Technologies, Cat #14265S). Samples with 20-25 ug of total protein were loaded into 4-12% Bis-Tris Mini-Gel with 1×MES Running buffer and run at 150V for 45 minutes. Gels were transferred using Trans-Blot® Turbo™ Transfer System (semi-dry) at 25V for 10 minutes (High MW setting) on nitrocellulose blots. Blots were blocked in 5% milk in TBST for 1 hour and probed with BRD9 antibody (Bethyl Labs, Cat #A303-781A, 1:750 for SYO1, and Cell Signaling Technologies, Cat #71232S for ASKA) and beta-Actin antibody (Cell Signaling Technologies, Cat #3700, 1:2,000) overnight at 4° C. The next day, blots were washed in TBST 3× and probed with 1:5,000 IRDye® 680LT Goat anti-Rabbit IgG Secondary Antibody (LiCOR, Cat #926-68021) and 1:10,000 IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (LiCOR, Cat #926-32210) in LiCOR Odyssey® Blocking Buffer (TBS) for 1 hour at room temperature. Blots were washed in TBST 3× and scanned at 700 nM and 800 nM wavelength using LiCOR Odyssey® CLx Imaging System. Western blot signal was quantified using same analyses program included in the same machine. BRD9 signal was quantified by normalizing to beta-actin signal and all samples were normalized to DMSO, set as 100% signal.
(347) For the assessment of interconversion between Enantiomer 1 and Enantiomer 2 in cell medium, the following test was performed. Enantiomer 1 and Enantiomer 2 (each was 40 μM in DMSO) was spiked into cell medium (DMEM+Glutamax+10% FBS) at a final concentration of 0.2 μM and incubated at 37° C. and 5% CO.sub.2 in duplicate. At designated time point, aliquot (50 μL) was taken and processed by the addition of 150 μL of acetonitrile containing 0.1% formic acid and internal standard for LC/MS-MS analysis. Peak areas of both Enantiomer 1 and Enantiomer 2 were determined for each sample using a chiral specific analytical method. The results are summarized in Table 5 below.
(348) Results. To assess BRD9 degradation activity of two enantiomers, degrader treatment and subsequent western-blot experiments were carried out using two synovial sarcoma cell lines (SYO-1 and ASKA). Significant more potent BRD9 degradation activity was observed with Enantiomer 2, with a fitted DC.sub.50 value of 0.092 nM, comparing to 2.8 nM for Enantiomer 1 in SYO-1 with 1 h treatment time (
(349) TABLE-US-00004 TABLE 4 Cell Line Fitted DC.sub.50 (nM) Enantiomer 1 Enantiomer 2 ASKA 0.5 h >10 0.34 SYO-1 1 h 2.8 0.092 ASKA 2 h 0.38 0.012 SYO-1 6 h 0.066 0.023
(350) Epimerization of the chiral center in thalidomide or other IMiD drugs and their derivatives is reported. The acidic hydrogen in the chiral center can be scrambled under physical or neutral pH conditions. To investigate the chiral stability under cell assay conditions for these degraders, we performed a time course study for Enantiomer 1 and Enantiomer 2 in cell culture medium at 37° C. There is no detectable Enantiomer 2 in Enantiomer 1 samples at time 0 or 0.5 h. But substantial Enantiomer 2 was detected at later time points, accounting for 12% and 30% of the total at 2 h and 6 h, respectively (Table 5). Similarly, Enantiomer 2 is converted to Enantiomer 1 overtime and its effective concentration was reduced to 63% at 6 h (Table 5). These data indicate that epimerization rate is relatively fast under the cell assay conditions, and suggest that the time-dependent BRD9 degradation activity for Enantiomer 1 is likely due to the converted Enantiomer 2. Overall, these data indicate that Enantiomer 2 is the active enantiomer in degrading BRD9 in cells.
(351) TABLE-US-00005 TABLE 5 Enantiomer 1 Dosing Enantiomer 2 Dosing Mean peak area Mean peak area ratio of ratio of Enantiomer 2 over % Enantiomer 1 over % Time Enantiomer 1 Enantiomer Enantiomer 2 Enantiomer (h) peak area ratio 2 peak area ratio 2 0 0.0 0.0 0.01 99 0.5 0.0 0.0 0.06 95 2 0.13 12 0.22 82 6 0.43 30 0.60 63
Example 14—the Effect of Compounds S-D1 and R-D1 on Synovial Sarcoma Cells
(352) Method. The SYO-1 tumor cells were maintained in vitro as adherent cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37° C. in an atmosphere of 5% CO2 in air. The cells growing in an exponential growth phase were harvested and counted for tumor inoculation. BALB/c Nude mice (Shanghai Lingchang biological science) were inoculated subcutaneously on the right flank with (5×10.sup.6) in 0.1 mL of phosphate buffered saline (PBS). The treatment—(described in the table below)—was started on day 19 after tumor inoculation, when the average tumor size reached 499 mm.sup.3.
(353) TABLE-US-00006 TABLE 6 Treatment information Number Antibody of mice Vehicle control, sulfobutylether-β-cyclodextrin 3 (SBECD), Single Dose, 10 μL/g Racemic D1, 0.5 mg/kg i.v., Single Dose, 12 10 μL/g (20% SBECD) Enantiomer 1, 0.25 mg/kg i.v., Single Dose, 18 10 μL/g (20% SBECD) Enantiomer 2, 0.25 mg/kg i.v., Single Dose, 18 10 μL/g (20% SBECD) Enantiomer 2, 1 mg/kg i.v., Single Dose, 18 10 μL/g (20% SBECD)
(354) Mice were treated with racemic D1, 1 mg/kg, i.p. for 4 weeks, mice were euthanized, and tumors collected 1, 4, 8, 24, 48 and 72-hour post last dose. Tumors were lysed with 1×RIPA lysis buffer (Boston BioProducts, BP-115D) with protease and phosphatase protein inhibitor (Roche Applied Science #04906837001 & 05892791001). Equal amount of lysate (30 μg) were loaded in in 4-12% Bis-Tris Midi Protein Gels in 1×MOPS buffer; samples ran at 120 V for 120 min. Proteins was transferred to membrane (NC) with TransBlot at 250 mA for 150 minute, then membranes were blocked with Odyssey Blocking buffer for 1 hour at room temperature. Membranes were hybridized overnight in cold room with primary antibodies. Images acquired using Li—COR imaging system (Li—COR Biotechnology, Lincoln, Nebr.)
(355) TABLE-US-00007 TABLE 7 Detection antibody information Antibody Vendor Cat# Species Dilution BRD9 Bethyl, (Montgomery, TX) A303-781A Rabbit 1:1000 GAPDH CST, (Danvers, MA) 97166 Mouse 1:2000
(356) Results. Pharmacodynamic activities of Enantiomer 1, Enantiomer 2, and racemic compound D1 were evaluated in SYO-1 Xenograft model. Enantiomer 2 demonstrated significant activity which was assessed by BRD9 protein level using western blot assay
Example 15—Forced Degradation Study of Compound D1
(357) Solution Preparation
(358) Diluent 1: H.sub.2O
(359) Diluent 2: dimethylsulfoxide (DMSO)
(360) Diluent 3: [0.05% formic acid (FA) in acetonitrile (MeCN)]: H.sub.2O=1:1 (v/v)
(361) 2N HCl was prepared by weighing 19.7076 g concentrated aq. HCl (37%) into a 100 mL volumetric flask containing approximately 20 mL diluent 1, diluting to volume with diluent 1, and mixing well.
(362) 0.002N HCl was prepared as follows. 1 mL of 2N HCl into was transferred into a 100 mL volumetric flask containing approximately 20 mL diluent 1, diluted to volume with diluent 1, and mixed well. Then, 1 mL of this solution was pipetted into a 10 mL volumetric flask, diluted to volume with diluent 1, and mixed well.
(363) Diluent 4: 0.001N HCl was prepared as follows. 1 mL of the 2N HCl was transferred into a 100 mL volumetric flask containing approximately 20 mL diluent 1, diluted to volume with diluent 1, and mixed well. Then, 5 mL of this solution were pipetted into a 100 mL volumetric flask, made up to volume with diluent 1 and mixed well.
(364) 2N NaOH was prepared as follows. 8.0248 g of NaOH were weighed into a 100 mL volumetric flask, diluted to volume with diluent 1, and mixed well.
(365) 0.1N NaOH was prepared as follows. 5 mL of the 2N NaOH were transferred into a 100 mL volumetric flask containing approximately 5 mL diluent 1, diluted to volume with diluent 1, and mixed well.
(366) 0.002N NaOH was prepared as follows. 1 mL of the 2N NaOH was transferred into a 100 mL volumetric flask containing approximately 20 mL diluent 1, diluted to volume with diluent 1, and mixed well. Then, 10 mL of this solution were pipetted into a 100 mL volumetric flask, diluted to volume with diluent 1, and mixed well.
(367) 6% Hydrogen peroxide was prepared as follows. 20 mL of concentrated H.sub.2O.sub.2 (30%) were transferred to a 100 mL volumetric flask, diluted to volume with diluent 1, and mixed well.
(368) 92% relative humidity control was achieved using a dryer containing a saturated potassium nitrate solution.
(369) 1N Citric acid was prepared as follows. 19.2032 g of citric acid were weighed into a 100 mL volumetric flask, diluted to volume with diluent 1, and mixed well.
(370) pH=4.0 50 mM Sodium citrate buffer was prepared as follows. 1.4814 g of sodium citrate were weighed into a 100 mL volumetric flask, diluted up to volume with diluent 1, and mixed well. pH was adjusted to 4.0 by 1N citric acid.
(371) pH=5.0 50 mM Sodium citrate buffer was prepared as follows. 1.4815 g of sodium citrate were weighed into a 100 mL volumetric flask, diluted to volume with diluent 1, and mixed well. pH was adjusted to 5.0 by 1N citric acid.
(372) pH=7.0 Phosphate buffer was prepared as follows. 0.6963 g of KH.sub.2PO.sub.4 were weighed into a 100 mL volumetric flask, 29.1 mL of the 0.1N NaOH were transferred into the same volumetric flask, diluted to volume with diluent 1, and mixed well.
(373) Sample Preparation
(374) Sample stock solution was prepared by weighing 60.74 mg of compound D1 to a 100 mL volumetric flask, dissolving by ultrasonic in 50 mL of diluent 2, diluting to volume with diluent 2, and mixing well.
(375) Unstressed sample solution was prepared by weighing 21.32 mg of compound D1 into a 100 mL volumetric flask, dissolving by ultrasonic in 50 mL of diluent 3, diluting to volume with diluent 3, and mixing well.
(376) Sensitivity solution (LOQ) was prepared as follows. 1.0 mL of unstressed sample solution was pipetted into a 100 mL volumetric flask, diluted to volume with diluent 3, and mixed well. Then, 5 mL of this sample solution were pipetted into a 100 mL volumetric flask, diluted to volume with diluent 3, and mixed well.
(377) Sample solution for pH=4.0 solution stability was prepared as follows. 20.96 mg of compound D1 were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of pH=4.0 50 mM sodium citrate buffer, diluted to volume with pH=4.0 50 mM sodium citrate buffer, and mixed well.
(378) Sample solution for pH=5.0 solution stability was prepared as follows. 20.75 mg of compound D were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of pH=5.0 50 mM sodium citrate buffer, diluted to volume with pH=5.0 50 mM sodium citrate buffer, and mixed well.
(379) Sample solution for pH=7.0 solution stability was prepared as follows. 19.91 mg of compound D1 and 684.71 mg KH.sub.2PO.sub.4 were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of diluent 1, transferred 29.1 mL of 0.1N NaOH into the same volumetric flask, diluted to volume with diluent 1, and mixed well.
(380) Forced Degradation
(381) Stress Conditions
(382) TABLE-US-00008 TABLE 8 Stress Conditions and Sampling time points Degradation type Stress Solvent/Conditions Time Point Acid Hydrolysis 1N HCl at RT 0 h, 1 d, 2 d, 3 d Basic Hydrolysis 0.001N NaOH at RT 0 h, 1 h Oxidation 3% Hydrogen Peroxide at RT 0 h, 8 h Photolysis 1.10 W/m.sup.2/25° C. for solid 13 h, 26 h, 39 h (1.10 W/m.sup.2/25° C. with 13 hours equal to visible 1.2M lux hrs and UV 647 Wh/m.sup.2) Thermal 80° C. for solid 1 d, 3 d, 7 d 80° C. for solution 1 d, 7 d Humidity 92% RH 1 d, 3 d, 7 d Solution pH = 4.0, 50 mM Sodium 0 h, 1 d, 3 d, 7 d Stability citrate buffer at RT pH = 5.0, 50 mM Sodium 0 h, 1 d, 3 d, 7 d citrate buffer at RT pH = 7.0 Phosphate buffer at RT 0 h, 1 d, 3 d, 7 d Notes: 1. The target endpoint of a stress study was to form approximately 5-15% of total degradation product. 2. Based on actual degradation of the sample, the stress conditions including concentration of sample and reagent, and temperature, humidity, light may be adjusted. 3. After stressing samples in acid and base, neutralize them before placing into freezer. 4. All degradable samples before analysis must be placed into the 2° C. to 8° C. condition.
(383) Acid degradation (1N HCl at RT). 3 mL of the sample stock solution (see above) were transferred into an 8 mL vial, 3 mL of 2N HCl solution were added, and the resulting mixture was mixed well. Samples were prepared in quadruplicate and kept at RT. At the sampling point, 2 mL of the sample were transferred into an 8 mL vial and neutralized with 1 mL of 2N NaOH.
(384) Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound D1.
(385) Basic degradation (0.001N NaOH at RT). 3 mL of the sample stock solution (see above) were transferred into an 8 mL vial, 3 mL of 0.002N NaOH solution were added, and the resulting mixture was mixed well. Samples were prepared in triplicate and kept at RT. At the sampling point, 2 mL of the solution were transferred into an 8 mL vial and neutralized with 1 mL of 0.002N HCl.
(386) Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound D1.
(387) Oxidation degradation (3% H.sub.2O.sub.2 at RT). 3 mL of the sample stock solution (see above) were transferred into an 8 mL vial, 3 mL of 6% H.sub.2O.sub.2 solution were added, and the resulting mixture was mixed well. Samples were prepared in quadruplicate and kept at RT. At the sampling time point, 2 mL of the solution were transferred into an 8 mL vial, neutralized with 1 mL of diluent 3, and mixed well.
(388) Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound D1.
(389) Photolysis degradation (solid). About 60 mg of compound D1 were placed onto a watch glass. Samples were prepared in triplicate and placed in a photo chamber (see Table 8). At the sampling time point, the sample was placed in a vial for analysis. About 20.0±2.0 mg of the sample into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of diluent 3, diluted to volume with diluent 3, and mixed well.
