Patent Application
20230073512
It is provided the use of Pyrimido[4,5-B]indole derivatives as anti-cancer compounds.
Humans suffer from various types of cancer, including lymphoma and leukemia, which are very aggressive tumors and result in high mortality rates. In the majority of the cases, the currently existing treatment modalities (chemotherapy, radiotherapy, surgery, certain additional anticancer drugs and bone marrow transplantation) are far from satisfactory, and only a relatively small proportion of for example lymphoma and leukemia patients can survive for many years.
The ubiquitin-proteasome system (UPS) promotes the timely degradation of short-lived proteins with key regulatory roles in cell cycle progression, oncogenesis and genome integrity. Abnormal regulation of UPS disrupts the protein homeostasis and causes many human diseases, particularly cancer. Bortezomib is a FDA therapeutic proteasome inhibitor approved drug for the treatment of relapsed multiple myeloma and mantle cell lymphoma. Accordingly, modulators of the ubiquitin-proteasome system are anti-cancer targets. The normal cell toxicity associated with bortezomib, resulting from global inhibition of protein degradation, promotes the focus of drug discovery efforts on targeting enzymes upstream of the proteasome for better specificity. E3 ubiquitin ligases, particularly those known to be activated in human cancer, become an attractive choice. Cullin-RING Ligases (CRLs) with multiple components are the largest family of E3 ubiquitin ligases and are responsible for ubiquitination of ˜20% of cellular proteins degraded through the ubiquitin-proteasome system (Zhao and Sun, 2013, Curr Pharm Des, 19: 3215-3225).
Pevonedistat (MLN4924), currently ongoing clinical trials, is a selective inhibitor of NEDD8. The inhibition of NEDD8-activating enzyme (NAE) prevents activation of cullin-RING ligases (CRLs), which are critical for proteasome-mediated protein degradation CRLs.
Indisulam is an anti-cancer agent mediating the interaction between RBM39 (a splicing factor) and the E3 ligase DCAF15 leading to RBM39 poly-ubiquitination and proteasomal degradation which acts as a molecular glue degrader that binds to DCAF15, creating a novel ligase surface that enhances RBM39 binding (Bussière et al., 2019, bioRxiv, 737510).
Thus, there is still a need to be provided with novel drugs and/or treatment alternatives that can kill selectively cancer cells.
It is provided a method of treating cancer in a patient comprising the step of administering to said patient at least one compound of formula I:
or a salt or a prodrug thereof,
wherein:
each Y is independently selected from N and CH;
m is an integer from 0 to 3 (or 0 to 4 when Y is CH in the ring comprising substituent Z);
Z is each time independently selected from:
RB is
In an embodiment, the compound of formula I is
or a pharmaceutically acceptable salt thereof.
In another embodiment, the compound of formula I is a hydrobromide salt of
In a further embodiment, the compound of formula I is
or a pharmaceutically acceptable salt thereof.
In an embodiment, the compound of formula I is as defined herein, especially as defined in Table 3.
In an additional embodiment, the patient is a human or an animal.
In a further embodiment, the animal is a mouse.
In an embodiment, the cancer is a cancer based on K27 mutation, EZH2 or PRC2 mutation.
In an embodiment, the compound is formulated for an administration orally, intramuscularly, intravenously or subcutaneously.
In a further embodiment, the compound degrades at least one of LSD1, RCOR1, HDAC2 and CoREST.
In another embodiment it is provided a method of inhibiting proliferation of cancerous cells ex vivo comprising administering the compound described herein into the medium containing the cells in proliferation, wherein the compound has anantineoplastic on the proliferation of the cancerous cells.
It is also provided a compound of formula I:
or a salt or a prodrug thereof,
wherein:
each Y is independently selected from N and CH;
Reference will now be made to the accompanying drawings.
It is provided pyrimido[4,5-B]indole derivatives, as well as 5H-pyrido[4,3-b]indole derivatives and pyrido[2′,3′:4,5]pyrrolo[2,3-d]pyrimidine derivatives as anti-cancer compounds.
One example of such pyrimido[4,5-B]indole derivative is UM171 (see U.S. Pat. Nos. 9,409,906 and 10,336,747, the content of which is incorporated by reference), which has an activity on primitive cells which is rapidly reversible if the compound is washed out from culture. UM171 does not independently trigger cell proliferation in the absence of growth factors; it is not mitogenic but rather prevents cell differentiation. The molecule was reported to preferentially expand long-term hematopoietic stem cells (HSC) in ex vivo cultures, and this expansion was maximal by day 7. Results initially described in NSG mice were confirmed in a clinical trial in which 22 patients were transplanted with a single UM171-expanded cord blood graft. These patients showed rapid neutrophil recovery and less fever than control transplants. Most interestingly, the UM171 patients never developed extended chronic graft versus host disease and presented a very low rate of transplantation-related mortality and disease relapse. It was found that UM171 induced an important expansion of CD86+ dendritic progenitors and mast cells in addition to CD201+ (EPCR) primitive HSCs (see WO 2019/161494, the content of which is incorporated by reference). In addition, the molecule was also shown to rapidly induce the expression of numerous pro- and anti-inflammatory genes and of several essential stem cell marker genes including CD201 and CD86. Other related compounds that are similarly contemplated herein are referred to as 5H-pyrido[4,3-b]indole derivatives and pyrido[2′,3′:4,5]pyrrolo[2,3-d]pyrimidine derivatives. Those compounds are disclosed for example in U.S. Pat. No. 10,647,718 B, the content of which is incorporated by reference.
These results prompted the investigation to determine if UM171 can have a broad anti-cancer use.
Hematopoietic cell lines such as OCI-AML5 exposed to UM171 upregulate more than 400 genes within 6 hours with less than a dozen genes repressed. This nearly exclusive effect on gene upregulation pointed to a potential transcriptional repressor being targeted by the molecule. CRISPR screens in 2 different cells lines over a range of UM171 dose (
While the function of KBTBD4 is unknown to date, Kelch BTB domain proteins are known to bridge CULLIN3-RING ubiquitin ligases (CRL3 complex) to their substrate (Genschik et al., 2013, EMBO J, 32: 2307-2320), thereby dictating specificity in targeting proteins for proteolysis. In line with this observation, several core and key components of the CRL3 complex were strong suppressors of UM171 in the different CRISPR screens, namely CULLIN3 itself and its essential regulator Cullin-associated NEDD8-dissociated protein 1 (CAND1) (
Directed mass spectrometry analysis of BirA-KBTBD4 proximity labeling also readily identified several components of CRL3 or associated proteins such as CULLIN3, CSN4, UBC12 and the KELCH-domain-directed HSP90 adaptor NUDCD3 (
It is provided that histone 3 lysine 27 acetylation (H3K27ac) mark is rapidly enhanced upon exposure to UM171 and of the essential role of KBTBD4 for the rapid impact of UM171 on this epigenetic mark (
Moreover, reduction in RCOR1 levels further enhance the impact of HDAC or LSD inhibitors on CD201 and CD86 expression (
In further support to this model, it is shown that the induction of CD201 and CD86 by UM171 is strictly dependent on KBTBD4 in OCI-AML1 cells engineered by CRISPR/Cas9 to lack this gene (see sgKBTBD4 panels in
These results were validated in primary CD34+ human cord blood cells to show that CD34+CD201+ hematopoietic stem/progenitor cells (HSPC) exposed to UM171 show higher levels of both H3K27ac and H3K4me2 activation marks (
LSD1 is a member of a corepressor complex composed of LSD1, CoREST and HDAC1/2. This complex represses hematopoietic stem and progenitor cell transcriptional gene signatures likely by coupling LSD1 (H3K4me1/2 demethylase) and HDAC1/2 (e.g. H3K27ac deacetylase) activities. Recent studies showed that CoREST (or RCOR1) has a bi-lobed structure with the two enzymes located at opposite ends, a conformation which allows extensive crosstalk for cis activation or inhibition. As such, LSD1 inhibition is associated with some level of HDAC1/2 inhibition and vice-versa. Notably, HDAC inhibitors, in particular those inhibiting class I HDACs (including HDAC1/2) such as valproic acid, are capable of reversing the ex vivo dysfunction of human HSCs to some extent.
Consistent with the hypothesis presented in
In summary, and focusing on absolute CD34+CD201+ cell counts, it is disclosed that shRCOR1 or shLSD1 could at least partly mimic the impact of UM171 and that shKBTBD4 completely abrogates the UM171-induced HSPC phenotype (see
It is provided that chemical inhibition of LSD1 (partner to RCOR1 in CoREST complex) induced the surface expression of CD201 and CD86 independently of KBTBD4.
To further understand the epistatic relationship between KBTBD4 and the reported RCOR1-HDAC1/2-LSD1 complex, it is first demonstrated that RCOR1 levels are reduced in AML1 cells engineered to express high levels of KBTBD4 and that both Kelch (substrate-binding) and BTB (CUL3-binding) domains are essential (
In summary, using UM171 CRISPR-based screens and BioID-directed proteomics, it is provided a novel CRL3 complex that uses the hitherto uncharacterized KBTBD4 adaptor leading to RCOR1 protein loss. This, in turns, promotes the upregulation of several HSC essential genes including CD201 and CD86 through the downstream modulation of chromatin-bound LSD1-RCOR1-HDAC epigenetic modifiers (
Using purified human CD34+ cells, UM171 treatment reduces RCOR1 and LSD1 levels within 4 hours. HDAC2 levels were less affected in these cells at this time point (
Together these results document that UM171 operates through KBTBD4 to rapidly (within 4 hours) induce the degradation of CoREST members.
