NZ749533A - Compounds and methods for modulating rna function - Google Patents
Compounds and methods for modulating rna functionInfo
- Publication number
- NZ749533A NZ749533A NZ749533A NZ74953317A NZ749533A NZ 749533 A NZ749533 A NZ 749533A NZ 749533 A NZ749533 A NZ 749533A NZ 74953317 A NZ74953317 A NZ 74953317A NZ 749533 A NZ749533 A NZ 749533A
- Authority
- NZ
- New Zealand
- Prior art keywords
- rna
- target
- compound
- mrna
- ring
- Prior art date
Links
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Abstract
The present invention provides compounds, compositions thereof, and methods of using the same.
Description
COMPOUNDS AND METHODS FOR MODULATING RNA FUNCTION
CROSS-REFERENCE T0 RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. § 119(e) of US Provisional
Application nos. 62/357,654, filed July 1, 2016, and ,487, filed February 1, 2017, the
contents of all of which are incorporated herein in their entireties by reference.
CAL FIELD OF THE INVENTION
The present invention relates to compounds and methods of use thereof for
ting the biology of RNA transcripts to treat s diseases and conditions by the
binding of such compounds to trans or cis three-way junctions. The invention also provides
methods of identifying RNA transcripts that can form new three-way junctions stabilized by
small molecule binders, thus rendering such three-way junctions druggable, and designing and
screening drug candidates that will bind to RNA three—way ons.
BACKGROUND OF THE INVENTION
Mammalian diseases are ultimately mediated by the transcriptome, while viruses and
other ens depend on RNA for s aspects of infection, reproduction, and survival.
Insofar as messenger mRNA (mRNA) is part of the transcriptome, and all protein expression
derives from mRNAs, there is the potential to intervene in protein-mediated diseases by
modulating the expression of the relevant protein and by, in turn, modulating the translation of
the corresponding upstream mRNA. But mRNA is only a small portion of the transcriptome:
other transcribed RNAs also regulate cellular biology either directly by the structure and function
of RNA ures (e.g., ribonucleoproteins) as well as via protein expression and action,
including (but not limited to) miRNA, lncRNA, lincRNA, snoRNA, snRNA, scaRNA, piRNA,
ceRNA, and pseudo-genes. RNA folds into secondary and tertiary structures in which portions
of the nucleic acid form double-stranded segments through Watson-Crick base g while
other segments remain unpaired (single-stranded). One common structure is a three-way
on (3WJ). In this ce, the nucleic acid forms three double helices in a Y-shaped
pattern, with the central point of intersection of the three helices termed the 3WJ. Small
molecules can bind with high affinity to the 3WJ and stabilize this structure, thus affecting the
function of the target RNA by altering its structural preferences and hence ting its
downstream biological activity. However, the discovery and use of small molecules as ligands
for RNA to treat RNA-mediated diseases has received little attention from the pharmaceutical
industry.
Accordingly, targeting the RNA transcriptome and 3WJ in particular with small
molecules represents an ed therapeutic approach to treat RNA-mediated diseases.
Furthermore, there remains a high, unmet need for improved treatments of RNA-mediated
diseases, including mammalian diated diseases, viruses, tes, and microbes.
SUMMARY OF THE INVENTION
The present invention provides compounds and s of use thereof for the
modulation of the levels and/or activities of nucleic acid, e. g. RNA or DNA, les by drug-
like small molecules, The present invention also provides methods of screening nucleic acid
sequences for cis or trans sequence complementarity likely to form a 3WJ or likely to form a
3WJ in the ce of a disclosed compound. Furthermore, the present invention es
methods of screening small molecules for binding to a 3WJ. The present invention takes
advantage of 3WJ ion by identifying these structures and screening for small molecules
that selectively bind them to stabilize the 3WJ in cells and animals. By stabilizing the 3WJ, the
small molecule can modulate the stability or activity of the nucleic acid.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1A shows a fully complementary interaction between an miRNA and mRNA
(i.e. the ces share full sequence complementarity and form a RNA-RNA duplex). Figure
1B shows an interaction in which the miRNA is splinted discontinuously across the flank region
of the mRNA located at the base of the stem-loop element. This interaction creates a 3WJ at the
base of the stem—loop element.
Figure 2 shows a flowchart g a hypothetical target RNA of interest to an
or RNA to 3WJ-binding small molecules to lar mechanism in cells. Importantly,
using the disclosed methods it is possible to begin with either a known RNA target and fy
or/target pairs that form 3WJs using a bioinformatic screen, or perform a bioinformatic
screen of the entire transcriptome to identify all effector/target pairs that form 3WJs, followed by
selection of an effector/target RNA pair of interest. ing the remaining steps of the
flowchart identifies small molecules that bind 3WJs on targets of high therapeutic value, thereby
modulating the target to treat or ameliorate a disease or disorder.