(390) TABLE-US-00009 TABLE 9 Photolysis degradation of samples' mass weight Sampling Name* Weight (mg) PSD-13h 20.44 PSD-26h 19.93 PSD-39h 19.43 *PSD-13h, PSD-26h and PSD-39h in category mean the compound is degradation product under photolysis solid stress condition in 13 h, 26 h, and 39 h, respectively.
(391) Dark control sample: one sample was prepared as described above for this test, but the watch glass was covered with aluminum foil, and processed as described above.
(392) Thermal Stress Degradation (80° C. for Solid and Solution).
(393) Solid: about 60 mg of compound D1 were weighted into an 8 mL vial. Samples were prepared in triplicate and placed in an oven at 80° C. At the sampling time point, about 20.0±2.0 mg of the sample were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of diluent 3, diluted to volume with diluent 3, and mixed well.
(394) TABLE-US-00010 TABLE 10 Thermal degradation of samples' mass weight Sample Name* Weight (mg) T-1d 19.82 T-3d 22.07 T-7d 21.68 *T-1d, T-3d and T-7d in category mean the compound is degradation product under thermal solid stress condition in 1 d, 3 d and 7 d, respectively.
(395) Solution: 3 mL of the sample stock solution (see above) were transferred into an 8 mL vial. Samples were prepared in triplicate and placed them in an oven at 80° C. At the sampling time point, 1 mL of the solution was transferred into an 8 mL vial, neutralized with 2 mL of diluent 3, and mixed well.
(396) Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound D1.
(397) Humidity degradation (92% RH for solid). About 60 mg of compound D1 were transferred into a 20 mL vial (without cap). Samples were prepared in triplicate and placed in a desiccator at 92% RH. At the sampling time point, about 20.0±2.0 mg of the sample were transferred into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of diluent 3, diluted to volume with diluent 3, and mixed well.
(398) TABLE-US-00011 TABLE 11 Humidity degradation of samples' mass weight Sample Name* Weight (mg) H-1d 21.02 H-3d 21.41 H-7d 20.11 *H-1d, H-3d and H-7d in category mean the compound is degradation product under humidity solid stress condition in 1 d, 3 d and 7 d, respectively.
(399) Solution Stability. Samples were prepared tested under the solution stability conditions noted in Table 8. The sampling time points were as noted in Table 8.
(400) Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound D1.
(401) Sample Analysis
(402) TABLE-US-00012 TABLE 12 HPLC Method 1 Mobile Phase A H.sub.2O + 0.1% TFA Mobile Phase B MeCN + 0.1% TFA Column Poroshell 120 SB-AQ (3.0*150 mm, 2.7 μm) Needle Wash MeCN:H.sub.2O = 1:1 Injection volume 3 μL Column Temperature 40° C. Flow Rate 0.8 mL/min Detection 210 nm Time (min) % A % B Gradient 0.0 98 2 7.0 70 30 12.0 50 40 15.0 5 95 17.0 5 95 Post time 5 min Run time 17 min
(403) TABLE-US-00013 TABLE 13 Summary Degradation Stress Solvent/ Sample Sample Area % % Mass Type Conditions Name Number Time % Degradation Balance Unstressed N/A N/A 0 N/A 94.172 N/A N/A Acid 1N HCl RT A 0 1 0 h 93.734 0.438 108.2 Hydrolysis A 1 2 1 d 89.391 4.781 106.5 A 2 3 2 d 85.180 8.992 107.6 A 3 4 3 d 82.047 12.125 106.9 Base 1N NaOH RT B 0 5 0 h 68.962 25.210 104.0 Hydrolysis B 1 6 1 h 17.331 76.841 98.0 Oxidation 3% H.sub.2O.sub.2 RT O 0 7 0 h 77.988 16.184 106.8 O 8 8 8 h 33.755 60.417 105.2 Photolysis 1.10 W/m.sup.2/25° C. P 1 9 13 h 91.084 3.088 101.0 for solid with P 2 10 26 h 90.916 3.256 92.1 13 hours equal P 3 11 39 h 90.736 3.436 105.7 to visible 1.2 M lux hrs and UV 647 Wh/m.sup.2 Thermal 80° C. Solid TSD 1 12 1 d 93.448 0.724 105.7 TSD 3 13 3 d 93.194 0.978 97.6 TSD 7 14 7 d 82.192 11.980 92.7 80° C. Solution TSN 1 15 1 d 94.053 0.119 100.5 TSN 7 16 7 d 31.937 62.235 104.2 Humidity 92% RH H 1 17 1 d 92.927 1.245 98.8 H 3 18 3 d 92.081 2.091 99.5 H 7 19 7 d 90.890 3.282 99.8 Solution pH = 4.0, 50 mM pH4 0 20 0 h 93.516 0.656 90.1 Stability Sodium citrate pH4 1 21 1 d 93.331 0.841 93.7 buffer at RT pH4 3 22 3 d 92.873 1.299 93.9 pH4 7 23 7 d 92.130 2.042 93.7 pH = 5.0 50 mM pH5 0 24 0 h 92.498 1.674 95.3 Sodium citrate pH5 1 25 1 d 92.250 1.922 103.1 buffer at RT pH5 3 26 3 d 92.039 2.133 100.0 pH5 7 27 7 d 89.514 4.658 100.8 pH = 7.0 pH7 0 28 0 h 92.638 1.534 97.1 Phosphate pH7 1 29 1 d 85.252 8.920 102.6 buffer at RT pH7 2 30 2 d 79.002 15.170 100.6
(404) TABLE-US-00014 TABLE 14 Summary result of mass balance, resolution and purity factor Mass Balance *Resolution Purity Factor Sample Purity Pass/ Pass/ Pass/ Number % Result Criteria Fail Result Criteria Fail Result Criteria Fail 4 82.047 106.9 90%- Pass 2.0 ≥1.2 Pass 999.943 >990 Pass 6 17.331 98.0 110% 1.8 996.409 8 33.755 105.2 1.0 Fail 998.510 11 90.736 105.7 1.8 Pass 999.780 14 82.192 92.7 1.8 999.941 16 31.937 104.2 1.8 999.708 19 90.890 99.8 1.9 999.438 23 92.130 93.7 2.0 999.830 27 89.514 100.8 2.0 999.639 30 79.002 100.6 2.0 998.160 *Resolution was the adjacent impurity with the main peak. According to the result above, samples 6 and 8 were used to method optimized.
Example 16—Forced Degradation Study of Compound S-D1
(405) Solutions Preparation
(406) Mobile phase: methanol (MeOH): acetonitrile (MeCN)=3:7 (v/v)+25 mM formic acid (FA)+25 mM NH.sub.3. Prepared by transferring 300 mL MeOH, 700 mL MeCN, 970 μL FA, and 12.5 mL Ammonia in to 1 L bottle, mixed well.
(407) Needle wash solution: MeOH
(408) Diluent 1: H.sub.2O
(409) Diluent 2: 0.05% FA in MeCN:THF=1:1 (v/v)
(410) 2N HCl was prepared by weighing 19709.02 mg concentrated aq. HCl (37%) into a 100 mL volumetric flask containing approximately 20 mL diluent 1, diluting to volume with diluent 1, and mixing well.
(411) 0.3N HCl was prepared by transferring 15 mL of the 2N HCl (see above) into a 100 mL volumetric flask containing approximately 20 mL diluent 1, diluting to volume with diluent 1, and mixing well.
(412) 2N NaOH was prepared by weighing 7996.77 mg NaOH into a 100 mL volumetric flask, made up to volume with diluent 1 and mixed well.
(413) 0.3N NaOH was prepared by transferring 15 mL of the 2N NaOH (see above) into a 100 mL volumetric flask containing approximately 5 mL diluent 1, diluting to volume with diluent 1, and mixing well.
(414) 0.1N NaOH was prepared by transferring 5 mL of the 2N NaOH (see above) into a 100 mL volumetric flask containing approximately 5 mL diluent 1, diluting to volume with diluent 1, and mixing well.
(415) 1N Citric acid was prepared by weighing 19193.47 mg citric acid into a 100 mL volumetric flask, diluting to volume with diluent 1, and mixing well.
(416) pH=4.0 50 mM Sodium citrate buffer was prepared by weighing 1474.25 mg sodium citrate into a 100 mL volumetric flask, diluting to volume with diluent 1, and mixing well. pH was adjusted pH to 4.0 by 1N citric acid.
(417) pH=5.0 50 mM Sodium citrate buffer was prepared by weighing 1474.11 mg sodium citrate into a 100 mL volumetric flask, diluting to volume with diluent 1, and mixing well. pH was adjusted to 5.0 by 1N citric acid.
(418) Sample solution stock was prepared by weighing 501.53 mg of compound S-D1 to a 100 mL volumetric flask, dissolving by ultrasonic in 50 mL of diluent 2, diluting to volume with diluent 2, and mixing well.
(419) Unstressed sample solution was prepared by weighing 50.83 mg of compound S-D1 into a 100 mL volumetric flask, dissolving by ultrasonic in 50 mL of diluent 2, diluting to volume with diluent 2, and mixing well.
(420) Sensitivity solution (LOQ) was prepared as follows. 1.0 mL of unstressed sample solution were pipetted into a 100 mL volumetric flask, diluted to volume with diluent 2, and mixed well. Then 2 mL of this sample solution were pipetted into a 10 mL volumetric flask, diluted to volume with diluent 2, and mixed well.
(421) Sample solution for pH=4.0 solution stability was prepared as follows. 50.79 mg of compound S-D1 were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of pH=4.0 50 mM sodium citrate buffer (see above), diluted to volume with pH=4.0 50 mM sodium citrate buffer, and mixed well.
(422) Sample solution for pH=5.0 solution stability was prepared as follows. 50.66 mg of the compound of Formula I were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of pH=5.0 50 mM sodium citrate buffer (see above), diluted to volume with 50 mL of pH=5.0 50 mM sodium citrate buffer and mixed well.
(423) Sample solution for pH=7.0 solution stability was prepared as follows. 50.78 mg of compound S-D1 and 680.42 mg KH.sub.2PO.sub.4 were weighed into a 100 mL volumetric flask, dissolved by ultrasonic in 50 mL of diluent 1, transferred 29.1 mL of the 0.1N NaOH (see above), diluted to volume with diluent 1, and mixed well.
(424) TABLE-US-00015 TABLE 15 Stress Conditions and Sampling Time Points Sampling Degradation type Stress Solutions/Conditions Time Point Acid Hydrolysis 1N HCl at RT 0 h, 1 d, 2 d, 3 d Basic Hydrolysis 0.15N NaOH at RT 0 h, 1 h Thermal 50° C. for solid 1 d, 3 d, 7 d Solution Stability pH = 4.0, 50 mM Sodium 0 h, 1 d, 2 d, 3 d citrate buffer at RT pH = 5.0, 50 mM Sodium 0 h, 1 d, 2 d, 3 d citrate buffer at RT pH = 7.0 Phosphate buffer at RT 0 h, 1 d, 2 d, 3 d Note: After stressing samples in acid or base, the samples were neutralized before placing into freezer.
(425) Acid degradation (1N HCl at RT). 3 mL of the sample stock solution (see above) into an 8 mL vial, added 3 mL of 2N HCl solution, and mixed well. Samples were prepared in quadruplicate and kept at room temperature (RT). At the sampling time point, 1 mL of the sample was transferred into a 5 mL volumetric flask, neutralized with 0.5 mL of 2N NaOH, diluted to volume with diluent 2, and mixed well.
(426) Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound S-D1.
(427) Basic degradation (0.15N NaOH at RT). 3 mL of the sample stock solution (see above) into an 8 mL vial, added 3 mL of 0.3N NaOH solution, and mixed well. Samples were prepared in quadruplicate and kept at room temperature (RT). At the sampling time point, 1 mL of the sample was transferred into a 5 mL volumetric flask, neutralized with 0.5 mL of 2N NaOH, diluted to volume with diluent 2, and mixed well.
(428) Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of compound S-D1.
(429) Thermal stress degradation (50° C. for solid). Approximately 150 mg of compound S-D1 were weighed into an 8 mL vial. Multiple samples were prepared and placed in an oven at 50° C. At the sampling time points, each of the 50.57 mg, 50.84 mg, and 50.84 mg samples were transferred into their respective 100 mL volumetric flasks, dissolved by ultrasonic in 50 mL of diluent 2, diluted to volume with diluent 2, and mixed well.
(430) Solution Stability. The study was performed according to the conditions outlined in Table 15 using sample solution for pH=4.0 solution stability, sample solution for pH=5.0 solution stability, and sample solution for pH=7.0 solution stability (see above).
(431) Blank: a blank has been prepared following the same procedure as described above, only without the inclusion of sample.
(432) Sample Analysis
(433) TABLE-US-00016 TABLE 16 HPLC Method Mobile Phase MeOH:MeCN = 3:7 (v/v) + 25 mM FA + 25 mM NH.sub.3 Column Cellulose SB, 100*4.6 mm, 3.0 μm Needle Wash MeOH Injection volume 1 μL Column Temperature 30° C. Flow Rate 0.8 mL/min Diluent 0.05% FA in MeCN:THF = 1:1 (v/v) Autosampler Temperature RT Detection 280 nm Time (minute) Mobile Phase (%) Isocratic 0.0 100 8.0 100 Data Record Time 8 minutes
(434) TABLE-US-00017 TABLE 17 Summary Degra- Stress Area Area % dation Solvent/ Sample % % Degra- Type Conditions Time Number (R-D1) (S-D1) dation Un- N/A N/A 0 0.823 99.177 N/A stressed Acid 1N HCl 0 h 1 0.825 99.176 0.002 Hydrolysis RT 1 d 2 0.828 99.172 0.005 2 d 3 0.834 99.166 0.011 3 d 4 0.839 99.161 0.016 Base 0.15 N 0 h 5 19.083 80.917 18.260 Hydrolysis NaOH 1 h 6 53.033 46.967 52.210 RT Thermal 50° C. 1 d 7 1.001 98.999 0.178 3 d 8 1.154 98.847 0.331 7 d 9 1.203 98.797 0.380 Solution pH = 4.0, 0 h 10 0.921 99.079 0.098 Stability 50 mM 1 d 11 0.962 99.038 0.139 Sodium 2 d 12 1.124 98.876 0.301 citrate 3 d 13 1.305 98.695 0.482 buffer at RT pH = 5.0 0 h 14 1.471 98.529 0.648 50 mM 1 d 15 1.861 98.139 1.038 Sodium 2 d 16 2.230 97.770 1.407 citrate 3 d 17 2.825 97.175 2.002 buffer at RT pH = 7.0 0 h 18 11.555 88.445 10.732 Phosphate 1 d 19 19.745 80.255 18.922 buffer at 2 d 20 28.374 71.626 27.551 RT 3 d 21 35.926 64.074 35.103
(435) TABLE-US-00018 TABLE 18 Summary result of resolution Resolution between Resolution between compound R-D1 compound S-D1 peak and its peak and its adjacent peak adjacent peak Purity Factor Sample Pass/ Pass/ Pass/ Number Result Criteria Fail Result Criteria Fail Result Criteria Fail 4 1.3 ≥1.2 Pass 2.0 ≥1.2 Pass 999.917 >990 Pass 6 1.8 4.4 996.140 9 1.8 2.3 999.152 13 1.7 3.7 999.947 17 1.9 4.1 999.616 21 2.1 3.9 999.728
Example 17—Stability Studies of the Intravenous Formulations of the Compound of Formula I
(436) The data obtained in this study indicate that superior stability may be obtained at lower temperature 2-5° C. vs 2500 vs 4000, lower pH ˜5 vs ˜6, and higher concentration of sulfobutylether-β-cyclodextrin (SBECD) 20% vs 10% vs 0%. Samples stored at 2-5° C., pH ˜5 containing 20% SBECD showed the lowest amount of degradation. SBECD percentages in this Example are (w/w).