As seen in Table 1 below, UM171 and a variant (UM681) were tested to evaluate their ability in inhibiting cell proliferation in various cancerous cell line.
UM134 and UM681 have the following structure:
Further, as seen in Table 2, the antineoplastic effect of UM171, and two variants (UM681 and UM134) is also demonstrated in cancerous samples (acute myelogenous leukemia or “AML” samples) from patients.
As demonstrated herein, UM171 and its derivatives activate the CULLIN3-RING ubiquitin ligase complex (CRL3 complex described herein). The CRL3/KBTBD4 complex degrades RCOR1 which normally acts as the scaffolding protein for the RCOR1/LSD1 and HDAC2 complex, itself being dissociated in the presence of UM171.
Accordingly, it is encompassed a method of treating cancer in a subject following administration of the compound of general formula I as defined herein:
or a salt or a prodrug thereof,
wherein:
each Y is independently selected from N and CH;
Z is —CN; —C(O)OR1; —C(O)N(R1)R3; —C(O)R1; or -heteroaryl optionally substituted with one or more RA or R4 substituents, wherein, when (R1) and R3 are attached to a nitrogen atom, optionally they join together with the nitrogen atom to form a 3 to 7-membered ring which optionally includes one or more other heteroatom selected from N, O and S, optionally the ring is substituted with one or more RA or R4;
W is —CN; —N(R1)R3; —C(O)OR1; —C(O)N(R1)R3; —NR1C(O)R1; —NR1C(O)OR1; —OC(O)N(R1)R3; —OC(O)R1; —C(O)R1; —NR1C(O)N(R1)R3; —NR1S(O)2R1; -benzyl optionally substituted with 1, 2 or 3 RA or R1 substituents; —X-L-(X-L)n; —N(R1)R3; —X-L-(X-L)nheteroaryl optionally substituted with one or more RA or R4 substituents attached on either or both the L and heteroaryl groups; —X-L-(X-L)n—heterocyclyl optionally substituted with one or more RA or R4 substituents attached on either or both the L and heterocyclyl groups; —X-L-(X-L)n- aryl optionally substituted with one or more RA or R4 substituents; —X-L-(X-L)n-NR1RA or —(N(R1)-L)n- N+R1R3R5 R6−, wherein n is an integer equal to either 0, 1, 2, 3, 4, or 5,
and wherein, when R1 and R3 are attached to a nitrogen atom, optionally they join together with the nitrogen atom to form a 3 to 7-membered ring which optionally includes one or more other heteroatom selected from N, O and S, optionally the ring is substituted with one or more RA or R4;
each X is independently selected from C, O, S, and NR1;
L is each independently —C1-6 alkylene; —C2-6 alkenylene; —C2-6 alkynylene; —C3-7 cycloalkylene, which optionally includes one or more other heteroatom selected from N, O and S; or —C3-7 cycloalkenylene, which optionally includes one or more other heteroatom selected from N, O and S, wherein the alkylene, the alkenylene, the alkynylene, the cycloalkylene and the cycloalkenylene groups are each independently optionally substituted with one or two R4 or RA substituent;
R1 is each independently —H; —C1-6 alkyl; —C2-6 alkenyl; —C2-6 alkynyl; —C3-7 cycloalkyl; —C3-7 cycloalkenyl; —C1-6 perfluorinated; -heterocyclyl; -aryl; -heteroaryl; or -benzyl, wherein the alkyl, the alkenyl, the alkynyl, the cycloalkenyl, the perfluorinated alkyl, the heterocyclyl, the aryl, the heteroaryl and the benzyl groups are each independently optionally substituted with 1, 2 or 3 RA or Rd substituents;
R2 is —H; —C1-6 alkyl, optionally substituted with one more RA substituents; —C(O)R4; -L-heteroaryl optionally substituted with one or more RA or R4 substituents; -L-heterocyclyl optionally substituted with one or more RA or R4; or -L-aryl optionally substituted with one or more RA or R4 substituents;
R3 is each independently —H; —C1-6 alkyl; —C2-6 alkenyl; —C2-6 alkynyl; —C3-7 cycloalkyl; —C3-7 cycloalkenyl; —C1-6 perfluorinated; -heterocyclyl; -aryl; -heteroaryl; or -benzyl, wherein the alkyl, the alkenyl, the alkynyl, the cycloalkyl, the cycloalkenyl, the perfluorinated alkyl, the heterocyclyl, the aryl, the heteroaryl and the benzyl groups are each independently optionally substituted with 1, 2 or 3 RA or Rd substituents;
R4 is each independently —H; —C1-6 alkyl; —C2-6 alkenyl; —C2-6 alkynyl; —C3-7 cycloalkyl; —C3-7 cycloalkenyl; —C1-6 perfluorinated; -heterocyclyl; -aryl; -heteroaryl, or -benzyl; wherein the alkyl, the alkenyl, the alkynyl, the cycloalkyl, the cycloalkenyl, the perfluorinated alkyl, the heterocyclyl, the aryl, the heteroaryl and the benzyl groups are each independently optionally substituted with 1, 2 or 3 RA or Rd substituents;
R5 is each independently —C1-6 alkyl; —C1-6 alkylene-C2-6 alkenyl which optionally includes one or more other heteroatom selected from N, O and S; —C1-6 alkylene-C2-6 alkynyl which optionally includes one or more other heteroatom selected from N, O and S; -L-aryl which optionally includes one or more RA or R4 substituents; -L-heteroaryl which optionally includes one or more RA or R4 substituents; —C1-6 alkylene-C(O)O—; —C1-6 alkylene-C(O)OR1; —C1-6 alkylene-CN; —C1-6 alkylene-C(O)NR1R3, wherein R1 and R3 optionally they join together with the nitrogen atom to form a 3 to 7-membered ring which optionally includes one or more other heteroatom selected from N, O and S; or —C1-6 alkylene-OH;
R6 is halogen; —OC(O)CF3; or —OC(O)R1;
RA is each independently -halogen; —CF3; —OR1; -L-OR1; —OCF3; —SR1; —CN; —NO2; —NR1R3; -L-NR1 R1; —C(O)OR1; —S(O)2R4; —C(O)N(R1)R3; —NR1C(O)R1; —NR1C(O)OR1; —OC(O)N(R1)R3; —OC(O)R1; —C(O)R4; —NHC(O)N(R1)R3; —NR1C(O)N(R1)R3; or —N3; and Rd is each independently —H; —C1-6 alkyl; —C2-6 alkenyl; —C2-6 alkynyl; —C3-7 cycloalkyl; —C3-7 cycloalkenyl; —C1-5 perfluorinated; -benzyl; or -heterocyclyl.
In one embodiment, the compound has the formula:
In one embodiment, in any formula herein comprising m substituents Z, m is an integer of 1 or 2.
In one embodiment, Z is —C(O)OR1, or -heteroaryl optionally substituted with one or more RA or R1 substituents, R2 is H, —C1-6 alkyl optionally substituted with one or more RA substituents or -L-aryl optionally substituted with one or more RA or R4 substituents, W is —N(R1)R3 wherein R1 is C3-7 cycloalkyl substituted by RA and R3 is H.
In one embodiment, Z is —C(O)O—C1-4 alkyl or 5-membered ring heteroaryl, the heteroaryl comprising 2-4 heteroatoms (N or O), R2 is H, or -L-aryl optionally substituted by halogen, OR1, C1-6 alkyl optionally substituted by RA, C(O)R4, -heterocyclyl, C(O)OR4 OR C2-6 alkynyl, W is —N(R1)R3 wherein R1 is cyclohexyl substituted by RA, and R3 is H.
In accordance with another embodiment,
Z is CO2Me or 2-methyl-2H-tetrazol-5-yl;
R2 is benzyl, or H; and
W is NH-L-N(R1)R3 wherein L is C2-4 alkylene or C3-7 cycloalkylene and R1 and R3 is C1-4 alkyl or H; or R1 and R3 join together with the nitrogen atom to which they are attached to form a 3 to 7-membered ring, which optionally includes one or more other heteroatom selected from N, O and S, optionally the ring is substituted with one or more RA or R4.
In accordance with another embodiment,
Z is —C(O)O—C1-4 alkyl or 5-membered ring heteroaryl, the heteroaryl comprising 2-4 heteroatoms (N or O) or an aryl comprising 6 carbon atoms;
R2 is =H, or —CH2-aryl optionally substituted by substituted by halogen, OR1, C1-6 alkyl optionally substituted by RA, C(O)R4, -heterocyclyl, C(O)OR4 OR C2-6 alkynyl, wherein the aryl is phenyl; or R2 is —C1-6 alkylene-heteroaryl or —C1-6 alkylene-aryl, optionally substituted with one or more RA or R4 substituents; and
W is —X-L-(X-L)n-N(R1)R3, X=NR1 wherein R1 is H or C1-6 alkyl (such as a methyl), n=0, (R1) and R3 are attached to the nitrogen atom to form a 3 to 7-membered ring (such as a pyperidinyl or pyrrolidinyl ring) and L is —C1-6 alkylene (such as a propyl or ethyl chain), or W=—X-L-(X-L)n-NR1RA, X=NR1, wherein R1 is H or C1-6 alkyl (such as a methyl), n=0, R1 and RA=respectively —H, or —C1-6 alkyl (for R1) and RA is as described herein, and for example as illustrated in table 3 herein.