Figure 3 shows exemplary effector RNA/target RNA pairings. For example, miRNA
and mRNA could form a 3W1 in trans, priRNA could form a cis 3WJ, and so on.
Figure 4 shows the mechanism of 3WJ g a small molecule (represented by a
triangle) with a stem-loop (3WJ) in the effector RNA. The RNA may or may not be bound to an
RNA binding protein (RBP, represented by oval). Binding of the small molecule may (or may
not) influence binding of any RBP that normally binds to the RNA/RNA duplex.
Figure 5 shows the mechanism of 3WJ binding a small molecule with a stem-loop
(3WJ) in the target nucleic acid. The RNA may or may not be bound to an RNA binding protein
(RBP; represented by oval). Binding of the small molecule may (or may not) influence binding
of any RBP that ly binds to the RNA/RNA duplex.
Figure 6 shows two strategies for targeting trans-acting 3-way junctions. In one
strategy (top), first one identifies a “natural” miRNA/mRNA pair that yields a 3WJ, then
identifies small molecule ligands that lock in the miRNA/mRNA trans-regulatory structure to
y the miRNA regulatory . Notably, the small molecule can thus act as either a
ational agonist (amplify translation & n expression) or a translational antagonist
(suppress translation & protein sion). In a second strategy (bottom), first one identifies
“candidate” miRNA/mRNA pairs that will present a 3W]; then identifies SM ligands that lock in
mRNA trans-regulatory structure to amplify the miRNA regulatory effect. Notably, the
small molecule can thus act as either translational agonist (amplify translation & protein
expression) or a translational antagonist (suppress translation & protein expression).
Figure 7 shows a scheme demonstrating how small molecules could act to stabilize
trans-acting 3WJs. Using the sed methods, for example, one could ate ca. 1000
miRNAs with 20,000 mRNAs for 20,000,000 potential pairs — optionally with the simplifying
assumption that each mRNA has only one hybridizable site. This catalog can be assembled
without nce to the actual endogenous regulatory role of a given miRNA. Insofar as the
small molecule can stabilize the ternary complex, this may render moot the contributions of
Argonaute and/or RBPs to regulation of mRNA on.
Figure 8 shows exemplary binding modes of nding small molecules. Small
molecules might bind the 3WJ only, the 3WJ plus g the major or minor groove nt to
one base pair, the 3WJ plus binding the major or minor groove adjacent to one base pair in two
separate duplexes, and so on.
Figure 9 shows the topology of 3WJs. Note: (A) X, Y, and Z vary independently as
O, l, 2, 3, 4, 5, or 6. (B) Nucleotides in the loops that define the periphery of the 3WJ vary
independently as A, C, G, or U. (C) Inside the cells those nucleotides may have undergone posttranscriptional
modifications. (D) The stems are minimally 4 base pairs each, but can n
bulges and loops. (E) The loop at the end of the stem-loop can vary from 3 on up.
Figure 10 shows a cis model system that may be used to understand a corresponding
trans 3WJ system. Synthetic single-stranded RNA can recapitulate key binding events. It can
link via 5’-to-3’ or 3’-to-5’. The length and structure of the linker will be selected to favor but
not e 3WJ formation. Ligands that bind to 3WJs in the cis model must induce formation
of ternary complex in the trans model. This format enables rapid and combinatoric selectivity
screening. The binding free energy here is expected to be predictive of cellular outcome. Some
features of this method: the structure to be analyzed is small — small enough to screen by NMR;
easy to make; screenable via DEL (DNA encoded libraries); ically relevant — models the
regulatory interaction of miRNA with its e mRNA; biologically specific — focus on single
mRNA; immediate focus on one subsite, so one can design structure—informed displacement
assays; inherent connection between target/subsite and biological function; and the potential for
co-opting non-cognate miRNAs to tackle arbitrary mRNAs at arbitrary sites.
Figure 11 shows how the PEARL-seq method (Hook the Worm, or HTW; see USSN
62/289,671, which is hereby orated by reference) can reveal endogenous mRNA/miRNA
pairs. WH = warhead moiety that reacts with proximate 2’-OH sites on an RNA after binding of
a small molecule ligand that is covalently attached to the WH. Separate sequencing of
RNA/RNA pairs such as mRNA reveals which nucleotides are proximate to each other
and to the binding site of the HTW molecule under the assay conditions and/or in vivo.
Figure 12 shows exemplary triptycene scaffolds with l auxiliary (i.e. group e
of binding to a nucleic acid feature, such as the major or minor groove, outside of the central
cavity of the 3WJ).