(437) HPLC Method. Key method parameters are listed in Table 19.
(438) TABLE-US-00019 TABLE 19 Key Method Parameters HPLC Column Waters RP Shield C18, 4.6 × 250 mm, 5 mm % A 10 mM ammonium bicarbonate, Time adjusted to pH 6.5 % B (min) with formic acid Acetonitrile Mobile Phase 0 90 10 25 20 80 30 20 80 30.1 90 10 35 90 10 Column Temp. 40° C. Autosampler Room Temperature Temp. Flow Rate 1.0 mL/min Injection Volume 5 μL Detection UV 254 nm Run Time 35 min Target Conc. 0.5 mg/mL Standard Prep Dissolved in H.sub.2O (1 drop of 0.1N HCl added to solubilize if needed) Sample Prep Dilute 20 mg/mL solution 40 folds in DI water to obtain 0.5 mg/mL concentration The chromatogram of the racemate of the free base of the compound of Formula I is described below. The retention time of the parent compound peak is approximately 18 min (Peak #11 below). The two major degradant peaks have retention times of 12.2 and 12.4 min (RRT's 0.67 and 0.68, respectively). The HPLC purity of the racemate of the free base of the compound of Formula I as provided was found to 97.1 % Peak # RT RRT Area % Typical 1 12.172 0.67 0.02 Chromatograms 2 12.407 0.69 0.04 3 13.545 0.75 0.67 4 14.391 0.80 0.19 5 14.558 0.81 0.44 6 14.784 0.82 0.25 7 15.6 0.86 0.17 8 15.758 0.87 1.05 9 16.32 0.90 0.07 10 16.904 0.94 0.04 11 18.066 1.00 97.06 Total 100
Stability Studies
(439) Summary of Experiments. It was found that complete dissolution of the racemate of the free base of the compound of Formula I may be achieved at pH 3 (e.g., by lowering the pH with a hydrochloric acid solution). The pH of the compounding solution would later be adjusted by sodium hydroxide solution to the target pH. It was found that formulations containing 0% or 10% SBECD showed diminished solubility at both pH 5 and pH 6 compared (less than 20 mg/mL). Only samples containing 20% SBECD (samples S-1.1 and S-2.1) were fully soluble at the desired pH. While the free-base of the compound of Formula I had high solubility at pH 3 in all formulations, significant precipitation was observed upon adjusting the pH when the formulation contained <20% SBECD. Formulations containing 0% SBECD or 10% SBECD showed significant decomposition at both pH 5 and pH 6 over 1 week ranging from ˜10-43% at 40° C. In general, decomposition was found to be lower at lower temperatures.
(440) Sample Preparation. To a 20 mL scintillation vial was added 60 mg of the racemate of the free base of the compound of Formula I and a magnetic stir bar. 3 mL of formulation solution (either 0%, 10% or 20% w/w SBECD in H.sub.2O) was added. The pH of the mixture was adjusted to approximately pH 3 by the addition of 1N HCl. Upon full dissolution (with magnetic stirring, sonication if necessary), the IV formulation was adjusted to the final pH by the addition of 1N NaOH solution. The IV formulation was filtered through a syringe filter, separated into 3 samples of approximately equal volume, and the samples were transferred to a stability chamber at 2-5° C., 25° C., or 40° C. At the specified time point the sample was removed from the stability chamber, an aliquot of the IV formulation was removed and diluted to 0.5 mg/mL with H.sub.2O for HPLC analysis.
(441) Stability Data. IV formulations of free-base compound D1 were prepared at different concentrations of SBECD and different pH levels as shown in Table 20.
(442) TABLE-US-00020 TABLE 20 Sample ID API pH SBECD w/w C-1 20 mg/mL 5.23 — S-1.1 20 mg/mL 5.04 20% S-1.2 20 mg/mL 5.33 10% C-2 20 mg/mL 6.33 — S-2.1 20 mg/mL 6.19 20% S-2.2 20 mg/mL 6.40 10%
(443) As noted above, significant precipitation of compound D1 occurred upon adjusting the IV formulation to the final pH level of 5 or 6 for IV formulations containing <20% SBECD. IV formulations containing %20 SBECD showed no precipitation. IV formulations were filtered prior to conducting the stability studies.
(444) The stability data (in Area % of main API HPLC peak relative to total Area %) at 2-5° C., 25° C., and 40° C. are included in Table 21.
(445) TABLE-US-00021 TABLE 21 Area % 2-5° C. 25° C. 40° C. Area Area Area % of % of % of t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API t = 0 h h week lost h h week lost h h week lost C-1 97.06 69.33 69.01 52.04 45.02 87.23 74.64 65.71 31.35 83.28 83.10 80.88 16.18 S-1.1 96.05 95.94 95.93 1.14 95.44 95.16 94.93 2.13 93.72 94.32 93.46 3.60 S-1.2 92.52 92.28 92.21 4.85 91.17 90.14 91.03 6.03 88.98 88.85 85.17 11.89 C-2 79.02 78.822 63.91 33.15 86.99 85.85 75.73 21.33 81.01 80.79 77.04 20.02 S-2.1 95.45 95.27 95.50 1.56 94.27 93.90 93.30 3.76 92.73 92.43 88.69 8.37 S-2.2 89.52 89.19 87.04 10.02 83.05 78.58 68.16 28.90 71.26 64.91 54.85 42.21
(446) The stability data (in Area % of main API HPLC peak relative to Area % of major degradants only) at 2-5° C., 25° C., and 40° C. are included in Table 22.
(447) TABLE-US-00022 TABLE 22 Area % 2-5° C. 25° C. 40° C. Area Area Area % of % of % of Peak t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API C-1 # RRT h h week lost h h week lost h h week lost 1 0.67 0.54 2.37 * — 0.81 3.35 3.93 11.38 1.92 2.92 4.09 12.17 2 0.68 1.25 5.89 * 1.79 7.85 7.46 3.98 6.95 8.08 3 1.00 98.21 91.74 * 97.41 88.81 88.62 94.09 90.13 87.83 Total 100 100 100 100 100 100 100 100 Area Area Area % of % of % of Peak t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API S-1.1 # RRT h h week lost h h week lost h h week lost 1 0.67 0.10 0.10 0.10 <0.5% 0.14 0.23 0.30 0.95 0.35 0.46 1.06 3.31 2 0.68 0.23 0.31 0.22 0.27 0.59 0.65 0.7 91.15 2.25 3 1.00 99.67 99.59 99.68 99.59 99.19 99.05 98.87 98.39 96.69 Total 100 100 100 100 100 100 100 100 100 Area Area Area % of % of % of Peak t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API S-1.2 # RRT h h week lost h h week lost h h week lost 1 0.67 0.18 0.28 0.23 0.85 0.38 0.56 1.08 3.73 0.94 1.56 3.09 9.94 2 0.68 0.47 0.80 0.62 0.90 1.60 2.65 2.22 4.07 6.85 3 1.00 99.35 98.92 99.15 98.72 97.84 96.27 96.84 94.37 90.06 Total 100 100 100 100 100 100 100 100 100 Area Area Area % of % of % of Peak t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API C-2 # RRT h h week lost h h week lost h h week lost 1 0.67 1.80 1.258 * — 2.18 2.09 5.06 15.08 3.49 4.26 6.19 18.32 2 0.68 4.04 3.223 * 4.85 5.26 10.01 7.25 10.40 12.13 3 1.00 94.17 95.52 * 92.98 92.65 84.93 89.26 85.35 81.69 Total 100 100 100 100 100 100 100 100 Area Area Area % of % of % of Peak t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API S-2.1 # RRT h h week lost h h week lost h h week lost 1 0.67 0.16 0.25 0.20 0.70 0.27 0.45 0.81 2.88 0.63 1.11 2.34 7.78 2 0.68 0.36 0.64 0.50 0.65 1.29 2.07 1.47 3.02 5.44 3 1.00 99.49 99.11 99.30 99.07 98.27 97.12 97.90 95.88 92.22 Total 100 100 100 100 100 100 100 100 100 Area Area Area % of % of % of Peak t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API t = 24 t = 48 t = 1 API S-2.2 # RRT h h week lost h h week lost h h week lost 1 0.67 0.99 1.51 2.04 8.02 2.92 4.40 8.22 28.98 7.16 8.73 13.18 43.55 2 0.68 2.61 4.52 5.98 7.35 12.67 20.77 16.75 23.08 30.36 3 1.00 96.40 93.97 91.98 89.74 82.93 71.02 76.09 68.19 56.46 Total 100 100 100 100 100 100 100 100 100 *Sample for analysis contained minimal API due to precipitation of sample overtime
(448) In the data table above only the Area % values for the API and the major degradation products (RRT's 0.67 and 0.68) are provided. It is important to note that the initial ˜3% impurity in the initially provided sample of free-base compound D1 is present in the sample throughout the stability studies and in some cases (particularly at 40° C.) these initial impurities are further degraded to a variety of new peaks with ˜3% combined area in overall intensity.
(449) As an example, the HPLC trace of IV formulation S-1.1 after 1 week at 40° C. is included below. With consideration to only the 2 major degradants (RRT's 0.67 and 0.68) the Area % of the API in the sample is approximately 96.7% (as reported in the full data table above). With consideration of the additional impurities present in the initial sample the Area % of the API in the sample is approximately 93.5%
(450) In Vitro Dissolution Test of IV Formulation. The in vitro dissolution test method on IV formulation samples has been completed in accordance with the method as provided by Foghorn. IV formulation samples were prepared as above. Both IV formulations C-1 and C-2 showed initial precipitation upon dilution of the IV formulation with diluent (step 1 in procedure provided, left-most vial in pictures below) but eventually became fully homogenous with full dilution (step 4 in procedure provided; right-most vial in pictures below). All other IV formulations did not show any precipitate throughout the test or become cloudy.
Example 18—Lyophilized Formulations of the Compound of Formula I
(451) General Procedure
(452) 1) An appropriately sized beaker and stir bar was obtained for drug product formulation.
(453) 2) An 80% charge of WFI was dispensed into the formulation beaker, which was then placed on a stir plate and mixing was initiated.
(454) 3) A 20 mM citrate buffer at pH 4.5 was compounded by adding citric acid, monohydrate and sodium citrate, dihydrate at proper concentrations and dissolving them.
(455) 4) The required amount of SBECD (Captisol) was added to the formulation vessel, and mixed until fully dissolved.
(456) 5) The formulation vessel, DS, and magnetic stir plate was moved inside an isolator.
(457) 6) The required amount of compound S-D1 was measured, added to the formulation vessel, and mixed until fully dissolved. The solution and stir bar were then removed from the isolator.
(458) 7) The pH of the solution was determined, and adjusted to 4.5±0.1, if necessary.
(459) 8) The solution was then QSed with WFI using an appropriately sized volumetric flask or graduated cylinder. The solution was then dispensed back into the vessel and mixed for an additional five minutes.
(460) 9) The solution was then filtered with a 0.22 μm PES/PVDF filter.
(461) 10) A sample post-filter for density was removed. The fill weight and fill range were calculated based on a 5 mL fill volume.
(462) 11) A 3.2 mm tubing set was installed onto a peristaltic pump, and the pump was set to dispense the required mass per vial of product. Three consecutive passing weight checks were performed before continuing.
(463) 12) The filtered solution was dosed into vials and a fill check performed every 50 vials.
(464) 13) Once filling was complete, the vials were stoppered with 20 mm stoppers in the lyophilization position. Thermocouples were added to vials as necessary.
(465) 14) Vials were unloaded onto a lyophilizer shelf. The lyophilizer door was then sealed and the cycle initiated.
(466) 15) Once the cycle was complete, vials were backfilled with filtered nitrogen to 600,000 mTorr and stoppered. Once stoppered, the vacuum was broken to atmospheric pressure the vials were removed from the lyophilizer.
(467) 16) Vials were capped with 20 mm caps and visually inspected for any defects.
(468) 17) Acceptable finished product samples were then sampled for finished product testing, including appearance, reconstitution time, pH, osmolality, residual moisture, assay/related substances, X-Ray diffraction, and particulate matter. 18) Vials were then packaged and stored at 2-8° C.
(469) Experiment 1
(470) The pH screening study formulations are summarized in Table 23.
(471) TABLE-US-00023 TABLE 23 Formulation ID R1 R2 R3 R4 Buffer N/A Citrate Citrate Citrate 20 mM 20 mM 20 mM SBECD 200 mg/mL 200 mg/mL 200 mg/mL 200 mg/mL pH 4.0 4.0 4.5 4.5 Compound 5 mg/mL 5 mg/mL 5 mg/mL 10 mg/mL S-D1
(472) Formulation, Filtration, and Filling. The formulation was performed as described in the General Procedure section above. Excipients were added based on their proposed concentration for each sublot. The density of each sublot was taken to determine the fill volume on a mass basis, and a sample from Sublot R1 was taken for thermal analysis (see Section 13.0). Each solution was then filtered with a 0.22 μm PVDF Stericup filter into a HEPA-filtered hood. Filtration was slow, taking approximately 15 to 20 minutes for each sublot. Each sublot was filled into respectively labelled 20R vials at 5 mL/vial for sublots R1 to R3 while sublot R4 was filled at 2.5 mL per vial. Thermocouples were inserted into a vial in each sublot and subsequently loaded into the lyophilizer. The product was lyophilized using a conservative cycle to ensure lyophilized product quality.