In accordance with another embodiment,
Z is COOMe, COOEt, methyl-tetrazole, ethyl-tetrazole or methyl-oxadiazole, thiophenyl, or phenyl;
R2 is H, —CH3, —CH2N(CH3)2, —CH2-phenyl, —CH2CH2-phenyl, —CH2-thiophenyl, —CH2-pyridyl, CH2-cyclohexyl, —NH-phenyl, —CH(Obenzyl)-phenyl, —CH(OH)-phenyl, —CH(CH3)-phenyl, —C(O)-phenyl, —CH2naphthyl, optionally substituted with one or more RA or R4 substituents; and
W is —X-L-(X-L)n-N(R1)R3, X=NR1 wherein R1 is H or C1-6 alkyl (such as a methyl), n=0, (R1) and R3 are attached to the nitrogen atom to form a 3 to 7-membered ring (such as a pyperidinyl or pyrrolidinyl ring) and L is —C1-6 alkylene (such as a propyl or ethyl chain), or W=—X-L-(X-L)n-NR1RA, X=NR1, wherein R1 is H or C1-6 alkyl (such as a methyl), n=0, R1 and RA=respectively —H, or —C1-6 alkyl (for R1) and RA is as described herein, and for example as illustrated in table 3 herein, and in particular W=
In one embodiment, Z is —C(O)O—C1-4 alkyl or 5-membered ring heteroaryl, the heteroaryl comprising 2-4 heteroatoms (N or O) or an aryl comprising 6 carbon atoms.
In one embodiment, Z is COOMe, COOEt, methyl-tetrazole, ethyl-tetrazole or methyl-oxadiazole, thiophenyl, or phenyl.
In one embodiment, Z is COOMe, COOEt, tetrazole or oxadiazole.
In one embodiment, R2 is =H, or —CH2-aryl optionally substituted by substituted by halogen, OR1, C1-6 alkyl optionally substituted by RA, C(O)R4, -heterocyclyl, C(O)OR4 OR C2-6 alkynyl, wherein the aryl is phenyl.
In one embodiment, R2 is H, —C1-6 alkylene-heteroaryl or —C1-6 alkylene-aryl, optionally substituted with one or more RA or R4 substituents.
In one embodiment, R2 is H, —CH3, —CH2N(CH3)2, —CH2-phenyl, —CH2CH2-phenyl, —CH2-thiophenyl, —CH2-pyridyl, CH2-cyclohexyl, —NH-phenyl, —CH(Obenzyl)-phenyl, —CH(OH)-phenyl, —CH(CH3)-phenyl, —C(O)-phenyl, —CH2naphthyl, optionally substituted with one or more RA or R4 substituents.
In accordance with another embodiment, W=—X-L-(X-L)n-N(R1)R3, X=NR1 wherein R1 is H or C1-6 alkyl (such as a methyl), n=0, (R1) and R3 are attached to the nitrogen atom to form a 3 to 7-membered ring (such as a pyperidinyl or pyrrolidinyl ring) and L is —C1-6 alkylene (such as a propyl or ethyl chain).
In one embodiment, W=—X-L-(X-L)n-NR1RA, X=NR1, wherein R1 is H or C1-6 alkyl (such as a methyl), n=0, R1 and RA=respectively —H, or —C1-6 alkyl (for R1) and RA is as described herein, and for example as illustrated in table 3 herein.
In accordance with another embodiment, the compound is of Formula I wherein W is
The compounds of formula I (including the representative compounds set forth below) disclosed herein, including the preparation and characterization thereof, are described in PCT publication No. WO 2013/110198, the content of which is incorporated by reference in its entirety as well as in the synthetic methodology section found below.
As used herein, the term “alkyl” is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, for example, C1-C6 in C1-C6 alkyl is defined as including groups having 1, 2, 3, 4, 5 or 6 carbons in a linear or branched saturated arrangement. Examples of C1-C6 alkyl as defined above include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, and hexyl.
As used herein, the term “cycloalkyl” is intended to mean a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms therein, for example, C3-C7 in C3-C7 cycloalkyl is defined as including groups having 3, 4, 5, 6 or 7 carbons in a monocyclic saturated arrangement. Examples of C3-C7 cycloalkyl as defined above include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
As used herein, the term, “alkenyl” is intended to mean unsaturated straight or branched chain hydrocarbon groups having the specified number of carbon atoms therein, and in which at least two of the carbon atoms are bonded to each other by a double bond, and having either E or Z regiochemistry and combinations thereof. For example, C2-C6 in C2-C6 alkenyl is defined as including groups having 2, 3, 4, 5 or 6 carbons in a linear or branched arrangement, at least two of the carbon atoms being bonded together by a double bond. Examples of C2-C6 alkenyl include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl, 1-butenyl and the like.
As used herein, the term “alkynyl” is intended to mean unsaturated, straight chain hydrocarbon groups having the specified number of carbon atoms therein and in which at least two carbon atoms are bonded together by a triple bond. For example C2-C4 alkynyl is defined as including groups having 2, 3 or 4 carbon atoms in a chain, at least two of the carbon atoms being bonded together by a triple bond. Examples of such alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl and the like.
As used herein, the term “cycloalkenyl” is intended to mean a monocyclic saturated aliphatic hydrocarbon group having the specified number of carbon atoms therein, for example, C3-C7 in C3-C7 cycloalkenyl is defined as including groups having 3, 4, 5, 6 or 7 carbons in a monocyclic arrangement. Examples of C3-C7 cycloalkenyl as defined above include, but are not limited to, cyclopentenyl, cyclohexenyl and the like.
As used herein, the term “halo” or “halogen” is intended to mean fluorine, chlorine, bromine or iodine.
As used herein, the term “haloalkyl” is intended to mean an alkyl as defined above, in which each hydrogen atom may be successively replaced by a halogen atom. Examples of haloalkyl include, but are not limited to, CH2F, CHF2 and CF3.
As used herein, the term “aryl,” either alone or in combination with another radical, means a carbocyclic aromatic monocyclic group containing 6 carbon atoms which may be further fused to a second 5- or 6-membered carbocyclic group which may be aromatic, saturated or unsaturated. Examples of aryl include, but are not limited to, phenyl, indanyl, 1-naphthyl, 2-naphthyl, tetrahydronaphthyl and the like. The aryl may be connected to another group either at a suitable position on the cycloalkyl ring or the aromatic ring.
As used herein, the term “heteroaryl” is intended to mean a monocyclic or bicyclic ring system of up to 10 atoms, wherein at least one ring is aromatic, and contains from 1 to 4 hetero atoms selected from the group consisting of O, N, and S. The heteroaryl may be attached either via a ring carbon atom or one of the heteroatoms. Examples of heteroaryl include, but are not limited to, thienyl, benzimidazolyl, benzo[b]thienyl, furyl, benzofuranyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, napthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, isothiazolyl, isochromanyl, chromanyl, isoxazolyl, furazanyl, indolinyl, isoindolinyl, thiazolo[4,5-b]-pyridine, tetrazolyl, oxadiazolyl, thiadiazolyl, thienyl, pyrimido-indolyl, pyrido-indolyl, pyrido-pyrrolo-pyrimidinyl, pyrrolo-dipyridinyl and fluoroscein derivatives.
As used herein, the term “heterocycle,” “heterocyclic” or “heterocyclyl” is intended to mean a 3, 4, 5, 6, or 7 membered non-aromatic ring system containing from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Examples of heterocycles include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, piperidyl, 3,5-dimethylpiperidyl, pyrrolinyl, piperazinyl, imidazolidinyl, morpholinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, tetrahydro-1H-thieno[3,4-d]imidazole-2(3H)-one, diazirinyl, and the like, where the attachment to the ring can be on either the nitrogen atom or a carbon atom of the ring such as described hereafter:
As used herein, the term “optionally substituted with one or more substituents” or its equivalent term “optionally substituted with at least one substituent” is intended to mean that the subsequently described event of circumstances may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. The definition is intended to mean from zero to five substituents.
As used herein, the term “subject” or “patient” is intended to mean humans and non-human mammals such as primates, cats, dogs, swine, cattle, sheep, goats, horses, rabbits, rats, mice and the like.
If the substituents themselves are incompatible with the synthetic methods described herein, the substituent may be protected with a suitable protecting group (PG) that is stable to the reaction conditions used in these methods. The protecting group may be removed at a suitable point in the reaction sequence of the method to provide a desired intermediate or target compound. Suitable protecting groups and the methods for protecting and de-protecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which may be found in T. Greene and P. Wuts, “Protecting Groups in Chemical Synthesis” (4th ed.), John Wiley & Sons, NY (2007), which is incorporated herein by reference in its entirety. Examples of protecting groups used throughout include, but are not limited to, Fmoc, Bn, Boc, CBz and COCF3. In some instances, a substituent may be specifically selected to be reactive under the reaction conditions used in the methods described herein. Under these circumstances, the reaction conditions convert the selected substituent into another substituent that is either useful in an intermediate compound in the methods described herein or is a desired substituent in a target compound.