Figure 13 shows exemplary triptycene scaffolds with 2 auxiliaries.
Figure 14 shows exemplary triptycene scaffolds with 3 auxiliaries.
Figure 15 shows ary trityl scaffolds with l auxiliary.
Figure 16 shows exemplary trityl scaffolds with 2 auxiliaries.
Figure 17 shows exemplary l-azabicyclooctane scaffolds with l auxiliary.
Figure 18 shows exemplary l-azabicyclooctane scaffolds with 2 auxiliaries.
Figure 19 shows exemplary trioxabicyclooctane scaffold with l auxiliary.
Figure 20 shows exemplary bicyclooctane scaffolds with 2 auxiliaries.
Figure 21 shows exemplary adamantane scaffolds with 2 auxiliaries.
Figure 22 shows exemplary adamantane scaffolds with 2 auxiliaries.
Figure 23 shows general molecular scaffolds that may bind the l cavity of 3WJs
and also bind to double stranded RNA grooves by cting with exposed edges of base pairs
when substituted with one or more functional groups or edge binders as bed in various
embodiments herein.
Figure 24 shows structures of DNA groove binders that provide a variety of synthons
for edge interactions.
Figure 25 shows structures of exemplary small molecule scaffolds that feature
functional groups capable of base-pairing with c acid target 3WJs, as well as functional
groups capable of binding ctions with edge features such as the minor groove.
Figure 26 shows pictorial representations and an exemplary compound structure of a
designed small molecule that features a nucleobase capable of base-pairing with nucleic acid
target 3WJs, as well as onal groups capable of g interactions with edge features such
as the major or minor groove. H—bond donor and acceptor functionalities are spaced to optimize
interactions (including sequence-specific interactions) with the stem of a nucleic acid stem-loop
structure.
Figure 27 shows exemplary g modes for designed small molecules optimized
for geometries of given nucleic acid junction ures. Binding focuses not only on the central
cavity but also es “arms” that bind with stem structures away from the central cavity,
optionally including functionality capable of binding in distant secondary loops,
Figure 28 shows ary g modes for ed small molecules optimized
for geometries of given nucleic acid duplexes that have one or more bulges (unpaired
nucleotide(s)). Here, hydrogen bond donors and acceptors are spaced to interact with the duplex,
such as in the major or minor groove, with one or more nucleobases placed to interact with the
bulge(s).
Figure 29 shows exemplary binding modes for ed small molecules optimized
for geometries of given nucleic acid duplexes that have one or more bulges (unpaired
nucleotide(s)). Here, hydrogen bond donors and acceptors are spaced to interact with the duplex,
such as in the major or minor groove, with one or more nucleobases placed to interact with the
bulge(s).
Figure 30 shows exemplary g modes for designed small molecules optimized
for geometries of given nucleic acid 3WJs. Here, hydrogen bond donors and ors are
spaced to interact with one or more duplex arms protruding from the 3WJ, such as cting
with the major or minor groove, with one or more nucleobases placed to interact with distant
loops and a nucleobase (left structure) or chemical scaffold such as a compound of Formula I, II,
etc. optionally placed for binding in the central cavity of the 3WJ (three rightmost ures).
Figure 31 shows a list of exemplary target mRNAs that, in certain embodiments, are
down-regulated by a disclosed nd.
Figure 32 shows a list of exemplary target mRNAs that, in certain embodiments, are
up-regulated by a disclosed nd.
Figure 33 shows structures of exemplary DNA groove binders that provide a variety
of synthons for edge interactions.
Figure 34 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 35 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 36 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the t invention.
Figure 37 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present ion.
Figure 38 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 39 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 40 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 41 shows exemplary compound scaffolds, small les, and synthetic
methods of preparing same, for use in the present invention.
Figure 42 shows exemplary compound scaffolds, small molecules, and tic
methods of preparing same, for use in the present invention.
Figure 43 shows exemplary compound scaffolds, small les, and synthetic
methods of preparing same, for use in the present invention.
Figure 44 shows exemplary compound lds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 45 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 46 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 47 shows exemplary compound scaffolds, small molecules, and synthetic
methods of ing same, for use in the present invention. In (A), ribozymes were selected
from a combinatorial library of random-sequence ther-anthracene conjugates by on
with biotin maleimide and subsequent isolation of biotinylated molecules. (B) ary
ure motif shared by most sequences. N = any nucleotide; N’ = nucleotide complementary
to N. (C) Bimolecular cycloaddition catalyzed by D- or L-ribozymes. R1 = H or larger, R2 =
ethyl or larger.
Figure 48 shows exemplary compound lds, small molecules, and tic
methods of preparing same, for use in the present invention.