(473) Lyophilization. Vials were loaded onto a shelf controlled at 5.0° C. Once thermocouples were inserted, a slight vacuum was applied to the system to seal the door from the external environment. The loading temperature was held at 5.0° C. for 30 minutes to allow all vials to reach equilibrium. The shelf temperature was ramped to −45.0° C. over 150 minutes and held for 180 minutes to ensure all vials were thoroughly frozen. The lyophilizer condenser was then chilled, and vacuum was pulled to 50 mTorr. Once the vacuum set-point was achieved, the shelf temperature was ramped to −20.0° C. over 50 minutes and held constant for 4,350 minutes. Completion of primary drying was indicated by the convergence of the Pirani gauge with the capacitance manometer, as shown in
(474) TABLE-US-00024 TABLE 24 Step # Operation Temperature (° C.) 1 Load Set Point 5.0 Door Seal Step # Operation Pressure (mTorr) 2 Seal Door ~550,000 Thermal Treatmen Shelf Duration Step # Operation Temperature (° C.) (min) 3 Hold 5.0 30 4 Ramp −45.0 150 5 Hold −45.0 180 Shelf Vacuum Duration Step # Operation Temperature (° C.) (mTorr) (min) Primary Drying 10 Hold −45.0 50 30 11 Ramp −20.0 50 50 12 Hold −20.0 50 4,350 Secondary Drying 13 Ramp 25.0 50 180 14 Hold 25.0 50 1,410 Shelf Step # Operation Temperature (° C.) Shut Down 15 Backfill with nitrogen 25.0 gas to 11.6 PSIA = ~600,000 mTorr 16 Stopper vials. 25.0 Unloading 17 Unload 20.0 Total Time 6,380 minutes (106.3 hrs).sup.1
(475) Packaging and Storage. Lyophilized vials were capped with flip-off 20 mm caps and inspected. Thermocouple vials were rejected. A total of 701 vials were acceptable (approximately 175 per sublot). Vials were sampled for t=0 testing, which included appearance, reconstitution time, pH, osmolality, residual moisture, assay/related substances, X-Ray diffraction, and particulate matter. Once samples were taken, each sublot was stored at 2-8° C. until a final sublot was chosen to be placed on accelerated stability.
(476) Lyophilization Cycle Analysis. Analysis of the run chart demonstrates that shelf temperature was controlled to within 1.4° C. of the set point during all stages of the cycle. The condenser was maintained at low temperatures throughout the drying portion, and vacuum was controlled within less than 1 mTorr of the set-point.
(477) In the lyophilization process, sublimation occurs when the vapor pressure of the frozen solvent at a certain temperature is higher than the pressure of the environment. The difference between the vapor pressure of ice at a corresponding temperature and the subsequent vacuum level of the product chamber is the driving force behind sublimation. The product temperature during primary drying is also dictated by the shelf temperature and vacuum of the shelf chamber; higher product temperatures result in a higher vapor pressure of ice, and will provide more efficient sublimation at the same vacuum level. Care must be taken, however, that the product temperature does not exceed a critical temperature (glass transition temperature, eutectic melt, etc.) that will result in a collapsed product.
(478) Product temperatures were maintained below −24° C. during primary drying when the shelf temperature was held at −20.0° C. As the critical temperatures for each sublot were observed to be very low (see DSC and FDM Results, Section 11.0), the lyophilizer setpoints remained conservative throughout primary drying.
(479) Product temperatures were observed to converge with the shelf temperature at approximately the same time (except for Sublot R1) compared to the convergence of the Pirani vacuum gauge and the capacitance manometer. The vacuum gauge convergence is considered in the industry as the principal method for determining the end-point of primary drying. The difference in conversion times between vacuum gauges and thermocouples can be attributed to faster drying rates observed within vials containing thermocouple probes and the location of the probe within the cake. Therefore, for the purposes of future cycle development, the convergence of the vacuum gauges was used as the primary indicator for the completion of primary drying, and product temperature convergence acted as a secondary confirmation (
(480) When the cycle was advanced to secondary drying, an increase in the Pirani gauge reading was observed. This indicated additional moisture being removed from vials as bound water via desorption. This is consistent with amorphous products, which tend to bind or trap water molecules. Product temperatures followed closely with the shelf temperature through the ramp to secondary drying and throughout the secondary drying hold. This is consistent with a dried cake with no residual bound or un-bound water.
(481) Finished Product Testing Results. Acceptable finished product vials were analyzed by LSNE for t=0 finished product testing. Vials for HPLC and particulate matter were performed by LSNE QC Analytical, and X-Ray diffraction was performed at a third-party lab. The HPLC method was in development at the time this report was written; THE and 0.9% NaCl were used for dilution due to the method still being under development. The t=0 finished product results are shown in Tables 26-28.
(482) TABLE-US-00025 TABLE 26 Sublots Test Specification R1 R2 R3 R4 Appearance (Lyophilized) Report Acceptable Acceptable Acceptable Acceptable Appearance (Reconstituted) Report Acceptable Acceptable Acceptable Acceptable Reconstitution Time Report 34 sec 39 sec 38 sec 24 sec pH Report 4.15 3.94 4.43 4.44 Osmolality Report 576 600 606 448 Residual Moisture via KF NMT 3.0% 0.527% 0.557% 0.533% 0.469% Note: all vials were tested in duplicate - the average between the two runs is reported. Diluent used for reconstitution was 0.9% NaCl.
(483) TABLE-US-00026 TABLE 27 Sublots R2 R3 R4 R1 0.9% 0.9% 0.9% Test Specification THF THF NaCl THF NaCl THF NaCl Assay 93%-110% or 2.30 18.01 21.60 19.01 21.62 21.51 24.22 (mg/mL) 23.25 mg/mL- 27.5 mg/mL Related Purity-Report 25.71 96.96 97.69 97.69 97.79 97.44 97.72 Substances RRT 0.65 55.87 0.42 0.31 0.28 0.24 0.37 0.31 RRT 0.83 18.42 1.95 1.33 1.28 1.22 1.80 1.69 RRT 0.89 ND ND ND 0.09 0.08 ND ND RRT 1.23 ND 0.42 0.41 0.42 0.41 0.39 0.28 RRT 1.69 ND 0.24 0.26 0.25 0.27 ND ND Particulate NMT 6 K 284 357 397 401 Matter Particles ≥ 10 μm NMT 600 15 19 53 28 Particles ≥ 25 μm Note: all vials were tested in duplicate-the average between the two runs is reported. The HPLC method was performed twice with THF and 0.9% NaCl as the diluents. ND = Not Detected.
(484) TABLE-US-00027 TABLE 28 Sublots Test Specification R1 R2 R3 R4 Assay (mg/mL) 90%-110% or 11.3 24.0 24.6 24.1 23.25 mg/mL-27.5 mg/mL Note: all vials were tested in duplicate - the average between the two runs is reported. ND = Not Detected.
Critical Temperature Determination
(485) Differential Scanning Calorimetry. Each sample was tested by DSC to determine if any glass transition temperatures (Tg′), eutectic melt temperatures (Teu), or re-crystallization temperatures (Tcry) were present. Standard 10° C./min and 2° C./min heat cycles were performed. The results from DSC analysis are described in Table 29, and a representative figure of a thermogram is described in
(486) TABLE-US-00028 TABLE 29 Sublot R3 Tcry Tmelt Tnuc (° C.) Tg (° C.) (° C.) (° C.) Method 1, Run 1 (10° C./min) −21.4 −27.41 N/A −6.06 Method 1, Run 2 (10° C./min) −19.66 −27.49 N/A −5.61 Method 2, Run 1 (2° C./min) −23.16 −28.95 N/A −5.47 Method 2, Run 2 (2° C./min) −20.30 −29.08 N/A −5.27 Average: −21.13 −28.23 N/A −5.60
(487) DSC testing consisted of running two different methods. The methods consisted of standard freeze/heat DSC cycles, employing a heating rate of 10° C./min and 2° C./min, respectively. The higher heating rate method (Method 1, 10° C./min) is used to increase the amplitude of critical temperatures at the expense of temperature accuracy, while the lower ramp rate (Method 2) is used to define critical temperatures more accurately at the expense of the larger amplitude.
(488) Freeze-Drying Microscopy. A sample was retained and thermally characterized via freeze-drying microscopy (FDM). Additionally, an annealing FDM cycle was run to determine the effect of annealing on the collapse temperature. The annealing temperature was set above the glass transition temperature to drive crystallization during annealing. The results are described in Table 30, and a representative photo of the FDM process is shown in
(489) TABLE-US-00029 TABLE 30 Collapse Onset Average Collapse Total Collapse Method Run # (° C.) Onset (° C.) (° C.) 1 1 −23.7 −23.6 −22.6 2 −24.2 −20.1 3 −22.9 −21.6 2 1 −24.0 −24.0 −21.1
Low Robustness
(490) Formulation, Filtration. Filling. The formulation was performed as described in the General Procedure above, and a total batch size of 1,000 mL was compounded. An excipient solution was generated using the proposed concentrations. The solution was pH adjusted with 1N HCl to a post-QS pH of 4.52 and a 5 mL sample was taken for density in order to determine the fill volume on a mass basis. A pre and post filtration sample was taken for HPLC analysis; results can be seen in Table 32. Filtration was performed using a PES filter into the HEPA-filtered hood. Flow visibly slowed overtime. Upon completion of filtration it was noted that the filter membrane appeared yellow. One full tray of 20R vials were assembled, and the formulation was filled to a weight of 5.43 grams per vial. All fill weights were within specification. Thermocouples were inserted as required, and the trays were loaded into the lyophilizer. The cycle was initiated according to Table 31.
(491) Lyophilization. Vials were loaded onto a shelf controlled at 5.0° C. Once thermocouples were inserted, a slight vacuum was applied to the system to seal the door from the external environment. The loading temperature was held at 5.0° C. for 30 minutes to allow all vials to reach equilibrium. The shelf temperature was ramped to −45.0° C. over 100 minutes and held for 840 minutes to ensure all vials were thoroughly frozen. The shelves were then ramped to −50° C. over 10 minutes and held for an additional 60 minutes. The lyophilizer condenser was then chilled, and vacuum was pulled to 70 mTorr. Once the vacuum set-point was achieved (approximately 30 minutes), the shelf temperature was ramped to −20.0° C. over 60 minutes and held for 3,930 minutes. Completion of primary drying was indicated by the convergence of the Pirani gauge with the capacitance manometer. The vacuum gauges plateaued after approximately 3,600 minutes of primary drying time. Following completion of primary drying, the shelf temperature was ramped to 25.0° C. over 180 minutes and held for 1,450 minutes. Samples were taken over the course of secondary drying in order to map the residual moisture overtime (See Table 15). At the completion of secondary drying, the system was backfilled with nitrogen to ˜600,000 mTorr and vials were fully stoppered. The programmed lyophilization cycle is shown in Table 31.
(492) TABLE-US-00030 TABLE 31 Step # Operation Temperature (° C.) 1 Load Set Point 5.0 Door Seal Step # Operation Pressure (mTorr) 2 Seal Door ~550,000 Thermal Treatment Shelf Temperature Duration Step # Operation (° C.) (min) 3 Hold 5.0 30 4 Ramp −45.0 100 5 Hold −45.0 840 6 Ramp −50.0 10 7 Hold −50.0 60 Shelf Temperature Vacuum Duration Step # Operation (° C.) (mTorr) (min) Primary Drying 8 Hold −50.0 70 30 9 Ramp −20.0 70 60 10 Hold −20.0 70 3,930 Secondary Drying 11 Ramp 25.0 70 180 12 Hold 25.0 70 1,450 Shelf Step # Operation Temperature (° C.) Shut Down 13 Backfill with 25.0 nitrogen gas to ~600,000 mTorr 14 Stopper vials. 25.0 Unloading 15 Unload 25.0 Total Time 6,690 minutes (111.5 hrs).sup.1 .sup.1total cycle time does not account for the time required to reach vacuum or unloading.
(493) Lyophilization Cycle Analysis. During thermal treatment, the shelf temperature was lowered to −45° C. and held for several hours before being lowered to −50° C., which was held for 60 minutes. This ensures that the ramp rate matches the extended time it takes to get to the primary drying temperature. After thermal treatment of the product, run chart demonstrates that shelf temperature was controlled to within ±2° C. of the set point during all stages of the cycle. The condenser was maintained at low temperatures throughout the drying portion, and vacuum was controlled within ±2 mTorr of the set-point.
(494) Product temperatures were observed to reach a temperature of approximately −35.0° C. at the beginning of the primary drying hold. The product temperatures then increase until thermocouple break point at approximately 2,700 minutes of primary drying.
(495) The convergence of the Pirani vacuum gauge and the capacitance manometer occurred after the thermocouple gauge convergence. Vacuum gauge convergence for this cycle was allowed to fully complete to ensure that primary drying was completed since this cycle is used to set the primary drying time of the nominal cycle. Vacuum gauge convergence (±5 mTorr) occurred at 4,800 minutes from the start of the primary drying hold. The Pirani vacuum gradually dropped throughout primary drying. After convergence, the shelf temperature was ramped into secondary drying.
(496) When the cycle was advanced to secondary drying, an increase in the Pirani gauge reading was observed, indicating additional moisture being removed from vials as bound water via desorption. Product temperatures followed closely with the shelf temperature through the ramp to secondary drying and throughout the secondary drying hold. The secondary drying time was determined by leveraging the moisture content results from this run. Since the moisture results were passing after t=240 minutes of secondary drying, it was recommended that the secondary drying time be lengthened to further drive the moisture from 1.0% to 0.50%. At t=12 hours during secondary drying, this condition was met; the average residual moisture was 0.8216% as seen in Table 32 below.
(497) Intentional Collapse. Vials filled for the first cycle development study were subjected to an intentional collapse experiment to verify that product would not collapse, experience ablation or cake lift, and to detect any degradation of DS. Vials were loaded at 5.0° C., which was held for 5 minutes. The shelf was then ramped to −45.0° C. over 50 minutes and held for 30 minutes before a vacuum was applied at 250 mTorr. Once the vacuum was pulled, the shelf temperature was raised to 35.0° C. over 80 minutes and held for 795 minutes.
(498) The lyophilized cakes were dense, pale yellow cakes with no cracks and a porous surface with significant shrinkage; mild collapse was present. After reconstituting the cakes with 10 mL of 0.9% NaCl, the solution was a pale yellow, clear solution. Representative photos can be found in
(499) TABLE-US-00031 TABLE 32 t = 2 t = 4 t = 12 Pre- Post- Test Specification t = 0 Hours Hours Hours Filtration Filtration FP IC Appearance Conforms N/A Acceptable Porous (Lyophilized) surface with minor collapse. Appearance Conforms Acceptable Acceptable (Reconstituted) Reconstitution Report 41 23 Time Seconds Seconds (Average) pH (Average) Report 4.41 4.43 Osmolality Report 609 571 (Average) Residual In Process: 3.0180 1.5204 1.3091 0.8216 N/A 0.5580 1.6424 Moisture via Report KF (Average) Finished Product: NMT 3.0% Assay 90.0%-110.0% N/A 98.9 98.9 99.1 98.8 (Label Claim) Related RRT 0.543 0.63 0.60 0.56 0.56 Substances RRT 0.954 0.94 0.87 0.85 0.84 Particulate NMT 6 K N/A 772 667 Matter Particles ≥ 10 μm NMT 600 199 149 Particles ≥ 25 μm
Nominal Cycle and High Robustness
(500) Formulation, Filtration, Filling. The formulation was performed as described in the General Procedure above, and a total batch size of 2,000 mL was formulated to be split between both the nominal and high robustness cycles. All fill weight checks were within specification. Vials were stoppered in the lyophilization position, and thermocouples were inserted as needed.