As used herein, the term “pharmaceutically acceptable salt” is intended to mean both acid and base addition salts.
As used herein, the term “pharmaceutically acceptable acid addition salt” is intended to mean those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
As used herein, the term “pharmaceutically acceptable base addition salt” is intended to mean those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
The compounds encompassed herein or their pharmaceutically acceptable salts may contain one or more asymmetric centers, chiral axes and chiral planes and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms and may be defined in terms of absolute stereochemistry, such as (R)- or (S)- or, as (D)- or (L)- for amino acids. The present is intended to include all such possible isomers, as well as, their racemic and optically pure forms. Optically active (+) and (−), (R)- and (S)- or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as reverse phase HPLC. The racemic mixtures may be prepared and thereafter separated into individual optical isomers or these optical isomers may be prepared by chiral synthesis. The enantiomers may be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts which may then be separated by crystallization, gas-liquid or liquid chromatography, selective reaction of one enantiomer with an enantiomer specific reagent. It will also be appreciated by those skilled in the art that where the desired enantiomer is converted into another chemical entity by a separation technique, an additional step is then required to form the desired enantiomeric form. Alternatively specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts, or solvents or by converting one enantiomer to another by asymmetric transformation.
Certain compounds encompassed herein may exist as a mix of epimers. Epimers means diastereoisomers that have the opposite configuration at only one of two or more stereogenic centers present in the respective compound.
Compounds encompassed herein may exist in Zwitterionic form and the present includes Zwitterionic forms of these compounds and mixtures thereof.
In addition, the compounds encompassed herein also may exist in hydrated and anhydrous forms. Hydrates of the compound of any of the formulas described herein are included. In a further embodiment, the compound according to any of the formulas described herein is a monohydrate. In embodiments, the compounds described herein comprise about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, about 0.5% or less, about 0.1% or less by weight of water. In other embodiments, the compounds described herein comprise, about 0.1% or more, about 0.5% or more, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, or about 6% or more by weight of water.
It may be convenient or desirable to prepare, purify, and/or handle the compound in the form of a prodrug. Thus, the term “prodrug”, as used herein, pertains to a compound which, when metabolized (e.g., in vivo), yields the desired active compound. Typically, the prodrug is inactive, or less active than the desired active compound, but may provide advantageous handling, administration, or metabolic properties. Unless otherwise specified, a reference to a particular compound also includes prodrugs thereof.
In one embodiment, the compounds as defined herein or a pharmaceutically acceptable salt thereof, are provided in association with one or more pharmaceutically acceptable carrier.
Many pharmaceutically acceptable carriers are known in the art. It will be understood by those in the art that a pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and tolerated by a subject in need thereof. The pharmaceutical compositions can be formulated according to known methods for preparing pharmaceutically useful compositions. Carriers are described in a number of sources which are well known and readily available to those skilled in the art.
The proportion of each carrier is determined by the solubility and chemical nature of the agent(s), the route of administration, and standard pharmaceutical practice.
In order to ensure consistency of administration, in an embodiment, the pharmaceutical composition may be in the form of a unit dose.
The compounds or composition may be for oral administration as exemplified in
The solid oral compositions/dosage form may be prepared by conventional methods of blending, filling, tabletting, or the like. Repeated blending operations may be used to distribute the active agent(s) throughout those compositions employing carriers. Such operations are, of course, conventional in the art.
The solid oral compositions/dosage forms may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.
Oral liquid preparations may be in the form of emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicles before use. Such liquid preparations may or may not contain conventional additives.
The compounds or composition may be for parenteral injection; this being intramuscularly, intravenously (as exemplified in
For parenteral administration, the compounds or composition may be used in the form of sterile solutions, optionally containing solutes, for example sufficient saline or glucose to make the solution isotonic. The compounds or composition for parenteral injection may be prepared by utilizing the compounds and a sterile vehicle, and, depending on the concentration employed, the compounds may be either suspended or dissolved in the vehicle. Once in solution, the compounds may be injected and filter sterilized before filling a suitable vial or ampoule followed by subsequently sealing the carrier or storage package.
In one embodiment, the compound or pharmaceutical composition comprising said compounds can be further used in combination with at least one additional active ingredient.
It is encompassed that the compound encompassed herein be administered orally, intravenously, or subcutaneously.
It is also encompassed a method of treating cancer in a subject following transplantation of a cord blood graft obtained following expansion using the compound of formula I.
In a particular embodiment, the compound of formula I is UM171 which as the following structure:
In an embodiment, the compound of formula I is
or a pharmaceutically acceptable salt thereof.
In another embodiment, the compound of formula I is a hydrobromide salt of
In a supplemental embodiment, the compound of formula I is
or a pharmaceutically acceptable salt thereof.
The compounds pyrimido[4,5-B]indole derivatives disclosed herein may for example be prepared according to U.S. Pat. Nos. 9,409,906 and 10,336,747. The 5H-pyrido[4,3-b]indole derivatives and pyrido[2′,3′:4,5]pyrrolo[2,3-d]pyrimidine derivatives may for example be prepared according to U.S. Pat. No. 10,647,718 B, the contents of which are incorporated herein by reference.
Alternatively, representative compounds disclosed herein can be prepared according to the following chemistry examples. It will be apparent to the skilled person that the other compounds may be prepared using similar conditions, using different starting materials.
In a 100 mL round-bottomed flask were added methyl 4-chloro-9H-pyrimido[4,5-b]indole-7-carboxylate (0.1 g, 0.382 mmol) and N,N-Dimethylformamide dimethyl acetal (0.297 ml, 2.217 mmol) in toluene (35 ml) to give a tan suspension. Heated to 110° C. for 17 hours. The reaction mixture was cooled to 20° C. and concentrated to dryness to give 94 mg as a tan solid. The residue was purified on ISCO (RediSep 12 g column eluting with CH2Cl2/EtOAc) to give methyl 4-chloro-9-methyl-9H-pyrimido[4,5-b]indole-7-carboxylate (53 mg, 0.192 mmol, 50.3% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 3.96 (s, 3H) 4.03 (s, 3H) 8.08 (dd, J=8.2, 1.6 Hz, 1H) 8.39 (dd, J=1.6, 0.8 Hz, 1H) 8.46 (dd, J=8.2, 0.8 Hz, 1H) 8.93 (s, 1H). LCMS m/z 276.0 (M+H)+.
In a microwave vial was added methyl 4-chloro-9-methyl-9H-pyrimido[4,5-b]indole-7-carboxylate (0.02 g, 0.073 mmol), N-methyl-3-(piperidin-1-yl)propan-1-amine (0.024 g, 0.109 mmol) and triethylamine (0.020 ml, 0.145 mmol) in methanol (0.76 ml) to give a tan suspension. The vial was sealed and heated in the microwave to 140° C. for 15 minutes. The reaction mixture was concentrated to dryness to give a red oil. The residue was purified on ISCO (RediSep 4 g column eluting with CH2Cl2/MeOH/NH4OH) to give methyl 9-methyl-4-(methyl(3-(piperid in-1-yl)propyl)amino)-9H-pyrimido[4,5-b]indole-7-carboxylate (21 mg, 0.053 mmol, 73.2 yield) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.13-1.33 (m, 6H) 1.74-1.90 (m, 2H) 2.00-2.25 (m, 6H) 3.29 (s, 3H) 3.82 (t, J=6.8 Hz, 2H) 3.91 (s, 3H) 3.92 (s, 3H) 7.91 (dd, J=8.6, 1.6 Hz, 1H) 8.02 (d, J=8.6 Hz, 1H) 8.19 (d, J=1.6 Hz, 1H) 8.45 (s, 1H). HRMS m/z 396.2517 (M+H)+.
The title compound was obtained according to the procedure described in example 1. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.32-1.44 (m, 2H) 1.45-1.57 (m, 4H) 1.76-1.91 (m, 2H) 2.25-2.45 (m, 6H) 3.61-3.70 (m, 2H) 3.89 (s, 3H) 3.92 (s, 3H) 7.49 (t, J=5.3 Hz, 1H) 7.90 (dd, J=8.2, 1.6 Hz, 1H) 8.19 (d, J=1.6 Hz, 1H) 8.44 (s, 1H) 8.46 (d, J=8.2 Hz, 1H). HRMS m/z 382.2374 (M+H)+.
In a 200 mL round-bottomed flask was added potassium 1,3-dioxoisoindolin-2-ide (4.75 g, 25.6 mmol) in DMF (30.0 ml) to give a white suspension. 1,4-dichlorobut-2-yne (20.06 ml, 205 mmol) was added. Heated the brown suspension to 100° C. After 2 hrs, cooled to 20° C. Water (50.0 ml) was added and concentrated to dryness to give a brown solid. The solid was partitioned between water (75 mL) and CH2Cl2 (50 mL). Separated layers and extracted aqueous layer with CH2Cl2 (50 mL). The combined organic layer were washed with water (50 mL). The organic layer was dried over MgSO4, filtered and concentrated to give 6.8 g as a brown solid. The residue was purified on ISCO (RediSep 120 g column eluting with hexane/CH2Cl2) to give 2-(4-chlorobut-2-yn-1-yl)isoindoline-1,3-dione (3.90 g, 16.69 mmol, 65.1% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 4.44 (t, J=2.0 Hz, 2H) 4.47 (t, J=2.0 Hz, 2H) 7.81-7.96 (m, 4H). LCMS m/z 234.0 (M+H)+.