Figure 49 shows exemplary compound scaffolds, small molecules, and tic
methods of preparing same, for use in the present invention.
Figure 50 shows exemplary compound scaffolds, small molecules, and synthetic
methods of ing same, for use in the present invention. Cp = cyclopentadiene, cod = 1,5-
cyclooctadiene; TBAF = tetra-n-butylammonium fluoride; THF = tetrahydrofuran.
Figure 51 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 52 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 53 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 54 shows exemplary compound scaffolds, small molecules, and tic
methods of preparing same, for use in the present invention. The scheme on the left shows
oxidative degradation of aryladamantanes to the corresponding carboxylic acids. Ans: 2- or 4-
anisyl. Compound 16 was ed as a mixture of 2- and yl derivatives. The scheme on
the right shows synthesis of rigid adamantine scaffolds 20, 21, and 22.
Figure 55 shows exemplary compound lds, small molecules, and synthetic
methods of preparing same, for use in the present ion.
Figure 56 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 57 shows exemplary compound scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention. The compounds shown include
results of a competition binding is with TCT8—4 RNA ce 1994, vol. 263). The
chemical structures are shown for a series of tives used in competitive binding experiments
with TCT8-4 RNA. The right column represents the affinity of the competitor relative to
theophylline, Kd(c)/Kd(t), where Kd(c) is the individual competitor dissociation constant and
Kd(t) is the competitive iation constant of theophylline. Certain data denoted by > are
minimum values that were limited by the solubility of the competitor. Each experiment was
carried out in duplicate. The average error is shown.
Figure 58 shows exemplary compound lds, small molecules, and synthetic
methods of preparing same, for use in the present invention. See Lau, J. L. e1 61]., ACS Nano
2011, 5(10), 7722.
Figure 59 shows exemplary nd scaffolds, small molecules, and synthetic
methods of preparing same, for use in the present invention.
Figure 60 shows exemplary compound scaffolds and small molecules, for use in the
present invention, that bind various RNA aptamers taken from McKeague et al., Journal of
Nucleic Acids, Volume 2012 (2012), Article ID 748913, 20 pages.
Figure 61 shows exemplary compound scaffolds, small molecules, and synthetic
s of preparing same, for use in the present invention.
Figure 62 shows exemplary compound scaffolds, small molecules, and synthetic
s of preparing same, for use in the present invention.
Figures 63A and B show a synthetic route for compound ARK—132.
Figures 64A-D show a synthetic route for compound ARK-134.
Figures 65A—C show a synthetic route for compounds ARK-135 and 6.
Figure 66 shows a tic route for compound ARK-188.
Figure 67 shows a synthetic route for compound ARK—190.
Figure 68 shows a synthetic route for compound ARK-191.
Figure 69 shows a synthetic route for compound ARK-195.
Figure 70 shows a synthetic route for compound ARK-197.
Figures 71A and B show a synthetic route for compounds based on the ribocil
scaffold.
Figure 72 shows a calibration experiment to determine the dependence of
fluorescence on the tration of 3W1 RNA ucts.
Figure 73 shows the results of a fluorescence quenching experiment of compounds
Ark000007 and Ark000008 with two RNA 3WJ constructs at various concentrations.
Figure 74 shows likely structures for the following three RNA 3W1 constructs, with a
putative binding site for small molecule s shown as a le: A)
_1.0.0_SIB_3FAM (cis 3WJ with one unpaired nucleotide), B) Split3WJ.l_up_SIB +
2017/040514
Split3WJ.l_down_3FAM (trans 3WJ as 1:1 mix); and C) Split3WJ.2_up_5IB +
WJ.2_down_3FAM (trans 3WJ as 111 mix).
Figure 75 shows fluorescence quenching data measuring interaction of compounds
Ark0000013 and Ark0000014 with the following RNA constructs: A)
RNA3WJ_1.0.0_5IB_3FAM (cis 3WJ with one unpaired nucleotide); B) Split3WJ.l_up_5IB +
Split3WJ.1_down_3FAM (trans 3W] as 1:1 mix); and C) Split3WJ.2_up_5IB +
Split3WJ.2_down_3FAM (trans 3WJ as l:l mix).
Figure 76 shows l shift data for compounds Ark000007 and Ark000008 tested
with the 0.0_5IB_3FAM RNA uct. Data analysis shows significant effect for
Ark000007 with melting temperature shift of ~5 C’C (i.e. from 61.2 °C to 65.6 C’C). In contrast,
only a very small effect for Ark000008 was observed. These data suggest that the presence of
Ark000007 increases stability of the 3WJ.
Figure 77 shows thermal shift data for Ark0000013 and Ark0000014 in the presence
of _1.0.0_SIB_3FAM (cis 3W] with one unpaired nucleotide).