(501) Lyophilization—Nominal Cycle. Vials were loaded onto a shelf controlled at 20.000. Once thermocouples were inserted, a slight vacuum was applied to the system to seal the door from the external environment. The loading temperature was held at 20.0° C. for 30 minutes to allow all vials to reach equilibrium. The shelf temperature was ramped to −45.0° C. over 130 minutes and held for 180 minutes to ensure all vials were thoroughly frozen. The lyophilizer condenser was then chilled, and vacuum was pulled to 95 mTorr. Once the vacuum set-point was achieved the shelf temperature was ramped to −15.0° C. over 60 minutes and held constant for 4,770 minutes. Completion of primary drying was indicated by the convergence of the Pirani gauge with the capacitance manometer. Because of the need to pull samples during secondary drying, primary drying was extended an additional 840 minutes. Since the Low Robustness cycle showed that primary drying completed at 3,930 minutes, it was acceptable to extend primary drying to extend the cycle. The vacuum gauges converged at approximately 2,400 minutes. Following completion of primary drying, the shelf temperature was ramped to 30.0° C. over 180 minutes and held for 720 minutes. At the completion of secondary drying, the system was backfilled with nitrogen to ˜600,000 mTorr and vials were fully stoppered. The programmed lyophilization cycle is shown in Table 33.
(502) TABLE-US-00032 TABLE 33 Step # Operation Temperature (° C.) 1 Load Set Point 20.0 Door Seal Step # Operation Pressure (mTorr) 2 Seal Door ~550,000 Thermal Treatmen Shelf Temperature Duration Step # Operation (° C.) (min) 3 Hold 20.0 30 4 Ramp −45.0 130 5 Hold −45.0 180 Shelf Temperature Vacuum Duration Step # Operation (° C.) (mTorr) (min) Primary Drying 6 Hold −45.0 95 30 7 Ramp −15.0 95 60 8 Hold −15.0 95 4,770 Secondary Drying 9 Ramp 30.0 95 180 10 Hold 30.0 95 720 Shelf Step # Operation Temperature (° C.) Shut Down 15 Backfill with 30.0 nitrogen gas to ~600,000 mTorr 16 Stopper vials. 30.0 Unloading 17 Unload 30.0 Total Time 6,100 minutes (101.7 hrs).sup.1 .sup.1total cycle time does not account for the time required to reach vacuum or unloading.
(503) Lyophilization Cycle Analysis—Nominal Cycle. Analysis of the run chart demonstrates that shelf temperature was controlled to within 1.700 of the set point during all stages of the cycle. The condenser was maintained at low temperatures throughout the drying portion, and vacuum was controlled within ±1 mTorr of the set-point.
(504) Product temperatures were observed to reach a maximum temperature of −26.0° C. during primary drying when the shelf temperature was held at −20.0° C., which is within the 3° C. safe zone that was applied to the collapse onset temperature as described in section 11. This was expected, as the low robustness run demonstrates the worst-case sublimation rate and lowest temperature/vacuum combination.
(505) Product temperatures were observed to converge with the shelf temperature at a relatively similar time compared to the convergence of the Pirani vacuum gauge and the capacitance manometer. Vacuum gauge convergence occurred at approximately 2,400 minutes from the start of the primary drying hold as seen in
(506) When the cycle was advanced to secondary drying, an increase in the Pirani gauge reading was observed, indicating additional moisture being removed from vials as bound water was removed from the product via desorption. Product temperatures followed closely with the shelf temperature through the ramp to secondary drying and throughout the secondary drying hold.
(507) Packaging and Storage—Nominal Cycle. Lyophilized vials were capped with flip-off/tear-off 20 mm caps and inspected. Thermocouple vials were rejected. A total of 190 vials were acceptable. Vials were sampled for analytical testing and stored at 2-8° C.
(508) TABLE-US-00033 TABLE 34 Results (Average Test Specification n = 2 vials) Appearance Conforms Acceptable (Lyophilized) Appearance Conforms Acceptable (Reconstituted) Reconstitution Time Report 34 Seconds (Average) pH (Average) Report 4.38 Osmolality (Average) Report 588 Residual Moisture via KF NMT 3.0% 0.6269 (Average) Assay 93.0%-107.0% 23.85 mg/mL (Label Claim) Related Substances RRT (0.54) 0.52 RRT (0.96) 0.59 RRT (0.98) ND Particulate Matter NMT 6K Particles ≥10 μm 583 NMT 600 Particles ≥25 μm 19
(509) Lyophilization—High Robustness. Vials were loaded onto a shelf controlled at 25.0° C. Once thermocouples were inserted, a slight vacuum was applied to the system to seal the door from the external environment. The loading temperature was held at 25.0° C. for 30 minutes to allow all vials to reach equilibrium. The shelf temperature was ramped to −40.0° C. over 130 minutes and held for 180 minutes to ensure all vials were thoroughly frozen. The lyophilizer condenser was then chilled, and vacuum was pulled to 120 mTorr. Once the vacuum set-point was achieved, the shelf temperature was held at −40.0° C. for 30 minutes to allow all vials to equilibrate. The shelf was then ramped to −10.0° C. over 60 minutes and held constant for 4,770 minutes. Completion of primary drying was indicated by the convergence of the Pirani gauge with the capacitance manometer. The primary shelf temperature was held for an additional 840 minutes due to time constraints. This cycle was the high robustness condition for primary drying, the vacuum gauges were allowed to fully converge to set the timepoint for primary drying. The vacuum gauges converged at approximately 1,930 minutes of total drying. Following completion of primary drying, the shelf temperature was ramped to 35.0° C. over 180 minutes and held for 720 minutes. At the completion of secondary drying, the system was backfilled with nitrogen to ˜600,000 mTorr and vials were fully stoppered. The programmed lyophilization cycle is shown in Table 35.
(510) TABLE-US-00034 TABLE 35 Step # Operation Temperature (° C.) 1 Load Set Point 25.0 Door Seal Step # Operation Pressure (mTorr) 2 Seal Door ~550,000 Thermal Treatmen Shelf Temperature Duration Step # Operation (° C.) (min) 3 Hold 25.0 30 4 Ramp −40.0 130 5 Hold −40.0 180 Shelf Temperature Vacuum Duration Step # Operation (° C.) (mTorr) (min) Primary Drying 6 Hold −40.0 120 120 7 Ramp 25.0 120 60 8 Hold 25.0 120 4,770 Secondary Drying 9 Ramp 35.0 120 180 10 Hold 35.0 120 720 Shelf Step # Operation Temperature (° C.) Shut Down 15 Backfill with 35.0 nitrogen gas to 11.6 PSIA = ~600,000 mTorr 16 Stopper vials. 35.0 Unloading 17 Unload 35.0 Total Time 6,190 minutes (103.2 hrs).sup.1 .sup.1Total cycle time does not account for the time required to reach vacuum or unloading.
(511) Lyophilization Cycle Analysis. Analysis of the run chart demonstrates that shelf temperature was controlled to within 100C of the set point during all stages of the cycle. The condenser was maintained at low temperatures throughout the drying portion, and vacuum was controlled within ±1 mTorr of the set-point.
(512) Product temperatures were observed to reach a maximum temperature of −24.2° C. during primary drying when the shelf temperature was held at −10.0° C., which is within the safe zone of the collapse temperature of the product. This was expected, as the low robustness run demonstrates the worst-case sublimation rate and lowest temperature/vacuum combination.
(513) Due to issues with obtaining the individual product traces, it is unknown when, or at what time the product temperatures converged with the shelf set point. However, an estimate can be made with the thermocouple average temperature trace of approximately 1,700 minutes. Vacuum gauge convergence for this cycle was allowed to fully complete to ensure that primary drying was completed. Vacuum gauge convergence occurred at 2,885 minutes from the start of the primary drying hold.
(514) When the cycle was advanced to secondary drying, an increase in the Pirani gauge reading was observed, indicating additional moisture being removed from vials as bound water was removed from the product via desorption. Product temperatures followed closely with the shelf temperature through the ramp to secondary drying and throughout the secondary drying hold.
(515) In order to trace potential degradation during secondary drying, samples were taken at t=60 minutes, and t=360 minutes into secondary drying. The results can be seen in Table 36.
(516) TABLE-US-00035 TABLE 36 Test Specification t = 60 min at 35° C. t = 360 min at 35° C. FP Appearance Conforms N/A Acceptable (Lyophilized) Appearance Conforms Acceptable (Reconstituted) Reconstitution Report 30 Seconds Time (Average) pH (Average) Report 4.38 Osmolality Report 600 (mOsm/kg) Residual NMT 3.0% 0.5544 Moisture via KF (Average) Assay 93.0%-107.0% 23.3 mg/mL 23.7 mg/mL 23.7 mg/mL (Label Claim) Related RRT (0.54) 0.55 0.55 0.54 Substances RRT (0.96) 0.62 0.55 0.56 RRT (0.98) ND ND 0.12 Particulate NMT 6K Particles N/A 542 Matter ≥10 μm NMT 600 Particles N/A 23 ≥25 μm
(517) The final lyophilization cycle is outlined in Table 37 below.
(518) TABLE-US-00036 TABLE 37 Step # Operation Temperature (° C.) 1 Load Set Point 20.0 ± 5.0 Door Seal Step # Operation Pressure (mTorr) 2 Seal Door ~550,000 Thermal Treatmen Shelf Step # Operation Temperature (° C.) Duration (min) 3 Hold 20.0 ± 5.0 30 4 Ramp −45.0 ± 5.0 130 5 Hold −45.0 ± 5.0 180 Shelf Vacuum Duration Step # Operation Temperature (° C.) (mTorr) (min) Primary Drying 6 Hold −45.0 ± 5.0 95 ± 25 30 7 Ramp −15.0 ± 5.0 95 ± 25 60 8 Hold −15.0 ± 5.0 95 ± 25 3930 Secondary Drying 9 Ramp 30.0 ± 5.0 95 ± 25 180 10 Hold 30.0 ± 5.0 95 ± 25 720 Shelf Step # Operation Temperature (° C.) Shut Down 15 Backfill with nitrogen 30.0 ± 5.0 gas to 11.6 PSIA = ~600,000 mTorr 16 Stopper vials. 30.0 ± 5.0 Unloading 17 Unload 30.0 ± 5.0 Total Time 5,260 minutes (87.7 hrs).sup.1 .sup.1Total cycle time does not account for the time required to reach vacuum or unloading.
Example 19—Crystalline Feasibility Study of an Enantiomer of Compound D1
(519) Crystalline feasibility assessment of the citrate salt of compound R-D1 and salt screening were performed as follows. Crystalline feasibility assessment of citrate Freebasing and salt screening using 19 counter ions Crystalline feasibility of freebase
(520) Since properties described in this Example are same for enantiomers, the observations described in this Example for compound R-D1 are also applicable to compound S-D1.
(521) Crystalline feasibility studies of the citrate included long-term slurries in a wide range of solvents, vapor diffusion onto solid, antisolvent vapor diffusion, and slow evaporation. Most experiments did not yield crystalline patterns. However, after stirring for the duration of the project, a crystalline pattern was observed from MeOH slurry as wet cake. The solid became amorphous upon drying at 50° C. under vacuum and therefore was not characterized further.
(522) To generate material for salt screening, freebasing of the amorphous citrate was carried out in dichloromethane (DCM) with the slow addition of a solution of ammonium bicarbonate (5 wt. %) and subsequent washes with brine and water. The resulting amorphous freebase was fully characterized and used for salt screening. The salt screening was performed using 15 counterions and different solvents. The freebase did not produce salts with any of the counterions in the initial sampling. The experiments were left to stir for a few weeks and a salt was formed with toluenesulfonic acid (TSA) in acetonitrile (ACN). Although the salt was stable upon drying at 50° C., it resulted in an amorphous solid after exposure to >95% relative humidity (RH). Therefore, the salt was not considered a viable form.
(523) On the other hand, it was observed that the amorphous freebase converted to a crystalline form upon mixing in various solvents with the aid of sonication. The freebase formed crystalline Pattern FF-A in methanol (MeOH), while Pattern FF-B was produced in ethanol (EtOH) and acetone. Both patterns showed hygroscopicity and slight decrease in crystallinity upon drying and exposure to humidity, although Pattern FF-B regained some crystallinity after re-exposure to humidity. Slurries of crystalline FF-A were prepared in EtOH:water (95:5 vol) and MeOH at room temperature and after ˜1 h and both showed Pattern FF-B by XRPD. A selective salt formation using Pattern FF-A was performed using three counterions and two solvents. However, no new patterns were observed from these experiments.
(524) Based on the data collected (summarized in Table 38) the salts obtained in this work do not represent preferred forms of the compound R-D1 due to their instability upon humidification.
(525) TABLE-US-00037 TABLE 38 Purity by Purity Residual Hygro- HPLC (%) by HPLC solvents scopicity After 1 week DSC onsets (%) by by DVS 75% RH XRPD XRPD pattern (° C.) [enthalpy chemical .sup.1H-NMR (up to chemical pattern Wet Dry Humid (J/g)] [chiral] (wt. %) 95% RH) and [chiral] FF-A FF-A FF-A FF-A 190.4 [3.07] 98.50 BDL >10.70 98.28 [97.11] [94.55] FF-B FF-B FF-B FF-B 191.4 [20.57] 98.85 BDL >8.80 98.56 225.2 [9.38] [96.56] [96.13] citrate Am Am Am 154.11 [47.65] 99.46 2.68 (IPA) >25.55 99.12 (amorphous) 171.44 [45.43] [99.05] NA Note: Am, amorphous; BDL, below detection limit; NA, not applicable.
XRPD Patterns for XPRD Patterns FF-A and FF-B are in Tables 39 and 40, respectively.
(526) TABLE-US-00038 TABLE 39 d-spacing Rel. 2θ (deg.) (ang.) Intensity 5.26 16.79 57 9.04 9.77 38 10.41 8.49 40 13.41 6.60 14 13.80 6.41 100 14.82 5.97 16 15.26 5.80 15 15.54 5.70 9 16.12 5.49 14 17.32 5.11 11 17.71 5.00 71 17.91 4.95 99 18.45 4.80 21 18.71 4.74 10 20.54 4.32 11 20.82 4.26 45 21.48 4.13 13 22.57 3.94 10 22.78 3.90 4 23.74 3.75 10 24.12 3.69 11 26.34 3.38 10 26.85 3.32 24 27.91 3.19 6 28.34 3.15 5 Note. Only peaks with relative intensity ≥ 5 were reported.