In a 100 mL round-bottomed flask were added 2-(4-chlorobut-2-yn-1-yl)isoindoline-1,3-dione (1 g, 4.28 mmol) and piperidine (0.932 ml, 9.42 mmol) in acetonitrile (15 ml) to give a yellow solution. Heated to 80° C. and stirred the resulting suspension for 16.5 hours. Concentrated to dryness to give an orange solid. Partitioned the mixture between CH2Cl2 (30 mL) and water (20 mL). Separated layers. Extracted aqueous with CH2Cl2 (10 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to give 1.65 g as an orange oil. The residue was purified on ISCO (RediSep 40 g column eluting with CH2Cl2/EtOAc) to give 2-(4-(piperidin-1-yl)but-2-yn-1-yl)isoindoline-1,3-dione (752 mg, 2.66 mmol, 62.2% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.31 (s, 2H) 1.46 (quin, J=5.7 Hz, 4H) 2.33 (s, 3H) 3.18 (s, 2H) 4.40 (t, J=2.2 Hz, 2H) 7.84-7.89 (m, 2H) 7.89-7.94 (m, 2H). LCMS m/z 283.2 (M+H)+.
In a 50 mL round-bottomed flask were added 2-(4-(piperidin-1-yl)but-2-yn-1-yl)isoindoline-1,3-dione (0.752 g, 2.66 mmol) and hydrazine hydrate (0.152 ml, 2.80 mmol) in ethanol (13 ml) to give a yellow solution. Heated to reflux (ca. 80° C.) for 3 hours then added hydrazine hydrate (0.043 ml, 0.799 mmol) and continued heating to reflux for 1 hour. Cooled to 20° C. and stirred for 16 hours. Cooled to 0° C. and stirred the suspension for 1 hour. Filtered the solids over a Buchner funnel and rinsed solids with cold ethanol (2×1 mL). Concentrated the filtrate to dryness to give 615 mg as a yellow solid. Suspended the solid in Et2O (10 mL) and filtered over a Buchner funnel. Rinsed solids with Et2O (5 mL). Concentrated to dryness on rotovap to give 4-(piperidin-1-yl)but-2-yn-1-amine (315 mg, 2.069 mmol, 78% yield) as a yellow oil. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.29-1.41 (m, 2H) 1.49 (quin, J=5.6 Hz, 4H) 2.25-2.43 (m, 4H) 3.16 (t, J=2.1 Hz, 2H) 3.28 (t, J=2.1 Hz, 2H). LCMS m/z 153.2 (M+H)+.
The title compound was obtained according to the procedure described in example 1 using methyl 4-chloro-9H-pyrimido[4,5-b]indole-7-carboxylate and 4-(piperidin-1-yl)but-2-yn-1-amine. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.22-1.35 (m, 2H) 1.44 (quin, J=5.4 Hz, 4H) 2.28-2.40 (m, 4H) 3.17 (br. s., 2H) 3.90 (s, 3H) 4.43 (d, J=5.9 Hz, 2H) 7.81-7.89 (m, 2H) 8.06 (d, J=1.6 Hz, 1H) 8.43-8.48 (m, 2H) 12.24 (s, 1H). LCMS m/z 378.2 (M+H)+.
In a vial was added 7-bromo-N-(3-(piperidin-1-yl)propyl)-9H-pyrimido[4,5-b]indol-4-amine (0.04 g, 0.103 mmol), phenylboronic acid (0.025 g, 0.206 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.018 g, 0.015 mmol). The vial was sealed and evacuated with nitrogen (3 cycles vacuum+nitrogen refill). 1,4-dioxane (0.52 ml) and sodium carbonate 2M in water (0.309 ml, 0.618 mmol) were added. The vial was evacuated with nitrogen (3 cycles vacuum+nitrogen refill). Heated the biphasic mixture to 85° C. and stirred for 18 hrs. Cooled to 20° C. and diluted with methanol (2 mL) and CH2Cl2 (2 mL). Filtered the mixture over a 0.45 μm filter. Rinsed the vial and filter with methanol (2×2 mL) and CH2C12 (2×2 mL). Concentrated to dryness to give an orange solid. The residue was purified on ISCO (RediSep 12 g column eluting with CH2Cl2/MeOH/NH4OH) to give 7-phenyl-N-(3-(piperidin-1-yl)propyl)-9H-pyrimido[4,5-b]indol-4-amine (33 mg, 0.086 mmol, 83% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (br. s., 2H) 1.46-1.59 (m, 4H) 1.77-1.91 (m, 2H) 2.38 (br. s., 6H) 3.64 (q, J=6.5 Hz, 2H) 7.24 (t, J=4.9 Hz, 1H) 7.34-7.41 (m, 1H) 7.50 (t, J=7.6 Hz, 2H) 7.54 (dd, J=8.2, 1.2 Hz, 1H) 7.66 (d, J=1.2 Hz, 1H) 7.74 (d, J=7.4 Hz, 2H) 8.34 (s, 1H) 8.38 (d, J=8.2 Hz, 1H) 11.93 (s, 1H). LCMS m/z 386.2 (M+H)+.
In a 50 mL round-bottomed flask was added NaH 60% wt. in mineral oil (0.265 g, 6.62 mmol) in DMF (10 ml) to give a grey suspension. Cooled to 0° C. and added methyl 2-hydroxy-2-phenylacetate (1 g, 6.02 mmol) was added. The resulting orange suspension was stirred for 45 minutes. Benzyl bromide (0.787 ml, 6.62 mmol) was added. Warmed to 20° C. and stirred for 30 minutes. Quenched the reaction by adding sat. NH4C1 (40 mL)+water (10 mL). Extracted aqueous layer with EtOAc (2×50 mL). Washed the combined organic layer with water (2×50 mL) then with brine (35 mL). The organic layer was dried over MgSO4, filtered and concentrated to give 1.72 g as a light yellow oil. The residue was purified on ISCO (RediSep Gold 40 g column eluting with Hexane/Et2O). The ISCO was repeated two more times to give methyl 2-(benzyloxy)-2-phenylacetate (508 mg, 1.982 mmol, 32.9% yield) as a colorless oil. 1H NMR (400 MHz, DMSO-d6) δ ppm 3.64 (s, 3H) 4.49 (d, J=11.3 Hz, 1H) 4.59 (d, J=11.3 Hz, 1H) 5.11 (s, 1H) 7.04-7.19 (m, 1H) 7.23-7.45 (m, 9H).
In a microwave vial was added methyl 2-amino-3-carbamoyl-1H-indole-6-carbonate (0.185 g, 0.793 mmol), methyl 2-(benzyloxy)-2-phenylacetate (0.508 g, 1.983 mmol) and sodium methoxide 30% wt. in methanol (0.372 ml, 1.983 mmol) in methanol (2 ml). The vial was sealed and heated in the microwave to 140° C. for 1 h. Cooled to 20° C. and added AcOH (0.118 ml, 2.062 mmol). The resulting suspension was stirred at 20° C. for 1 hour. The solids were collected on Buchner. Cake was washed with methanol (3×0.5 mL). Dried the product at 20° C. under high vacuum until constant weight to give methyl 2-((benzyloxy)(phenyl)methyl)-4-hydroxy-9H-pyrimido[4,5-b]indole-7-carboxylate (238 mg, 0.542 mmol, 68.3% yield) as a tan solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 3.87 (s, 3H) 4.58 (d, J=11.7 Hz, 1H) 4.69 (d, J=11.7 Hz, 1H) 5.56 (s, 1H) 7.27-7.46 (m, 8H) 7.55-7.64 (m, 2H) 7.84 (dd, J=8.2, 1.2 Hz, 1H) 8.00-8.06 (m, 2H) 12.49 (br. s., 1H) 12.60 (br. s., 1H). LCMS m/z 440.2 (M+H)+.
In a 15 mL round-bottomed flask was added methyl 2-((benzyloxy)(phenyl)methyl)-4-hydroxy-9H-pyrimido[4,5-b]indole-7-carboxylate (0.234 g, 0.532 mmol) in phosphorus oxychloride (3.47 ml, 37.3 mmol) and heated to 85° C. for 16.5 hrs. Concentrated to dryness. The residue was suspended in sat. NaHCO3 (10 mL) and stirred for 1 hour. The solids were collected on Buchner. Cake was washed with water (3×2 mL). Dried the product at 40° C. under high vacuum until constant weight to give methyl 2-((benzyloxy)(phenyl)methyl)-4-chloro-9H-pyrimido[4,5-b]indole-7-carboxylate (228 mg, 0.498 mmol, 94% yield) as a tan solid. LCMS m/z 458.2 (M+H)+.