Figure 78 shows l shift data for Ark0000013 and Ark0000014 in the ce
of Split3WJ. l_up_5TB+Split3WJ. l_down_3FAM.
Figure 79 shows thermal shift data for Ark0000013 and Ark0000014 in the presence
of Split3WJ.2_up_5TB+Split3WJ.2_down_3FAM.
Figure 80 shows the ure of CPNQ, assigned proton resonances, NMR spectrum,
and epitope mapping results.
Figure 81 shows the structure of HP-AC008002-E01, assigned proton resonances,
NMR spectrum, and epitope mapping results. The scaled STD effect was plotted onto the
molecule according to the preliminary assignments. The data suggests for both RNA constructs
that protons of the pyridine ring are in closer proximity to RNA than the benzene ring. The
aliphatic CH2 group could not be observed due to buffer signal overlap in that region.
Figure 82 shows the structure of HP-AC008001-E02, assigned proton resonances,
NMR spectrum, and epitope mapping results. The scaled STD effect was d onto the
molecule according to the preliminary assignments. The data suggest for both RNA constructs
that aromatic protons closest to the heterocycle are in closer proximity to RNA protons.
Aliphatic proton resonances could not be ed by STD due to direct saturation
artifacts/buffer signal overlap in that region (epitope mapping by WaterLOGSY).
Figure 83 shows the ure of HP-AT005003-C03, assigned proton resonances,
NMR spectrum, and epitope mapping results. The scaled STD effect was d onto the
molecule according to the preliminary assignments. Due to signal overlap no individual
assignment of the CH2 groups was possible. The data suggest for both RNA constructs that
protons of the furan moiety are in closer proximity to RNA protons than the phenyl.
s 84A-E show steps for the production of Illumina small RNA-Seq library
preparation using T4 RNA ligase l adenylated adapters.
s 85A-E show steps for the production of Illumina small RNA-Seq library
preparation using T4 RNA ligase l adenylated adapters.
Figure 86 shows PAGE analysis of RNA target sequences for use in DEL
experiments. The gel lanes show: 1: HTTl7CAG in NMR buffer, 2: Before incubation with
Neutravidin resin; 3: Supernatant after incubation with Neutravidin resin; 4: RNA after
incubation with DEL compounds for 1 hour at RT. The RNA was recovered after heat release
from the resin.
Figure 87 shows exemplary steps of a Surface Plasmon Resonance (SPR) method for
use in the present invention.
Figures 88A and B shows exemplary steps of a Surface Plasmon Resonance (SPR)
method for use in the present invention.
DETAILED PTION OF THE INVENTION
The present invention es compounds and methods of use f for the
modulation of the levels and/or activities of nucleic acid, e. g. RNA or DNA, molecules by drug-
like small molecules. The present invention also provides methods of screening nucleic acid
sequences for cis or trans sequence complementarity likely to form 3WJ or likely to form 3WJ in
the presence of a sed compound. Furthermore, the present invention provides methods of
screening small molecules for binding to 3WJ. The present invention takes advantage of 3WJ
formation by fying these ures and screening for small molecules that selectively bind
them to stabilize the 3WJ in cells and animals. By stabilizing the 3WJ, the small le can
modulate the stability or activity of the nucleic acid.
In one , the present invention provides a compound or pharmaceutically
acceptable salt thereof that selectively binds to and stabilizes a 3WJ. In some embodiments, the
3WJ is the product of cis interactions, i.e. results from ctions within the same nucleic acid,
such as RNA, ce or . In some embodiments, the 3WJ is the product of trans
interactions i.e. results from interactions within two or more nucleic acid, such as RNA-RNA,
RNA-DNA, or DNA-DNA, sequences or strands. Thus, in a cis 3WJ, the sequences comprising
the 3WJ are all within one nucleic acid molecule. In contrast, in a trans 3WJ, the sequences
forming the 3WJ are derived from the interaction of two or three, most commonly two, different
nucleic acid molecules. For e, in some cis 3WJ, sequences in the 5’ untranslated region
(UTR) of a single mRNA transcript fold to form a 3WJ. In other embodiments, an exemplary
trans 3WJ is a 3WJ formed between a microRNA (miRNA) and an mRNA.
A y of RNAs could be targeted for 3WJ formation and modulation by a small
molecule that binds to the 3WJ. These e mRNA, long ing RNA (lncRNA), viral
RNA, and microbial RNA. These will be referred to in this patent application as “targets”. One
common use for this invention will be to target human mRNA in order to affect the level of the
protein expressed by translation of that mRNA. Another use will be to target the s of
RNA viruses to inhibit translation, block viral packaging, or inhibit other obligatory steps in the
virus life cycle.