(527) TABLE-US-00039 TABLE 40 d- spacing Rel. 2θ (deg.) (ang.) Intensity 5.11 17.29 26 5.39 16.37 46 8.81 10.03 28 9.05 9.77 39 10.15 8.71 4 10.47 8.44 8 10.74 8.23 11 13.10 6.75 10 13.38 6.61 53 13.58 6.52 100 13.97 6.33 8 14.59 6.07 9 15.19 5.83 31 15.76 5.62 33 17.41 5.09 93 18.10 4.90 78 18.62 4.76 31 19.37 4.58 6 20.36 4.36 7 21.00 4.23 69 21.59 4.11 4 22.30 3.98 4 22.65 3.92 6 23.06 3.85 8 23.51 3.78 10 24.25 3.67 11 24.76 3.59 5 25.80 3.45 15 26.33 3.38 32 27.47 3.24 8 28.09 3.17 7 Note. Only peaks with relative intensity 4 were reported.
Analytical Techniques
(528) Differential Scanning Calorimetry (DSC). DSC was performed using a Mettler Toledo DSC.sup.3+. The sample (1-5 mg) was weighed directly in a 40 μL hermetic aluminum pan with a pinhole and analyzed according to the parameters in Tables 41 and 42.
(529) TABLE-US-00040 TABLE 41 Parameters Method Ramp Sample size 3-5 mg Heating rate 10.0.sup.o C./min Temperature range 30 to 300° C. Method gas N.sub.2 at 60.00 mL/min
(530) TABLE-US-00041 TABLE 42 Parameters Method Modulation Sample size 5-10 mg Amplitude 1° C. Period 60 s Heating rate 2.0° C./min Temperature range 30 to 300° C. Method gas N.sub.2 at 60.00 mL/min
(531) Dynamic Vapor Sorption (DVS). DVS was performed using a Q5000SA. The sample (5-15 mg) was loaded into a metallic quartz sample pan, suspended from a microbalance, and exposed to a humidified stream of nitrogen gas. Weight changes were relative to a matching empty reference pan opposite the sample, suspended from the microbalance. One of two methods was used:
(532) Method 1: The sample was held for a minimum of 10 min at each level and only progressed to the next humidity level if there was <0.002% change in weight between measurements (interval: 5 s) or 60 min had elapsed. The following program was used:
(533) 1—Equilibration at 50% RH 2—50% to 2%. (50%, 40%, 30%, 20%, 10%, and 2%) 3—2% to 95% (2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95%) 4—95% to 2% (95%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, and 2%) 5—2% to 50% (2%, 10%, 20%, 30%, 40%, and 50%)
Method 2: The sample was held for a minimum of 10 min at each level and only progressed to the next humidity level if there was <0.002% change in weight between measurements (interval: 5 s) or 45 min had elapsed. The following program was used: 1—Equilibration at 50% RH 2—50% to 5%. (50%, 35%, 20%, and 5%) 3—5% to 95% (5%, 20%, 35%, 50%, 65%, 80%, and 95%) 4—95% to 5% (95%, 80%, 65%, 50%, 35%, 20%, and 5%) 5—5% to 50% (5%, 20%, 35%, and 50%)
(534) High Performance Liquid Chromatography (HPLC). HPLC was conducted using an Agilent 1220 Infinity LC or Agilent 1220 Infinity 2 LC equipped with diode array detector (DAD). Flow rate range of the instrument is 0.2-5.0 mL/min, operating pressure range is 0-600 bar, temperature range is 5° C. above ambient to 60° C., and wavelength range is 190-600 nm.
(535) The HPLC methods used in this study are shown in Tables 43 and 44.
(536) TABLE-US-00042 TABLE 43 Parameters for chemical purity Mobile phase A 10 mM NH.sub.4FA in distilled water (pH 4.0) Mobile phase B 0.02% formic acid (FA) acid in ACN:MeOH (3:2 vol.) Diluent 0.05% FA in ACN:water (1:1 vol.) Injection volume 5 μL Monitoring wavelength 210 nm Column Waters C-18, 4.6 × 150 mm, 3.5 μm Column temperature 45° C. Time (min) % A Flow rate (mL/min) Gradient method 0 95 1.0 10 60 1.0 14 15 1.0 17 15 1.0 17.10 95 1.0 26.00 95 1.0
(537) TABLE-US-00043 TABLE 44 Parameters for chiral purity Mobile phase A 25 mM FA and 25 mM NH.sub.3 in ACN: MeOH (7:3 vol.) Diluent 0.05% FA in ACN:THF (1:1 vol.) Injection volume 0.80 μL Monitoring wavelength 280 nm Column ChiralPak IB, 150 × 4.6 mm, 5 μm, chiral Column temperature 30° C. Time (min) % A Flow rate (mL/min) Gradient method 0 1.0 20 1.0
(538) Infrared (IR) Spectroscopy.
(539) Smart iTX. IR spectroscopy was performed using a Thermo Scientific Nicolet iS10 FTIR Spectrometer with a helium-neon laser. The beam splitter was potassium bromide/germanium optimized for mid IR. The source type was Ever-Glo and tungsten/halogen. The spectral range was 7800-350 cm.sup.−1 and spectral resolution was ≤0.4 cm.sup.−1. The detector type was deuterated triglycine sulfate. Data was analyzed using Thermo Scientific OMNIC software (Version 9.8). Samples were analyzed as solids on the Smart iTX accessory with high-efficiency optic reflectors and diamond ATR crystal attenuator.
(540) TGA-IR. IR spectroscopy was performed using a Thermo Scientific Nicolet iS10 FTIR Spectrometer with a helium-neon laser. The beam splitter was potassium bromide/germanium optimized for mid IR. The source type was Ever-Glo and tungsten/halogen. The spectral range was 7800-350 cm.sup.−1 and spectral resolution was ≤0.4 cm.sup.−1. The detector type was deuterated triglycine sulfate. Data was analyzed using Thermo Scientific OMNIC software (Version 9.8).
(541) The evolved gas emitted from samples upon TGA using the Mettler Toledo TGA/DSC.sup.3+ was analyzed with IR spectroscopy via a TGA-IR Module for Nicolet FTIR Spectrometers. The TGA-IR accessory facilitated time-based correlation of evolved gas emissions in IR analysis. The accessory was connected directly to the furnace of the TGA/DSC unit with 5 feet insulated, glass-lined, stainless steel transfer line (⅛″ O.D.) and compression fittings. The transfer line temperature was maintained at 250° C. with a digital controller. The flow cell was nickel-plated aluminum, with a volume of 22 mL and an optical path length of 10 cm.
(542) Karl Fischer (KF) Titration. KF titration for water determination was performed using a Mettler Toledo C20S Coulometric KF Titrator equipped with a current generator cell with a diaphragm, and a double-platinum-pin electrode. The range of detection of the instrument is 1 ppm to 5% water. Aquastar™
(543) CombiCoulomat fritless reagent was used in both the anode and cathode compartments. Samples of approximately 0.03-0.10 g were dissolved in the anode compartment and titrated until the solution potential dropped below 100 mV. Hydranal 1 wt. % water standard was used for validation prior to sample analysis.
(544) Microscopy. Optical microscopy was performed using a Zeiss AxioScope A1 digital imaging microscope equipped with 2.5×, 10×, 20×, and 40× objectives and polarizer. Images were captured through a built-in Axiocam 105 digital camera and processed using ZEN 2 (blue edition) software provided by Zeiss.
(545) Proton Nuclear Magnetic Resonance (.sup.1H NMR).
(546) Bruker. .sup.1H NMR was performed on a Bruker Avance 300 or 500 MHz spectrometer. Solids were dissolved in 0.75 mL deuterated solvent in a 4 mL vial, transferred to an NMR tube (Wilmad 5 mm thin wall 8″ 200 MHz, 506-PP-8) and analyzed according to the parameters in Tables 45 and 46.
(547) TABLE-US-00044 TABLE 45 Parameters - Bruker Avance 300 Instrument Bruker Avance 300 MHz spectrometer Temperature 300K Probe 5 mm PABBO BB-1H/DZ-GRD Z104275/0170 Number of scans 16 Relaxation delay 1.000 s Pulse width 14.2500 μs Acquisition time 2.9999 s Spectrometer 300.15 MHz frequency Nucleus .sup.1H
(548) TABLE-US-00045 TABLE 46 Parameters - Bruker Avance 500 Instrument Bruker Avance 500 MHz spectrometer Temperature 300K Probe 5 mm PABBO BB-1H/D Z-GRD Z113652/0159 Number of scans 32 Relaxation delay 1.000 s Pulse width 14.0000 μs Acquisition time 3.2506 s Spectrometer 500.13 MHz frequency Nucleus .sup.1H
(549) Nanalysis. .sup.1H NMR was performed on an Nanalysis 60e NMReady 60 MHz spectrometer. Solids were dissolved in 0.75 mL deuterated solvent in a 4 mL vial, transferred to an NMR tube (New Era NE-BT5-7, 4.95 mm+/−0.03 mm OD, 0.43 mm+/−0.02 mm wall, 7″ long, Type 1 Class B borosilicate glass) and analyzed according to the parameters in Table 47.
(550) TABLE-US-00046 TABLE 47 Parameters Instrument Nanalysis 60e NMReady 60 MHz spectrometer Temperature 33.00° C. Number of scans 256 Relaxation delay 1.0 s Pulse angle 78.88 Pulse width 14.45 μs Acquisition time 3.7 s Digital Resolution 0.04 Hz Spectrometer frequency 60 MHz Nucleus .sup.1H
(551) pH Measurement. InLab Expert NTC30 pH electrode. The electrode has two open junctions and U-glass membrane resistance of <250 MΩ. The electrode uses the ARGENTHAL™ reference system and XEROLYT® polymer reference electrolyte. The operating range is 0-14 pH units at 0-100° C.
(552) Simultaneous Thermogravimetric Analysis and Differential Scanning Calorimetry (TGA and DSC). TGA and DSC were performed on the same sample simultaneously using a Mettler Toledo TGA/DSC.sup.3+. Protective and purge gas was nitrogen at a flowrate of 20-30 mL/min and 50-100 mL/min, respectively. The desired amount of sample (5-10 mg) was weighed directly in a hermetic aluminum pan with pinhole and analyzed according to the parameters below:
(553) TABLE-US-00047 TABLE 48 Parameters Method Ramp Sample size 5-10 mg Heating rate 10.0.sup.o C./min Temperature range 30 to 300° C.
(554) X-Ray Powder Diffraction (XRPD). XRPD was performed using a Rigaku MiniFlex 600 in reflection mode (i.e., Bragg-Brentano geometry). Samples were prepared on Si zero-return wafers. The parameters for XRPD methods used are listed in Table 49.
(555) TABLE-US-00048 TABLE 49 Parameter Regular scan High resolution scan X-ray wavelength Cu Kα1, 1.540598 Å Cu Kα1, 1.540598 Å X-ray tube setting 40 kV, 15 mA 40 kV, 15 mA Slit condition 1.25° div., Ni kβ 1.25° div., Ni kβ filter, 0.3 mm rec. filter, 0.3 mm rec. Scan mode Continuous Continuous Scan range (°2θ) 4-30 4-40 Step size (°2θ) 0.05 0.05 Scan speed (°/min) 5 1.25 Spin No No
(556) XRPD was performed using a Bruker D8 Advance equipped with LYNXEYE detector in reflection mode (i.e., Bragg-Brentano geometry). Samples were prepared on Si zero-return wafers. The parameters for XRPD methods used are listed in Table 50.
(557) TABLE-US-00049 TABLE 50 Parameter Regular scan High resolution scan X-ray wavelength Cu Kα1, 1.540598 Å Cu Kα1, 1.540598 Å X-ray tube setting 40 kV, 40 mA 40 kV, 40 mA Slit condition 0.6 mm div. + 2.5° 0.6 mm div. + 2.5° soller soller Scan mode Step Step Scan range (°2θ) 4-30 4-40 Step size (°2θ) 0.03 0.02 Dwell time (s/step) 0.23 0.9 Spin Yes (0.5 Hz) Yes (0.5 Hz)
Citrate of Compound R-D1
(558) The X-ray powder diffraction (XRPD) revealed an amorphous pattern, as shown in
(559) Solubility in Process Solvents
(560) Qualitative solubility of the citrate salt of compound R-D1 at RT (22-24° C.) was assessed in 12 solvents having diverse properties. A summary of the results is shown in Table 51.
(561) In this procedure, the material was weighed in 2 mL vials, then a 6.3 mm stir bar was added to each vial. Three volumes (vol.) of solvent were added at first, followed by the addition in 1 vol. increments up until 12 vol., then 2 vol. increments up to 26 vol., followed by 5 vol. increments up to 44 vol., and finally 10 vol. increments until the solids dissolved. In the samples where solubility was low, the solvent was added until the vial filled. The slurries formed in CM and in dioxane:EtOH (1:1 vol.) thinned and formed a gel. However, they did not yield crystalline solids, as shown in
(562) The vials were monitored until the end of the project and it was noted that the yellow gum initially produced in MeOH turned into an off-white thick after about six weeks slurry that was filtered and analyzed. The solids collected from this sample produced a low crystalline Pattern, citrate-A. The material was dried at 50° C. for 3 h, followed by exposure to >95% RH for 5 h. The sample became amorphous after drying and exposure to RH, as shown in
(563) TABLE-US-00050 TABLE 51 Solubility Solvent (mg/mL) Observations TFE >333 Yellow clear solution. THF <16 White slurry. 1,4-Dioxane <16 White slurry, flowable but tended to settle partially. DMSO >333 Yellow clear solution. DCM <15 White slurry that thinned and formed a gel EtOH <15 White slurry, flowable but tended to settle partially. IPA <14 White slurry, flowable but tended to settle partially. MeOH <42 Insoluble yellow gum. Turned into a white slurry overtime (~6 weeks) 2-MeTHF <14 White slurry. EtOAc <14 White slurry. Acetone <14 White slurry, flowable but tended to settle partially. Dioxane:EtOH <14 White slurry that thinned and (1:1 vol.) formed a gel. DMAC >333 Yellow clear solution. Benzyl alcohol 200-333 Yellow clear solution.
Stability at 40° C./75% RH
(564) The stability at high RH was assessed for the citrate salt of compound R-D1. In this experiment, 30 mg of the citrate salt of compound R-D1 were weighed in a 4 mL vial and placed in a 20 mL vial set at 40° C. and 75% RH, with no stirring, for 1 week. The humid environment was generated with saturated sodium chloride in water in a sealed container. After one week, the sample appeared pale-yellow upon inspection. The XRPD analysis of this sample did not show conversion to a crystalline form.
(565) Amorphous to Crystalline Studies of Citrate
(566) Thermal Treatment of Amorphous Salt. Thermal treatment of the citrate salt of compound R-D1 was evaluated by subjecting the sample to high temperature, mainly 130, 175, and 205° C., followed by cooling down to RT. The thermal events were measured by TGA/DSC and XRPD was used to study the resulting materials. None of these experiments resulted in crystalline solids, as observed by XRPD and TGA/DSC.