In a microwave vial was added methyl 2-((benzyloxy)(phenyl)methyl)-4-chloro-9H-pyrimido[4,5-b]indole-7-carboxylate (0.228 g, 0.498 mmol), 3-(piperidin-1-yl)propan-1-amine (0.158 ml, 0.996 mmol) and triethylamine (0.173 ml, 1.245 mmol) in methanol (3.8 ml). The vial was sealed and heated in the microwave to 140° C. for 30 minutes. Concentrated to dryness. The residue was purified on ISCO (RediSep Gold 40 g eluting with CH2Cl2/MeOH/NH4OH). The ISCO purification was repeated two times to give methyl 2-((benzyloxy)(phenyl)methyl)-4-((3-(piperidin-1-yl)propyl)amino)-9H-pyrimido[4,5-b]indole-7-carboxylate (172 mg, 0.305 mmol, 61.3% yield) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.29-1.42 (m, 2H) 1.42-1.56 (m, 4H) 1.72-1.91 (m, 2H) 2.18-2.47 (m, 6H) 3.66 (tt, J=13.2, 6.6 Hz, 2H) 3.88 (s, 3H) 4.54 (d, J=11.7 Hz, 1H) 4.64 (d, J=12.1 Hz, 1H) 5.50 (s, 1H) 7.21-7.43 (m, 8H) 7.51 (t, J=5.9 Hz, 1H) 7.57 (d, J=7.0 Hz, 2H) 7.82 (dd, J=8.2, 1.2 Hz, 1H) 8.00 (d, J=1.2 Hz, 1H) 8.38 (d, J=8.2 Hz, 1H) 12.22 (s, 1H). HRMS m/z 564.2979 (M+H)+.
In a 10 mL round-bottomed flask were added methyl 4-((3-((3-aminopropyl)(methyl)amino)propyl)-amino)-2-benzyl-9H-pyrimido[4,5-13]indole-7-carboxylate (25 mg, 0.054 mmol) and tert-butylamine (8.63 μl, 0.081 mmol) in THF (2.7 ml). Propargyl bromide (7.26 μl, 0.065 mmol) was added and stirred at 20° C. for 69.5 hrs. Concentrated to dryness. The residue was purified on ISCO (RediSep 12 g column eluting with CH2Cl2/MeOH/NH4OH) to give methyl 2-benzyl-4-((3-(methyl(3-(prop-2-yn-1-ylamino)propyl)amino)propyl)amino)-9H-pyrimido[4,5-b]indole-7-carboxylate (3.2 mg, 6.42 μmol, 11.82% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.23 (s, 1H) 1.56 (dt, J=14.1, 7.0 Hz, 2H) 1.80 (dt, J=13.5, 6.6 Hz, 2H) 2.20 (s, 3H) 2.34-2.46 (m, 4H) 2.56 (t, J=7.0 Hz, 2H) 2.99 (t, J=2.2 Hz, 1H) 3.25 (d, J=2.2 Hz, 2H) 3.58-3.70 (m, 2H) 3.88 (s, 3H) 4.04 (s, 2H) 7.15-7.23 (m, 1H) 7.28 (t, J=7.6 Hz, 2H) 7.37 (m, J=7.4 Hz, 2H) 7.54 (t, J=5.5 Hz, 1H) 7.83 (dd, J=8.2, 1.2 Hz, 1H) 7.99 (d, J=1.2 Hz, 1H) 8.27 (d, J=8.2 Hz, 1H) 12.05 (s, 1H). HRMS m/z 499.2823 (M+H)+.
In a microwave vial was added 2-benzyl-4-chloro-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indole (0.100 g, 0.266 mmol) and N1-(3-aminopropyl)propane-1,3-diamine (0.375 ml, 2.66 mmol) in methanol (2 ml). The vial was sealed and heated in the microwave to 140° C. for 30 minutes. Concentrated to dryness. The residue was purified on ISCO (RediSep 12 g column eluting with CH2Cl2/MeOH/NH4OH) to give N1-(3-aminopropyl)-N3-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)propane-1,3-diamine (112 mg, 0.238 mmol, 89% yield) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.54 (quin, J=6.85 Hz, 2H) 1.79 (quin, J=6.26 Hz, 2H) 2.53-2.69 (m, 6H) 3.68 (q, J=6.26 Hz, 2H) 4.04 (s, 2H) 4.43 (s, 3H) 7.15-7.22 (m, 1H) 7.28 (t, J=7.43 Hz, 2H) 7.38 (d, J=7.43 Hz, 2H) 7.57 (t, J=4.89 Hz, 1H) 7.90 (dd, J=8.22, 1.17 Hz, 1H) 8.08 (s, 1H) 8.36 (d, J=8.22 Hz, 1H). HRMS m/z 471.2737 (M+H)+.
In a 15 mL round-bottomed flask were added N1-(3-aminopropyl)-N3-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)propane-1,3-diamine (57 mg, 0.121 mmol) and triethylamine (25.3 μl, 0.182 mmol) in DMF (2.8 ml). 2,5-dioxopyrrolidin-1-yl 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate (44.7 mg, 0.131 mmol) was added and stirred for 1 hr. Concentrated the reaction mixture to dryness. The residue was suspended in methanol (3 mL), water (2 mL) and Na2CO3 2 M in water (5 drops). Stirred for 30 minutes. The solids were collected on Buchner. Cake was washed with water (2×1 mL) then with methanol (1×1 mL). Dried the product at 40° C. under high vacuum until constant weight to give N-(3-((3-((2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)amino)propyl)amino)propyl)-5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamide (78 mg, 0.112 mmol, 92% yield) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.28 (m, J=13.79, 13.79, 6.85 Hz, 2H) 1.36-1.52 (m, 3H) 1.52-1.63 (m, 3H) 1.79 (dt, J=12.23, 5.82 Hz, 2H) 2.04 (t, J=7.24 Hz, 2H) 2.52-2.58 (m, 3H) 2.62 (t, J=6.26 Hz, 2H) 2.78 (dd, J=12.13, 5.09 Hz, 1H) 2.99-3.06 (m, 1H) 3.06-3.13 (m, 2H) 3.68 (q, J=6.26 Hz, 2H) 4.04 (s, 2H) 4.06-4.11 (m, 1H) 4.22-4.31 (m, 1H) 4.43 (s, 3H) 6.34 (s, 1H) 6.40 (s, 1H) 7.14-7.23 (m, 1H) 7.28 (t, J=7.43 Hz, 2H) 7.34-7.41 (m, 2H) 7.56 (t, J=5.28 Hz, 1H) 7.77 (t, J=5.48 Hz, 1H) 7.89 (dd, J=8.20, 1.20 Hz, 1H) 8.07 (d, J=1.20 Hz, 1H) 8.36 (d, J=8.22 Hz, 1H) 12.00 (br. s., 2H). HRMS m/z 697.3498 (M+H)+.
Intermediate 9A
A mixture of methyl 2-benzyl-4-chloro-9H-pyrimido[4,5-b]indole-7-carboxylate (0.100 g, 0.284 mmol), Et3N (0.079 ml, 0.569 mmol) and tert-butyl (3-aminopropyl)(methyl)carbamate (0.080 g, 0.426 mmol) in MeOH (1.0 ml) was heated in the microwave at 140° C. for 20 min. The reaction was not completed and more reagent were added and again heated at 140° C. for 20 min. The solvent was removed and the crude residue purified on a short pad of SiO2 with Hx-EA (65-35) to give 0.092 g of methyl 2-benzyl-4-((3-((tert-butoxycarbonyl)(methyl)amino)propyl)amino)-9H-pyrimido[4,5-b]indole-7-carboxylate. LRMS+H+: 504.2.
Intermediate 9B
TFA (1.0 ml, 12.9 mmol) was added dropwise to a cold (0-5° C.) suspension of the previous intermediate (0.092 g, 0.18 mmol) and then brought to rt. After 30 min it was diluted with some toluene and the solvent was removed. The residue was taken in more toluene and the solvent was removed. Finally it was taken in EA ant the solvent was removed to give 0.085 g of the trifluoroacetate salt of methyl 2-benzyl-4-((3-(methylamino)propyl)amino)-9H-pyrimido[4,5-b]indole-7-carboxylate. LRMS+H+: 404.2.
The previous TFA salt (0.020 g), sodium carbonate (8.81 mg, 0.083 mmol), sodium iodide (1.4 mg, 9.6 μmol) and 2-(2-(2-chloroethoxy)ethoxy)ethanol (6.5 μl, 0.04 mmol) was heated at 70° C. in acetone (0.2 ml) overnight. More 2-(2-(2-chloroethoxy)ethoxy)ethanol (6.4 μl, 0.04 mmol) were added and again stirred overnight at 70° C. It was then diluted with EA-water and the organic phase separated, dried over Na2SO4, filtered. The crude adduct was purified on combi-flash using DCM-MeOH (0-25%) to give 0.009 g of the title compound. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.74-1.85 (m, 2H) 2.25 (br. s., 3H) 2.55 (br. s., 2H) 3.32-3.36 (m, 4H) 3.39-3.46 (m, 6H) 3.51 (t, J=5.87 Hz, 2H) 3.64 (q, J=6.52 Hz, 2H) 3.88 (s, 3H) 4.04 (s, 2H) 4.53 (br. s., 1H) 7.18 (t, J=7.40 Hz, 1H) 7.28 (t, J=7.63 Hz, 2H) 7.37 (d, J=7.04 Hz, 2H) 7.55 (t, J=5.28 Hz, 1H) 7.82 (dd, J=8.22, 1.17 Hz, 1H) 7.99 (s, 1H) 8.27 (d, J=8.22 Hz, 1H) 12.05 (s, 1H). LRMS+H+: 536.3.