Small RNAs can act in trans to induce 3WJ formation. These RNAs will be referred
to as “effectors”. In some embodiments, effectors are selected from various natural forms of
small RNA such as miRNA, Piwi-interacting RNA (piRNA), and small nucleolar RNA
(snoRNA). These small RNAs can base pair with a target such as mRNA in a manner that
results in the ion of a 3WJ. The base pairing between the effector and target is often
incomplete, meaning that the two sequences have some, but not complete, complementarity.
Perfect complementarity would result in the formation of a fully double-stranded structure that
lacks a 3WJ. In order to form a 3WJ, the effector (e.g., a miRNA) and target (e. g., an mRNA)
would generally form a stretch of base pairing of at least 4 nucleotides (nt) followed by a base-
pairing stem of at least 4 nt and a loop of unpaired nt, followed by a second stretch of base
pairing between the effector and target ces. The stem-loop can be formed either in the
effector or the target RNA. ary 3WJ structures are shown in Figures 1, 7, and 9.
The process for the identification of the 3WJ first comprises a computational
approach to predict the 3WJ. Sequence databases can be searched for homology using a program
such as BLAST. In some embodiments, to identify effector miRNA to target mRNAs, a
homology search is med between a library of miRNA and one particular mRNA of interest
(or, in other embodiments, a known group of mRNAs ated with a particular miRNA, or, in
other embodiments, all mRNAs are searched) using an annotated database such as the UC Santa
Cruz Genome r. Public databases of miRNA are available (h_’g'_i_‘p_t__/_z'_3§f§gy_§§g_r_;;i__i__i;_h__;§§_e_._9;_rgg,
hit}; ://mirdb . orgx’miRDBX’; http:f/muv .mi cram-a . org/microma/hom e. do;
htt :.r"/mir1'arbase.mbc,i'ictucdu.tw htt 1/,"m ircancer htt :z’fwww
7 . ecu. ed u , . mi r2 di sease . ore“,
htt 3:.flszumm.uniuheidei a’a signifiminvallifo’; ht”: :.I‘x’muv.targetscanorg). The
effector1target identification search can be done by setting the parameters for the 3WJ including
minimal base-pairing of the effector and target, minimal size of the stem and maximum size of
the loop as well as the thermostability of the structure, Beyond this trans analysis, identification
of cis 3WJs could be done by searching a nucleic acid database (e.g., ted mRNA) for the
ability of individual RNAs to form 3WJs.
3WJs in nucleic acids form when, in a folded conformation, three double-helical
“stems” converge and the stems are connected with one or two loops of varying length. These
can be either “perfect” 3WJs in which all nucleotides in the junction take part in the base g
or can be reduced-symmetry 3WJs where one or more nt at the junction n the three
converging steps could be unpaired. The three positions — end of the first effectorztarget base
pairing (x), base of the stem (y) and start of the second effectorztarget base g (y) — can have
loops or bulges of unpaired nt (see Figure 9). In some embodiments, the loop lengths for X, y
and z in the 3WJ formed by binding of the small molecule to the target nucleic acid are
ndently selected from 0, l, or 2 nt. In some embodiments each loop is independently
selected from 0, l, 2, 3, or 4 nt. In some embodiments each loop is 0 nt. In some embodiments
each loop is 1 nt. In some embodiments each loop is 2 nt. In some embodiments one loop is 0
nt. In some embodiments one loop is 1 nt. In some embodiments one loop is 2 nt.
There have been reports of small molecules that bind to DNA and RNA 3WJs,
stabilizing that 3D structure (as evidenced by elevation of the melting temperature). Formation
of the 3WJ around the small molecule ligand has been reported for triptycenes with single-
stranded nucleic acids (Barros & Chenoweth, Angew. Chem. Int. Ed 2014, 53, 13746 —13750;
Barros & eth, Chem. Sci, 2015, 6, 4752—4755, Barros et al., Angew. Chem. Int. Ed.
2016, 55, 1—5), complementary hybridization of two strands around cocaine derivatives (Kent et
al., Anal. Chem. 2013, 85, 9916-9923; Sharma & Heemstra, J. Am. Chem. Soc. 2011, 133,
12426—12429; Heemstra, Beilstez'n J. Org. Chem. 2015, 11, 720), and the convergence of
three strands around symmetric metallic complexes (Phongtongpasuk et al., Angew.
Chem. Int. Ed 2013, 52, 11513 —11516, Oleksi et al., Angew. Chem. Int. Ed 2006, 45, 1227 —
1231).
Insofar as essentially all biology can be traced to the classes of targets under
consideration, a broad scope of diseases can ially be addressed by this invention.