(567) Long-Term Slurries. Long-term slurries were carried out for the purpose of producing crystalline forms. Slurries of the citrate salt of compound R-D1 were produced in 15 solvents (Chlorobenzene, Anisole, MEK, Methyl acetate, ACN, THF:DMSO (9:1 vol.), EtOH:DMSO (9:1 vol.), IPA:DMSO (9:1 vol.), 2-MeTHF:DMSO, (9:1 vol.), Acetone:DMSO, (9:1 vol.), IPAc:DMSO (9:1 vol.), ACN:DMSO (9:1 vol.), EtOH:water (95:5 vol.), n-Propanol, Toluene, THF, 1,4-Dioxane, DCM, EtOH, IPA, MeOH, 2-MeTHF, EtOAc, Acetone, Dioxane:EtOH, (1:1 vol.), Chlorobenzene:MeOH (8:2 vol.), Anisole:MeOH (8:2 vol.), MEK:MeOH (8:2 vol.), MeOAc:MeOH (8:2 vol.), ACN:MeOH (8:2 vol.), Acetone:MeOH (8:2 vol.), Toluene:MeOH (8:2 vol.), THF:MeOH (8:2 vol.), 1,4-Dioxane:MeOH (8:2 vol.), DCM:MeOH (8:2 vol.), 2-MeTHF:MeOH (8:2 vol.), EtOAc:MeOH (8:2 vol.), MtBE:MeOH (8:2 vol.), Chloroform:MeOH (8:2 vol.), and DMF:MeOH (8:2 vol.)) at RT in addition to the slurries continued from solubility experiments. In this procedure, 30 mg of solid was added to a 2 mL vial containing a 6.3 mm stir bar. Then, 3 vol. of solvent was added, followed by the addition of more solvent in 5 vol. increments until it formed a flowable slurry. After 10 days, the solids were filtered and transferred to an XRPD plate for further analysis on the wet cake. Overall, crystalline patterns were not observed after one week. However, a crystalline solid was observed after stirring in MeOH for about six weeks.
(568) Vapor-Diffusion Crystallization onto Solution and Solids. The vapor diffusion onto antisolvent solution was carried out by weighing 20 mg of the citrate salt of compound R-D1 in a 4 mL vial. A suitable solvent was added to completely dissolve the solid, while producing a solution of slightly undersaturated concentration. The open vial was placed inside a 20 mL scintillation vial and 3 mL of antisolvent was added to the 20 mL vial to diffuse into the solution. The 20 mL vial was capped, sealed with parafilm, and kept undisturbed at RT. A summary of the solvent systems used in this experiment is presented in Table 52.
(569) TABLE-US-00051 TABLE 52 Solvent Antisolvent added added Observations Pattern DMAC ACN Yellow-white solids Am Chlorobenzene Clear solution — Acetone Yellow-white solids Am EtOAc Yellow-white solids Am MEK Yellow-white solids Am IPA Yellow-white solids Am DCM Yellow-white solids Am DMSO 2-MeTHF Clear solution — Chlorobenzene Clear solution — Acetone Yellow-white solids Am MeOAc Yellow-white solids Am MEK Yellow-white solids Am EtOH Yellow-white solids Am DCM Yellow-white solids Am Benzyl can Clear solution — alcohol Chlorobenzene Clear solution — Acetone Yellow-white solids Am MeOAc Yellow-white solids Am THF Clear solution — EtOH Yellow-white solids Am Note. Am, amorphous.
(570) Vapor diffusion onto solids was also started. The procedure is very similar, with the only difference that the active pharmaceutical ingredient (API) is not dissolved in a solvent; the vapor diffused directly onto the solid. The samples were monitored for visual changes in the solid (color, texture, size, etc.) over 2 months. The samples that did not present a significant change were not analyzed further by XRPD. A summary of this experiment set up is presented in Table 53.
(571) TABLE-US-00052 TABLE 53 Antisolvent added Observations Pattern ACN — — Chlorobenzene — — Acetone — — EtOAc — — MeOAc — — MEK — — IPA — — EtOH — — MeOH Clear yellow gum Am DCM Clear yellow solids Am MeTHF — — THF — — MtBE White-yellow powder Am Hexanes White-yellow powder Am Diethyl ether White-yellow powder Am Methyl cyclohexane White-yellow powder Am Cyclohexane White-yellow powder Am Dioxane — — Toluene — — Chloroform Yellow hardened particles Am Note. Am, amorphous.
(572) Slow Evaporation. Approximately 5-10 mg of the citrate salt of compound R-D1 was weighed in a vial, and the solvent was added until the sample dissolved or up to 1.7 mL in total. The samples that did not dissolve were split up by half its volume, and a cosolvent was added to each part until the sample dissolved or thinned out. The samples that were thin slurries were filtered with a 45 μm syringe filter to obtain a clear solution. In some cases, cosolvent was added to obtain a solution. A needle was inserted in the cap septa to allow the solvents to evaporate slowly. The solutions were monitored frequently for precipitation. The experiments did not yield crystalline solids.
(573) Freebasing
(574) The freebasing of compound R-D1 from its citrate salt was carried out to produce a freeform of the compound. In this procedure, 250 mg of the citrate salt of compound R-D1 was weighed in a 20 mL vial. Twenty volumes of DCM were added with stirring on a chiller block set at 10° C. Then, 30 vol. of a base, either NH.sub.4HCO.sub.3 or NaHCO.sub.3 (5 wt. %), was added dropwise and the suspension was stirred for a few minutes. A yellow gum formed during this procedure; the suspension was vortexed vigorously. The undissolved gum was brought down to the DCM phase with the aid of a spatula and the suspension was left to stir in the chiller block for an additional 20 min. Subsequently, the stirring was stopped, and the suspension was left undisturbed to allow the separation of the phases. The aqueous phase was removed carefully, and 30 vol. of the basic solution was added dropwise to the DCM layer with stirring, at 10° C. The aqueous phase was removed again after allowing the suspension to stand undisturbed for a few minutes. The DCM phase was transferred to a clean vial, and 20 vol. of a saturated solution of NaCl (brine) was added. The suspension was stirred for 10 min. After this time, the aqueous phase was removed carefully with a pipette, and 20 vol. of distilled water was added to remove any residual salt. The suspension was left to stir for 5 min on the chiller block, and the water was removed carefully with a pipette. More water was added (20 vol.), and the suspension was left to stir for another 5 min, followed by the removal of the aqueous phase. Finally, the DCM phase was transferred to a clean vial and the solution was dried at RT overnight, followed by drying in a rotatory evaporator for 10 min.
(575) The resulting freebase was a yellow solid with glassy appearance. The XRPD analysis showed an amorphous material for the samples obtained in ammonium bicarbonate and sodium bicarbonate, as shown in
(576) To corroborate the removal of the citric acid, this compound was analyzed by .sup.1H NMR in deuterated methanol (MeOH-d.sub.4) (
(577) The amorphous freebase also showed the presence of residual DCM in 4.76 and 5.32 wt. %, for the solids prepared with ammonium bicarbonate and sodium bicarbonate, respectively, as measured by .sup.1H NMR in chloroform-d (300 MHz).
(578) Freebasing Scale Up. The scale-up of the amorphous freebase of compound R-D1 was performed using 5.0152 g of its citrate salt. About 5 g of the citrate salt were weighed in a 400 mL EasyMax vessel. Twenty vol. of DCM were added with stirring at 300 rpm (0.39 Watts/kg) at 10° C. A solution of ammonium bicarbonate (30 vol.) (5 wt. %) was added in volumes of 10 mL dropwise and the suspension was allowed to stir for a few minutes. A yellow gum formed after the addition of 20 mL; the suspension was left to stir vigorously to aid dissolving it. The undissolved gum was brought down to the DCM phase with the aid of a spatula. Subsequently, 40 mL of the base was added in vol. of 10 mL in a period of 30 min with stirring, followed by the addition of 1 mL of MeOH used as co-solvent. After this time, 90 mL of the base was added slowly during the period of 2.5 h to bring the volume of the base to a total of 30 vol. with respect to the weight of the citrate salt used. The suspension was left to stir for an additional 2 h. The gum was transferred to an amber vial, along with a portion of the DCM and the base used in the reaction in an approximately 2:3 vol. ratio. The gum was sonicated for 30 min in an ice bath to avoid heating up the mixture. After the amount of gum was brought to a minimum, the sonicated suspension was incorporated to the original bi-phasic solution, and it was stirred for additional 5 min at 300 rpm (0.16 Watts/kg). The suspension was left undisturbed to allow the separation of the phases. The aqueous phase was removed with the aid of a pipette. Additional 150 mL (30 vol.) of the ammonium bicarbonate solution (5 wt. %) was added in 10 mL vol., dropwise, over 50 min, with stirring at 0.16 Watts/kg at 10° C. After adding the base, the mixture was stirred for an additional 5 min, followed by the careful removal of the aqueous solution. The DCM layer was transferred to a clean 400 mL vessel. 100 mL (20 vol.) of a saturated solution of NaCl (brine) was added. The suspension was stirred for 10 min. Then, the aqueous phase was removed carefully with a pipette. 100 mL (20 vol.) of distilled water was added to remove any residual salt. The suspension was left to stir at 300 rpm (0.20 Watts/kg) for 5 min, and the water was removed carefully with a pipette. Distilled water was added again (20 vol.), and the suspension was left to stir for another 5 min, followed by the removal of the aqueous phase. The DCM phase was transferred to six clean scintillation vials, and the solution was dried at RT overnight. The residual DCM was dried using a direct flux of nitrogen until most of the solvent was evaporated. The vials were dried under vacuum (˜−29 inHg) at RT for 3 days. The yield of the reaction calculated after removing the residual solvent and water was 32%.
(579) The resulting freebase was a yellow solid with powdery appearance and irregular particles as shown in
(580) The analysis by .sup.1H NMR in MeOH-d.sub.4 corroborated the removal of the citric acid, as indicated by the lack of signals at 2.85 ppm in the spectrum. The sample also showed residual DCM (8.01 wt. %) after drying the sample for 1 h at RT under vacuum (˜29 inHg), as evidenced by .sup.1H NMR. The amount of solvent was partially removed to 4.48 wt. % after further drying for 3 h at RT under vacuum (˜29 inHg).
(581) Simultaneous TGA/DSC was also carried out to evaluate the residual content of DCM and water in the amorphous freebase after drying for 3 h under vacuum at RT. The simultaneous TGA/DSC thermogram showed two mass losses over the course of a broad temperature endotherm. The first weight loss of 2.93 wt. % corresponded to residual DCM, while the loss of 5.21 wt. % was related to water. After drying the amorphous freebase for over 3 days, the thermogram showed a partial removal of both the DCM and water, as indicated by the weight losses of 1.63 and 2.90 wt. %, respectively.
(582) Salt Screening Using the Amorphous Free-Base Compound R-D1
(583) The salt screening was initially set up with 15 counter ions (acetic acid, benzoic acid, citric acid, fumaric acid, gentisic acid, gluconic acid, glucuronic acid, malic acid, maleic acid, malonic acid, oxalic acid, sulfuric acid, succinic acid, phosphoric acid, and toluenesulfonic acid) in 3 solvents, using 1.1 eq. of counter ion to freebase; sulfuric acid was also set up using 0.55 eq.
(584) Fifty-one experiments were initially set up for the salt screening process in 2 mL vials containing 6.3 mm stir bars, including experiments involving the amorphous freebase in the absence of counter ions. A stock solution of the freebase obtained in DCM and ammonium bicarbonate was prepared in TFE at a concentration of 60.51 mg/mL. Stock solutions of the counter ions were prepared in EtOH at a concentration of 20 mg/mL. Each vial contained 495.8 μL of the freebase solution (30 mg in total), and 1.1 eq. of the counter ion stock solution was added. Three vials contained the freebase dissolved in TFE, only, with no counter ion used. The vials were heated at 45° C. with stirring at 540 rpm for 2 h, followed by evaporation at RT overnight.
(585) EtOH, THF, and IPAc, were chosen for the first round of solvent screening. The experiments produced solids in 40/51 vials. To sample these experiments, the vials were left to stand undisturbed for 30 min and an aliquot of the slurry was then filtered and analyzed. Crystalline salts were not observed for the first round of solvents. After sampling all the slurries, the vials were left uncapped to evaporate to be used in a second round of solvents using ACN, dioxane, and 2-methyltetrahydrofuran (2-MeTHF). The second round of solvents yielded 42/51 slurries that were analyzed following the same procedure mentioned above. Crystalline salts were initially not observed. The experiments were left to stir at 45° C. for 2 days and then at RT for approximately 3 weeks to be sampled again after this time.
(586) These screens afforded one crystalline compound, which did not correspond to either the counter ion or the crystalline freebase used for the screening. The compound was stable upon drying. However, it converted to an amorphous solid after exposure to high RH overnight.
(587) Crystalline Feasibility of Free-Base Compound R-D1
(588) For this experiment, 7.2 mg of the amorphous freebase prepared from DCM and ammonium bicarbonate were weighed in a 4 mL vial, and 0.75 mL of MeOH-d.sub.4 was added. The suspension was sonicated for thorough dissolution, and it was transferred to an NMR tube for .sup.1H NMR analysis. After approximately 1 h, the clear yellow solution became hazy pink, and white solids precipitated out of the solution. The solids showed crystallinity, as demonstrated by XRPD analysis (
(589) Crystalline Patterns Observed from Free-Base Compound R-D1 in MeOH and EtOH
(590) The amorphous freebase prepared in DCM and ammonium bicarbonate was weighed (15.5 mg) in a 2 mL vial, and 20 vol. of MeOH (ACS grade) or EtOH were added. The suspension was sonicated for thorough dissolution, which resulted in a yellow gum that dissolved in the solvent quickly, yielding a clear solution. After approximately 10 min under constant sonication, white solids formed in the solution, and the suspension was left to sonicate for some additional minutes. An aliquot of the sample was filtered for further XRPD analysis, while the remaining suspension was transferred to a stirring plate set at 500 rpm at RT. The resulting slurries were filtered to collect the solids for XRPD analysis on the wet cakes.
(591) XRPD analysis was done in three stages. XRPD of the wet cake was done for all samples where solids were observed. The samples that showed unique patterns were further dried on the XRPD plates at 50° C., under vacuum for 3 h, and were characterized with XRPD analysis.
(592) The solids were then exposed to >95% RH overnight. This humid environment was produced by placing a beaker of saturated potassium sulfate in water in a sealed container at RT. The materials exposed to these conditions were evaluated by XRPD, and their patterns were compared to those produced before.
(593) The solids collected from MeOH showed high crystallinity, the patterns did not agree with the low crystalline racemic mixture (
(594) The solids collected from EtOH showed low crystallinity (
(595) Preparation of Pattern FF-A in MeOH
(596) To produce crystalline freebase, 50 mg of the amorphous freebase of compound R-D1 were weighed in a 4 mL vial, and 20 vol. of MeOH was added. The amorphous freebase formed a yellow gum upon contact with the solvent. The mixture was vortexed vigorously to dissolve the gum, followed by sonication until the yellow clear solution turned into a white immobile slurry. More solvent was added (13 vol.) to produce a flowable slurry. A sample of the slurry was filtered, and analyzed by XRPD on the wet cake, which showed that the compound was initially low crystalline (by XRPD). The sample was seeded using 50 μL of a seed in acetone (Pattern FF-B) and it was left to stir at 540 rpm at RT using a 10 mm stir bar. An aliquot of the slurry was filtered for further XRPD analysis after 6, 18, and 24 h of seeding and slurring in MeOH. The analysis showed increased crystallinity after 6 h (by XRPD), showing the Pattern FF-A. The whole sample was filtered in a pre-weighed vial and filter paper. The solids collected were washed 3 times using 2 vol. of MeOH each time. The white solids were left to dry at RT in a vacuum oven (˜−29 inHg) for 24 h.