In a microwave vial was added methyl 2-benzyl-4-chloro-9H-pyrimido[4,5-b]indole-7-carboxylate (0.050 g, 0.142 mmol) and N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine (0.12 ml, 0.71 mmol) in MeOH (2.0 ml, 49.4 mmol). The vial was placed in the microwave and heated to 140° C. for 30 min. After 30 minutes it was concentrated to dryness. The residue was purified on ISCO RediSep Gold column using DCM-MeOH—NH4OH to give 0.044 g of the title compound. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.44 (dt, J=13.8, 6.6 Hz, 2H) 1.72 (dt, J=13.7, 6.8 Hz, 2H) 2.11 (s, 3H) 2.24-2.30 (m, 2H) 2.33 (t, J=6.7 Hz, 2H) 2.47 (br. s., 2H) 3.51-3.61 (m, 2H) 3.76-3.85 (m, 3H) 3.97 (s, 2H) 7.08-7.15 (m, 1H) 7.17-7.24 (m, 2H) 7.27-7.34 (m, 2H) 7.50 (t, J=5.3 Hz, 1H) 7.76 (dd, J=8.2, 1.6 Hz, 1H) 7.92 (d, J=1.6 Hz, 1H) 8.20 (d, J=8.2 Hz, 1H). LRMS+H+: 461.2.
To a mixture of pent-4-ynoic acid (2.56 mg, 0.026 mmol), EDC (6.24 mg, 0.033 mmol) and triethylamine (4.58 μl, 0.033 mmol) in DMF (0.12 ml) at 5° C. was added methyl 4-((3-((3-aminopropyl)(methyl)amino)propyl)amino)-2-benzyl-9H-pyrimido[4,5-13]indole-7-carboxylate (0.010 g, 0.022 mmol). After 5 min, bring to rt and stirred overnight. The mixture was diluted with DMSO and purified directly on the preparative HPLC (Zorbax SB-C18 PrepHT 5 um; 21.2×100 mm) with a gradient of 20% MeOH (0.065% TFA) to 100 MeOH (0.05% TFA) to give after lyophilization from ethanol 0.006 g of the title compound as the TFA salt. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.74 (quin, J=7.20 Hz, 2H) 2.02 (br. s., 2H) 2.20-2.29 (m, 2H) 2.31-2.38 (m, 2H) 2.68-2.74 (m, 3H) 2.76 (t, J=2.54 Hz, 1H) 3.07-3.13 (m, 3H) 3.69 (q, J=6.30 Hz, 2H) 3.88 (s, 3H) 4.08 (s, 2H) 7.21 (t, J=7.04 Hz, 1H) 7.30 (t, J=7.63 Hz, 2H) 7.34-7.40 (m, 2H) 7.51 (t, J=5.48 Hz, 1H) 7.84 (dd, J=8.22, 1.17 Hz, 1H) 7.98-8.02 (m, 1H) 8.05 (t, J=5.48 Hz, 1H) 8.37 (d, J=8.22 Hz, 1H) 9.18 (br. s., 1H) 12.15 (s, 1H). LRMS+H+: 541.3.
Following a similar procedure as previously described with 2-benzyl-4-chloro-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indole and N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine as the amine, the title compound was obtained. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.52 (quin, J=6.85 Hz, 2H) 1.80 (quin, J=6.85 Hz, 2H) 2.18 (s, 3H) 2.36 (t, J=7.24 Hz, 2H) 2.41 (t, J=6.65 Hz, 2H) 2.53-2.61 (m, 2H) 3.64 (q, J=6.52 Hz, 2H) 4.04 (s, 2H) 4.43 (s, 3H) 7.14-7.23 (m, 1H) 7.28 (t, J=7.43 Hz, 2H) 7.38 (d, J=7.43 Hz, 2H) 7.49 (t, J=5.09 Hz, 1H) 7.91 (d, J=8.22 Hz, 1H) 8.08 (s, 1H) 8.32 (d, J=8.22 Hz, 1H). LRMS+H+: 485.4.
Intermediate 13A
A mixture of methyl 2-amino-3-carbamoyl-1H-pyrrolo[3,2-b]pyridine-6-carboxylate (0.090 g, 0.384 mmol), methyl 2-(naphthalen-2-yl)acetate (0.269 g, 1.345 mmol) and sodium methoxide 5.4M (0.28 ml, 1.53 mmol) in MeOH (1.601 ml) was heated in the microwave for 75 min at 140° C. The mixture was diluted with MeOH—NH4Cl saturated and filtered. The solid was rinsed with MeOH-water and dried under high vacuum to give crude 0.120 g of methyl 4-hydroxy-2-(naphthalen-2-ylmethyl)-9H-pyrido[2′,3′:4,5]pyrrolo[2,3-d]pyrimidine-7-carboxylate.
Intermediate 13B
To this intermediate (0.060 g) in dioxane (0.41 ml)-DCE (0.62 ml) was added POCl3 (0.13 ml, 1.4 mmol) and the mixture was heated in the microwave for 10 min at 160° C. The reaction was not completed and more POCl3 (0.131 ml, 1.405 mmol) were added. Ok. Solvent removed. The solvent was removed and the residue was suspended in sat. NaHCO3 (10 mL) and stirred for 1 hour. The solids were collected on uchner and the cake was washed with water (3×2 mL) and dried at 40° C. under high vacuum to give 0.040 g of methyl 4-chloro-2-(naphthalen-2-ylmethyl)-9H-pyrido[2′,3′:4,5]pyrrolo[2,3-d]pyrimidine-7-carboxylate. LRMS+H+: 403.1.
Following a similar procedure as before with the previous intermediate and 3-(piperidin-1-yl)propan-1-amine as the amine, the title compound was obtained after purification on the preparative HPLC (Zorbax SB-C18 PrepHT 5 um; 21.2×100 mm) with a gradient of 20% MeOH (0.065% TFA) to 100 MeOH (0.05% TFA) to give 0.012 g of the title compound as the TFA salt. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.15-1.31 (m, 1H) 1.43-1.72 (m, 5H) 1.91-2.04 (m, 2H) 2.58 (q, J=11.00 Hz, 2H) 2.95 (br. s., 2H) 3.26 (d, J=11.35 Hz, 2H) 3.72 (q, J=5.50 Hz, 2H) 3.92 (s, 3H) 4.27 (s, 2H) 7.48 (quin, J=6.26 Hz, 2H) 7.58 (d, J=8.61 Hz, 1H) 7.64 (t, J=5.09 Hz, 1H) 7.78-7.93 (m, 4H) 8.22 (s, 1H) 8.94 (br. s., 1H) 9.01 (s, 1H) 12.32 (s, 1H). LRMS+H+: 509.3.
The compounds encompassed herein have been further tested for their ability of inhibiting cell proliferation in AML3, showing high potency (IC50=<500 nM) to less potency (IC50=2000-5000 nM). Accordingly, also encompassed are the following compounds with level of activity in AML3 cells as described above in parenthesis (wherein A: IC50=<500 nM; B: IC50=500-1000 nM; C: IC50=1000-2000 nM; and D: IC50=2000-5000 nM):
In an embodiment, the Pyrimido[4,5-B]indole derivatives described herein allow to treat cancer. Encompassed herein are any other cancer, based on similar mechanism of action (e.g. K27 mutation, EZH2 or PRC2 mutation).
UM171 by activating the CRL3 complex as described herein degrades RCOR1 which normally acts as the scaffolding protein for the RCOR1/LSD1 and HDAC2 complex, itself being dissociated in the presence of UM171. Thus UM171 acts like a molecular glue degrader, as an anti-cancer agent resulting in inhibition of HDACs and LSD1.
It is demonstrated herein that KBTB4 complex targets CoREST for degradation under UM171 treatment. The UM171 molecule leads to a clear target of CoREST for proteasome degradation, by increasing the interaction between the adaptor/receptor KBTBD4 and CoREST.
The Extended Knockout (EKO) pooled lentiviral library of 278,754 sgRNAs targeting 19,084 RefSeq genes, 3,872 hypothetical ORFs and 20,852 alternatively spliced isoforms was introduced within a clone of OCI-AML5 and OCI-AML1-cell lines engineered to express a doxycycline-inducible Cas9 as previously describe by Bertomeu et al. (2018, Mol Cell Biol, 38(1): e00302-1) The EKO library (kept at a minimum of 500 cells per sgRNA) was cultured in 10% FBS DMEM supplemented with 2 μg/mL doxycycline for a period of 7 days to induce knockouts. At day 7, 140 million cells were spun at 1,200 rpm for 5 min, washed with 1×PBS, pelleted and frozen. The library was left to expand 8 or 14 more days without doxycycline with 250 nM and 800 nM UM171 or DMSO only (250 cells per sgRNA on average). Cell concentration was assessed every 2 days. Genomic DNA was extracted from all samples using the QIAamp DNA blood maxi kit (Qiagen). SgRNA sequences were recovered and fitted with Illumina adaptors by PCR and NGS performed on an Illumina HiSeq 2000 device (IRIC) as previously described Bertomeu et al. (2018, Mol Cell Biol, 38(1): e00302-1). Synthetic rescue/positive selection and synthetic lethality/negative selection beta scores were determined using MAGeCK-VISPR (Li et al., 2015, Genome Biol, 16: 281).