In one aspect of the present invention, certain small molecules bring together effector
RNAs with regions of target nucleic acid, such as a target RNA, by inducing the formation of
and stabilization of 3WJs without the assistance or participation of RNA-binding proteins
(RBPs). Many /effector nucleic acid pairs have multiple potential 3W] interactions. Thus,
small molecule ligands can be “programmed” to modulate the ical functions of essentially
any targeted RNA in the cell. This recapitulates the original promise of antisense without the
pharmaceutical limitations imposed by administration of exogenous oligonucleotides.
ing Nucleic Acid 3WJs
RNA plays varied and important roles in the cell. Messenger RNAs ) are
transcribed from protein-encoding genes and are translated to proteins serving a wide range of
functions. Since mRNAs adopt structural features such as 3WJs that are al for their
function, some embodiments of the invention e compounds and methods in which one or
more mRNAs are the target nucleic acid. In other embodiments, other types of RNA are a target
nucleic acid. For example, mal RNA (rRNA) and transfer RNA (tRNA) function in
translation. Noncoding RNAs (ncRNAs) are diverse and abundant, playing regulatory roles in
many cellular processes. Long noncoding RNAs (lncRNAs) are RNAs of over 200 nt that do not
encode proteins (Morris & Mattick, Nature s Genetics 2014, 15, 423-437; Mattick &
Rinn, Nature Strucz‘. & M0]. Biol. 2015, 22, 5-7; Iyer et al., Nature Genetics 2015, 47, 199-208).
They can affect the expression of the protein-encoding mRNAs at the level of transcription,
splicing, and mRNA decay. lncRNA can regulate transcription by recruiting epigenetic
regulators that increase or se transcription by altering chromatin structure (Holoch &
Moazed, Nat. Rev. Genetics 2015, 16, 71-84). lncRNAs are associated with a wide range of
human diseases including cancer, inflammatory diseases, neurological diseases and
cardiovascular disease. The targeted tion of lncRNA could yield, depending on the site
of 3WJ formation, up-regulation or down-regulation of the expression of c genes and
ns for therapeutic benefit. RNA secondary and ry structures, in particular 3WJs, are
critical for these regulatory activities. Thus, the noncoding transcripts (the noncoding
transcriptome) represent a large group of new therapeutic targets. Some exemplary RNA targets
and diseases are described in greater detail below.
Targeting mRNA
mRNAs are ribed in the s by RNA polymerase II. There are roughly
,000 mRNAs in humans. However, there is ntial complexity in that each mRNA can
have several different isoforms that may vary in the 5’ start site, the length of the 3’ UTR, and
the polyAdenylation (polyA) site usage. Many mRNAs also have a wide variety of alternative
splice forms. Typical mRNAs have a cap at the 5’ end, a 5’ UTR, a range of exon and intron
numbers, a 3’ UTR and a polyA sequence at the 3’ end. The concentration or “level” of the
mRNA, and its resultant protein expression via translation, could be ted by small
molecules that affect the structure of the mRNA, splicing ncy, stability (half-life) of the
mRNA, transport of the RNA, and the efficiency of translation. 3WJ ion could be induced
and stabilized in either the coding or noncoding elements to affect mRNA and protein levels.
Depending upon the therapeutic goal, the e could be either down-regulation or up-
regulation. Accordingly, in some embodiments the present invention provides a method of
altering the concentration of a target mRNA, comprising the step of contacting the target mRNA
with a disclosed nd to form or stabilize a 3WJ comprising a portion of the target mRNA.
In some embodiments, forming or stabilizing a 3WJ sing a portion of the target mRNA
affects the splicing efficiency, stability (e.g., half-life), transport, or efficiency of translation of
the target mRNA. In some embodiments, the concentration of target mRNA is up—regulated‘ In
some embodiments, the concentration of target mRNA is down-regulated. In some
embodiments, the mRNA that is down-regulated is selected from those listed in Figure 31. In
some embodiments, the mRNA that is up-regulated is selected from those listed in Figure 32. In
some embodiments, the 3W] is cis. In some embodiments, the 3W1 is trans.
Within mRNAs, noncoding regions can affect the level of mRNA and protein
expression. Briefly, these include (a) internal ribosome entry sites (IRES) and (b) upstream open
reading frames (uORF) in the 5’ UTR that affect translation efficiency, (c) ic sequences
that affect splicing efficiency and alternative splicing patterns, ((1) 3’ UTR sequences that affect
mRNA and protein localization, and (e) elements in the body of the mRNA and/or the 3’ UTR
that control mRNA decay and half-life. (Komar and Hatzoglou, Frontiers Oncol. 5:233, 2015;
Weingarten-Gabbay ei al., Science 351:pii:aad4939, 2016, Calvo er al., Proc. Natl. Acad. Sci.