(597) The XRPD diffractogram of the dried sample revealed a crystalline Pattern FF-A. The HPLC analysis showed a chemical purity of 97.85% and a chiral purity of 96.80%, as compared to the chemical purities of 99.46% and 96.62% for the citrate salt of compound R-D1 and free-base compound R-D1, respectively, and to the chiral purities of 99.05% and 98.95% for the citrate salt of compound R-D1 and free-base compound R-D1, respectively.
(598) Preparation of Pattern FF-A has also been scaled up. HPLC showed a chemical and chiral purity of 98.50 and 97.11%, respectively. The wet cake of Pattern FF-A was dried at 50° C. under vacuum for 3 h. It was then exposed to >95% RH overnight. Reduction in the crystallinity of Pattern FF-A was observed, as indicated by a decrease in the intensity and broadening of some features in the XRPD trace.
(599) The one-week stability at 75% RH was also tested. In this experiment, 12 mg of the dried crystalline freebase from MeOH were weighted in a 4 mL vial and placed in a 20 mL vial set at 40° C. and 75% RH for 1 week. The humid environment was generated with saturated sodium chloride in water in a sealed container. The chemical and chiral purities of the resulting material, as measured by HPLC after this experiment, resulted in 98.27 and 94.55%, respectively.
(600) Finally, the DVS isotherm plot revealed that the compound is hygroscopic, according to the European Pharmacopeia standards. This was indicated by the high humidity absorption (10.70 wt. %, 5.5 eq.) over a 5-95% humidity range. However, equilibrium was not reached at high RH therefore the mass change was representative of kinetics and may be higher with longer time. The adsorption/desorption profile also showed that this process is reversible. The XRPD analysis of the compound collected after the DVS analysis showed a new signal at 26=5.8°. This signal was consistent upon subjecting the same sample to drying under vacuum at RT for 2 h. The chemical purity measured by HPLC for the compound after the DVS analysis resulted in 98.68%.
(601) To study the effect of the temperature, the compound was subjected to a thermal treatment consisting of heating up the sample from RT up to 100° C. at a 10° C./min, and holding this temperature for 5 min to subsequently cool down to RT. The sample was collected and further analyzed with XRPD. The resulting patterns from this sample showed loss of some crystallinity.
(602) Overall, these analyses showed that the crystalline freebase, Pattern FF-A, produced in MeOH loses some crystallinity upon drying and exposure to humidity. Additionally, an extra peak by XRPD was observed after humidity exposure in DVS.
(603) Preparation of Pattern FF-B
(604) Approximately 150 mg of the amorphous freebase were weighed in a 4 mL vial. EtOH (15 vol.) was added, resulting in a yellow gum that was dispersed by vigorous vortex, followed by sonication Preparation of Pattern FF-B in EtOH. for a prolonged time until producing a hazy yellow slurry. The suspension was seeded with 100 μL of the crystalline freebase obtained in acetone (Pattern FF-B). The slurry was left to stir overnight at 540 rpm, at RT using a 10 mm bar. The next day, the slurry appeared thicker with off-white color. The XRPD pattern on the wet cake showed an amorphous compound (
(605) Preparation of Pattern FF-B in Acetone. Approximately 200 mg of the amorphous freebase were weighed in a 20 mL vial. Acetone (20 vol.) was added, and the mixture was sonicated to produce a hazy yellow slurry mixed with a clear yellow solution, with off-white particles when left to stand. The suspension was seeded with dried solids of the crystalline freebase previously obtained in acetone (Pattern FF-B,
(606) Characterization of Pattern FF-B. The TGA/DSC thermograms showed a mass loss event of 3.9 wt. % over the course of a broad temperature endotherm. The analysis by KF showed a water content of 3.36 wt. % (1.6 eq.), in good agreement with the TGA/DSC result. In addition, the .sup.1H NMR in chloroform-d did not show the presence of additional solvent. Two subsequent melts with an onset at 195.7 and 226.7° C. were also observed without associated mass losses. The temperature of these thermal transitions was corroborated with stand-alone DSC analysis. Microscopy images revealed the morphology to be irregular particles of yellow color that showed birefringence under polarized light. The chemical and chiral purities evaluated by HPLC were 98.85 and 96.56%, respectively.
(607) The pattern FF-B was dried at 50° C. under vacuum for 3 h after filtration for further XRPD analysis. The solids were then exposed to >95% RH overnight. This humid environment was produced by placing a beaker of saturated potassium sulfate in water in a sealed container at RT. The materials exposed to these conditions were evaluated by XRPD, and their patterns were compared to those of the crystalline freebase along each step. This experiment showed that the crystallinity of the compound decreased after drying the sample under vacuum, as indicated by the loss of intensity and broadening of some features in the diffractogram (
(608) The one-week stability at 75% RH was also tested. In this experiment, 12 mg of the crystalline freebase from MeOH was weighted in a 4 mL vial and placed in a 20 mL vial set at 40° C. and 75% RH for 1 week. The humid environment was generated with saturated sodium chloride in water in a sealed container. The XRPD analysis showed the splitting of the signal at 5.3 and 13.7°, as shown in
(609) The DVS isotherm plot revealed that the compound is hygroscopic, according to the European Pharmacopeia standards. This was indicated by the high humidity absorption (8.80 wt. %, 4.0 eq.) over a 5-95% humidity range. However, equilibrium was not reached at high RH. Therefore, the mass change was representative of kinetics and may be higher with longer time. The adsorption/desorption profile also showed that this process is reversible. The XRPD analysis of the compound collected after the DVS analysis showed a slight loss in crystallinity, indicated by the decreased intensity of some signals (
(610) The sample was subjected to a thermal treatment consisting of heating the sample gradually (10° C./min) up to the first two thermal events shown by TGA/DSC analysis, i.e., 130 and 225° C. In each case, the temperature was held for 5 min, followed by cooling down to RT. The sample was collected and further analyzed with XRPD. The patterns of the collected sample showed decreased crystallinity after exposure to 130° C., possibly by releasing water. Overall, the crystalline freebase Pattern FF-B obtained from acetone lost some crystallinity upon drying and exposure to humidity and heat.
Example 20—Preparation of the Compounds of Formula A
(611) ##STR00248##
(612) A solution of FLE (5.0 g, 6.68 mmol, 1.00 equiv) and SM7S (2.2 g, 13.41 mmol, 2.00 equiv) in THE (90 ml) and MeOH (10 mL) was cooled to 0-10° C. and AcOH (0.5 g, 8.33 mmol, 1.25 equiv) was added. The reaction mixture was stirred at 0-10° C. for 0.5-6 hrs. Then NaCNBH.sub.3 (1.25 g, 19.89 mmol, 3.00 equiv) was added as solution (10 ml of THF:MeOH 9:1) to the above resulting solution, and the resulting mixture was stirred at 0-10° C. for 2 h and then warmed to room temperature overnight. The solution was cooled 0-15° C. reaction and was quenched by the addition of DCM (37.5V), DMSO (12.5V), and 5% Na.sub.2CO.sub.3 aqueous solution (30V), and then the solution was warmed to room temperature. The phases were separated and to the organic phase was added 5% Na.sub.2CO.sub.3 aqueous solution (30V). The phases were separated and to the organic phase was added a brine solution (25V). The phases were separated and to the organic phase was added 5% Na.sub.2CO.sub.3 aqueous solution (30V). The phases were separated and to the organic phase was added a brine solution (25V). The organic phase is concentrated to 7.5 V and MeCN (10V) is added. The reaction mixture is then filtered to remove solids and the filter cake is washed with DCM/MeCN (1:10). The filtrate is then concentrated to 7.5V and DMF (7V) is added. The solution is concentrated to 8V and cooled to room temperature. To the concentrated is added water (3V) and the reaction mixture is stirred. The reaction mixture is then filtered and the filter cake was wash by water. The filtrate was dried in vacuo. This provided 3-[6-(7-[[1-([4-[6-(azetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2,6-dimethoxyphenyl]methyl)piperidin-4-yl]methyl]-2,7-diazaspiro[3.5]nonan-2-yl)-1-oxo-3H-isoindol-2-yl]piperidine-2,6-dione (2.77 g, 50%) as a yellow solid.
(613) TABLE-US-00053 TABLE 54 Relative Type δ.sub.C.sup.b (ppm) δ.sub.H.sup.c (ppm) Intensity C 173.4 N/A N/A CH2 31.7 2.91(o), 2.54(o) 2H CH2 23.0 2.38(o), 1.98(m) 2H CH 52.2 5.07(dd, J = 13.20 Hz, 1H 5.00 Hz) C 171.6 N/A N/A NH N/A 10.96(s) 1H C 169.0 N/A N/A C 132.9 N/A N/A C 130.7 N/A N/A CH2 47.2 4.25(o) 2H CH 104.7 6.67(o) 1H C 152.4 N/A N/A CH 115.9 6.69(o) 1H CH 124.2 7.37(d, J = 8.80 Hz) 1H CH2 62.1 3.60(br) 4H C 34.4 N/A N/A CH2 34.9 1.77(o) 4H C 106.3 N/A N/A C 159.7 N/A N/A CH3 56.8 3.89(s) 6H CH 105.6 6.85(s) 2H C 139.4 N/A N/A C 115.3 N/A N/A CH 138.0 7.60(s) 1H C 161.3 or N/A N/A 161.4 C 111.9 N/A N/A C 142.3 N/A N/A CH3 36.3 3.48(s) 3H CH 151.7 9.01(s) 1H C 161.4 or N/A N/A 161.3 CH 94.2 6.20(s) 1H CH2 50.7 4.00(m) 4H CH2 16.5 2.34(o) 2H C 177.1 N/A N/A C 72.0 N/A N/A C 171.9 N/A N/A CH2 44.5 2.54(o) 4H LCMS (ESI) m/z: [M + H].sup.+ = 829.55.
(614) ##STR00249##
(615) Compound FPS has also been prepared with NHC-borane as described above in example 20, with the exception that NHC-borane was used instead of NaCNBH.sub.3. The chiral purity of FPS was assessed at each stage of the purification, and the results are summarized in Table 55.
(616) TABLE-US-00054 TABLE 55 Crude NHC- Reaction Conditions Result Borane (temp/time) IPC Chiral Starting Material (~45% Temp (%) purity FLE SM7S AcOH assay) Solvent (° C.) Time (h) FPS of FPS 4.7 g 3.0 eq. 5% 1.5 eq. THF/ 20-30 12 50.7% 89.1% net MeOH 9:0.5 (v/v) Charge 30 V DCM and 10 V DMSO into the Organic 69.7% 87.1% reaction mixture layer Charge 30 V 5% NaHCO.sub.3 into R1 Separated to obtain organic layer Wash the organic layer with another 5% NH.sub.4HCO.sub.3 twice Concentrated and slurry the residual in Wet cake 91.7% 65.6% DCM/MeCN to improve chiral purity Mother 59.0% 99.8% Filter to obtain mother solution solution Concentrate the mother solution to dryness supernatant 7.2% 81.6% Dissolved the dryness in 7 V DMF and Wet cake 77.0% 3 V MeCN Charge ~3 V water slowly into the solution, and charge seed into the solution Stir the reaction mixture for 16 hrs Slurry the wet cake in Wet cake2 77.3% DMF/MeCN/Water (7 V/3 V/3 V) product 79.2% 98.4% Dry to obtain 2.6 g crude FPS chiral
(617) ##STR00250##
Preparation of (3R)-3-{6-[(3R)-3-{4-[6-(3,3-difluoroazetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2-fluoro-6-methoxyphenyl}pyrrolidin-1-yl]-1-oxo-3H-isoindol-2-yl}piperidine-2,6-dione
(618) To a stirred solution of acetic acid; methyl methyl 5-[(3R)-3-{4-[6-(3,3-difluoroazetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2-fluoro-6-methoxyphenyl}pyrrolidin-1-yl]-2-formylbenzoate (100 mg, 0.150 mmol, 1 equiv) and (3R)-3-aminopiperidine-2,6-dione hydrochloride (49.38 mg, 0.300 mmol, 2 equiv) in MeOH (0.5 mL) and THF (5 mL) were added AcOH (11.71 mg, 0.195 mmol, 1.3 equiv) in THF (1 mL) dropwise at room temperature under nitrogen atmosphere, The mixture was stirred for 0.5 h at 0° C., then the mixture was added NaBH.sub.3CN (28.28 mg, 0.450 mmol, 3 equiv) in MeOH (0.1 mL) and THF (1 mL) dropwise at 0° C. under nitrogen atmosphere. The resulting mixture was stirred for overnight at room temperature under nitrogen atmosphere. The residue was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, MeCN in Water (0.1% TFA), 10% to 50% gradient in 10 min; detector, UV 254 nm. to afford (3R)-3-{6-[(3R)-3-{4-[6-(3,3-difluoroazetidin-1-yl)-2-methyl-1-oxo-2,7-naphthyridin-4-yl]-2-fluoro-6-methoxyphenyl}pyrrolidin-1-yl]-1-oxo-3H-isoindol-2-yl}piperidine-2,6-dione (38.5 mg) as a white solid. LCMS (ESI) m/z: [M+H].sup.+=687. 1H NMR (300 MHz, DMSO-d6) δ 10.98 (s, 1H), 9.09 (d, J=0.7 Hz, 1H), 7.70 (s, 1H), 7.40 (d, J=8.3 Hz, 1H), 7.04-6.93 (m, 2H), 6.88 (dd, J=8.4, 2.3 Hz, 1H), 6.82 (d, J=2.2 Hz, 1H), 6.45 (s, 1H), 5.10 (dd, J=13.2, 5.1 Hz, 1H), 4.51 (t, J=12.4 Hz, 4H), 4.39-4.16 (m, 2H), 4.12-3.94 (m, 1H), 3.89 (s, 3H), 3.66-3.38 (m, 7H), 2.92 (ddd, J=17.7, 13.4, 5.3 Hz, 1H), 2.60 (d, J=16.8 Hz, 1H), 2.50-2.20 (m, 3H), 2.07-1.94 (m, 1H).
(619) A modified version of the procedure described above has also been used in the stereoretentive preparation shown below.
(620) ##STR00251##
Other Embodiments
(621) All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.
(622) While the invention has been described in connection with specific embodiments thereof, it will be understood that invention is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
(623) Other embodiments are in the claims.