Mouse anti-human antibodies were used to detect CD34 (APC or BV421-BD Biosciences), CD45RA (PE-BD Biosciences), CD86 (PerCP-eFluor710-eBioscience), CD90 (PECY7- BioLegend), and CD201 (APC-BioLegend). Flow cytometry acquisitions were performed on a Canto II cytometer (BD Biosciences) and data analysis was performed using FowJo software (Tree Star, Ashland, Oreg., USA) and GraphPad Prism software. Dead cells were excluded using 7AAD staining.
For intracellular staining, cells were washed in phosphate-buffered saline, pelleted, and fixed using True Nuclear fixation kit (BioLegend, Cat #424401). Cells were then stained with mouse monoclonal anti-H3K27Ac (Cell Signaling Technology, Catalog #15562), rabbit anti-H3K4me2 (Abcam, Catalog #7766) and rabbit anti-H3 (Cell Signaling Technology, #12167S) and FACS analysis was performed on a BD Biosciences Canto II cytometer.
Umbilical cord blood units were collected from consenting mothers according to ethically approved protocol at Charles-Lemoyne Hospital, Montreal, QC, Canada. Human CD34+ cord blood (CB) cells were isolated using The EasySep™ positive selection kit (StemCell Technologies Cat #18056). Sorting for more primitive phenotypes was done in additional step using BD Aria II sorter.
Human CD34+ cells were cultured in HSC expansion media consisting of StemSpan SFEM (StemCell Technologies) supplemented with human 100 ng/ml stem cell factor (SCF, R&D Systems), 100 ng/ml FMS-like trysine kinase 3 ligand (FLT3, R&D Systems), 50 ng/ml thrombopoietin (TPO, R&D Systems), and 10 μg/ml low-density lipoproteins (StemCell Technologies).
All experiments with animals were conducted under protocols approved by the University of Montreal Animal Care Committee. Fresh CD34+CB cells or progeny were transplanted by tail vein injection into sub-lethally irradiated (250 cGy, <24 hr before transplantation) 8 to 10-week-old female NSG (NOD-Scid IL2Rγnull, Jackson Laboratory) mice. Human cells NSG-BM cells were collected by femoral aspiration or by flushing the two femurs, tibias and hips when animals were sacrificed at week 26.
HDAC inhibitors (panobinostat (Adooq Bioscience, #A10518), Valproic acid (Sigma, #P4543), M344 (Sigma, #M5820), LMK235 (Sigma, #SML1053) were used in CD34+ cord blood cells at 5,1000, 500 and 500 nM respectively. LSD1 inhibitor (tranylcypromine (TCP)) was used at 5 and 20 μM. iBET (Selleckchem, #S7189) was used at 50 and 100 nM in CD34+ cord blood cells. NEDD8 E1 Activating Enzyme (NAE) Inhibitor MLN4924 (Adooq Bioscience, #A11260-5) was used at 100 nM. Proteasome inhibitor MG132 (Adooq Bioscience, #A11043) was used at 10 μM.
Lentiviral vectors carrying shRNAs (KBTBD4, CUL3, NUDCD3, CAND1, RCOR1, LSD1) were generated by cloning appropriate shRNA sequences as described in Fellmann et al. (2013, Cell Rep, 5: 1704-1713) into MNDU vectors comprising miR-E sequences as well as GFP. Control vectors contained shRNA-targeting Renilla luciferase (shLuc).
Lentiviral vectors carrying sgRNA (KBTBD4, NUDCD3) were generated by cloning appropriate sequences (KBTBD4: GGCTAGCATGGAATCACCAG; SEQ ID NO: 1; NUDCD3: GGAGCGCTCCATGGCCACCG; SEQ ID NO: 2) into pLKO5.sg.EFS.tRFP647 lentiviral vector. Control vector contained sgRNA-targeting AAVS1 locus (sgAAVS1).
KBTBD4 cDNA (Dharmacon, #MHS6278-202829492) was subcloned into MNDU-eGFP lentiviral vector. BTB and Kelch mutants were generated by deleting the BTB domain and the first 3 kelch domains, respectively.
Lentiviruses were produced in HEK-293 cells and primary CD34+ cells or AML cell lines were infected with lentiviruses in media supplemented with 10 ng/mL polybrene for 24 hours. Infection efficiency, as determined by the percentage of GFP positive cells, was monitored by flow cytometry using a BD FACSCantoll flow cytometer. When needed, infected cells were sorted using a BD Aria II cell sorter and knockdown efficiency was determined by Western blotting using standard methods.
Total protein extraction was performed in lysis buffer (25 mM tris ph7.5, 150 mM NaCl, 1% NP-40, protease inhibitors). Chromatin-bound (CB) and cytoplasmic/nucleoplasmic (CN) fractionation were performed as described by Mladenov and colleagues (2007, J Cell Physiol, 211: 468-476). Cytoplasmic fractionation was performed in lysis buffer (10 mM pipes ph6.8, 0.3M sucrose, 0.1M NaCl, 3 mM MgCl2, 5 mM EGTA, 0.015% digitonin and protease inhibitors).
Anti-human antibodies were used to detect KBTBD4 (Novus Biologicals, #NBP1-88587), NUDCD3 (Novus Biologicals, #NBP1-82940), CUL3 (Novus Biologicals, #NB100-58788), RCOR1 (Novus Biologicals, #NB600-240), LSD1 (Cell Signaling Technology, #2139S), HDAC2 (Bethyl Laboratories, #A300-705A), H3K27Ac (Cell Signaling Technology, #8173S), CUL1 (Santa Cruz Biotechnology, #sc-17775), NCL (Cell Signaling Technology, #14574S), TUBA (Cell Signaling Technology, #2144) and H3 (Millipore, #06-755).
BioID pulldown experiments were performed as described previously (Comartin et al., 2013, Curr Biol, 23: 1360-1366). In brief, full-length human KBTBD4 coding sequences were amplified by PCR and cloned into pcDNA-FRT/TO-FLAGBirA expression vector. 293T cells stably expressing FLAGBirA or FLAGBirA*-KBTBD4 were generated and incubated for 24 hrs in complete media supplemented with 1 μg/ml tetracycline (Sigma), 50 μM biotin (BioShop, Burlington, ON, Canada) and 10 μM MG132. Cells were collected and lysed in lysis buffer. Protein extracts were incubated with streptavidin-Sepharose beads.
Peptides were analyzed by LC-MS/MS using a Proxeon nanoflow HPLC system coupled to a tribrid Fusion mass spectrometer (Thermo Fisher Scientific). Each sample was loaded and separated on a reverse-phase analytical column (18 cm length, 150 mmi.d.) (Jupiter C18.3 mm, 300 A°, Phenomenex) packed manually. LC separations were performed at a flow rate of 0.6 mL/min using alinear gradient of 5-30% aqueous ACN (0.2% FA) in 106 min. MS spectra were acquired with a resolution of 60,000. “TopSpeed” (maximum number of sequencing events within 5 s window) method was used for data dependent scans on the most intense ions using high energy dissociation (HCD). AGC target values for MS and MS/MS scans were set to 5e5 (max fill time 200 ms) and 5e4 (maxfill time 200 ms), respectively. The precursor isolation window was set to m/z 1.6 with a HCD normalized collision energy of 25. The dynamic exclusion window was set to 30 s. MS data were analyzed using MaxQuant software version 1.3.0.3 and searched against the SwissProt subset of the H. Sapiens uniprot database.
5×105 OCI-AML5 cells were exposed or not to UM171 (250 nM) for 6 to 72 h and preserved at −80C in TRIzol Reagent (Thermo Fisher Scientific cat #15596026). cDNA libraries were constructed according to TruSeq Protocols (Illumina) and sequencing was performed using an Illumina HiSeq 2000 instrument.
Gene expression statistics were obtained using the kallisto/sleuth analysis pipeline and the GRCh38 version 84 annotation. TPM values were loaded into R and differential expression was tested using Wilcoxon rank sum statistics. Differentially expressed genes were selected based on significance (p 0.01, Mann-Whitney test).
PK assays were performed in mice (n=3). The compounds were intravenously administered at 1 mg/kg (compound in
The compounds were orally administered at 20 mg/kg and plasma samples were collected at 15, 30 minutes and 1, 2, 4, 6 and 8 hours after administration. The following pharmacokinetic parameters were calculated. The maximal concentration (Cmax) of UM729 was 0.2 uM, the maximal time (tmax) was determined to be 0.8 hours, the area under the curve (AUC) was 1.2 uM*h and the bioavailability (% F) was 19% (see
While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations, including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2020/051755 | 12/18/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62949678 | Dec 2019 | US |