USA 106:7507-7512, Le Quesne ez‘ al., J. Paihol. 220:140-151, 2010; Barbosa et al., PLOS
cs 9:e10035529, 2013). For example, nearly half of all human mRNAs have uORFs, and
many of these reduce the translation of the main ORF. A vast number of single-nucleotide
rphisms (SNPs) associated with humans are d in these noncoding regions of
mRNA, suggesting that they have critical tory functions and their targeting by 3WJ
formation could impact expression levels. Accordingly, in some embodiments the present
invention provides a method of altering the concentration of a target mRNA, sing the step
of contacting the target mRNA with a disclosed compound to form or stabilize a 3W] comprising
a noncoding portion of the target mRNA. In some embodiments, the noncoding n of the
target mRNA is selected from: an al ribosome entry site (IRES), upstream open reading
frames (uORF) in the 5’ UTR, an intronic sequence that affects splicing efficiency and
alternative splicing patterns, a 3’ UTR sequence that affects mRNA and protein localization, or
an t in the body of the mRNA and/or the 3’ UTR that controls mRNA decay and half-life.
] In other embodiments, RNA structures in the 5’ UTR are targeted to inhibit
translation. RNA ures such as hairpins in the 5’ UTR have been shown to affect
translation. In some embodiments, the invention provides a method of modulating ribosome
recognition and/or progression or inhibiting translation in order to decrease the production of a
protein, comprising contacting an RNA ure in the 5’ UTR with a disclosed compound. In
some embodiments, ribosome recognition and/or progression is modulated or translation is
inhibited by formation or stabilization of a 3W] at or just upstream of the AUG at the start of
translation.
The half-life of mRNA is tightly controlled in cells (Palumbo et al., Wiley Interdiscip.
Rev. RNA, 2015, 6, 327-336). The concentration of mRNA in cells is a product of tight
regulation and the balance between transcription and mRNA decay. dual mRNAs have
their own distinct half-lives, but degradation can be accelerated when mRNA is misprocessed or
ation is blocked. The decay of individual mRNAs can be ated or inhibited by
translational impairment, and, likewise, changes in the half-life of mRNA can alter translational
efficiency (Roy & on, Trends Genet. 2013, 29, 9). mRNA stability and
degradation can also be controlled by environmental stimuli and biological processes such as cell
cycling, cell differentiation, and immune response. mRNA stability is conserved n cell
types and s. A majority of human mRNAs exhibit half-lives of less than 8 hours, with a
ntial proportion having half-lives of less than 4 hours. Formation and stabilization of a
3WJ in mRNA could significantly increase or decrease mRNA stability. Most mRNA decay
takes place by processive 3’ to 5’ and 5’ to 3’ degradation of the RNA by exoribonucleases that
degrade mRNA from its ends. These exoribonucleases degrade single-stranded, but not double-
stranded, RNA. Accordingly, in some embodiments, the present invention provides a method of
increasing the half-life of an mRNA or modulating the level of a protein regulated by the mRNA,
comprising contacting the mRNA with a disclosed compound. In some embodiments, the
compound induces the formation of or stabilizes a 3WJ in the mRNA, or between the mRNA and
a complementary nucleic acid such as another RNA. Without g to be bound by theory, the
presence of the 3WJ may block the exoribonuclease activity to increase the mRNA half-life and
increase the resulting protein level of a protein that would be therapeutically beneficial. In some
embodiments, a disclosed compound induces formation of a 3W] or izes a 3WJ in the 3’
UTR upstream of the polyA site. This may t a target mRNA from degradation.
atively, a 3WJ is introduced in the 5’ UTR ed that it does not t translation.
Termination of mRNA transcripts can occur at different positions, thus forming
mRNAs with distinct properties including alterative protein coding. mRNA processing occurs
co-transcriptionally, such that slowdown of RNA polymerase II processivity is linked to 3’ end
cleavage and polyadenylation. Protein—coding mRNAs of varying lengths can be created by
alternative termination and polyA site usage. These distinct RNAs can have different half-lives,
translational efficiencies or subcellular localization. In some embodiments, a disclosed
compound binds to and/or stabilizes a 3WJ at a site of RNA polymerase II termination and/or
polyA addition. In some embodiments, such 3WJ formation and/or stabilization alters the
function of the mRNA.
One consideration for the selection of s and effectors is their relative expression
levels. In some embodiments, the effector RNA concentration is similar to the target nucleic
acid (e.g., a target mRNA) concentration. If the abundance of the target greatly exceeds the
effector, then a functional impact on the RNA target is rendered more challenging. In certain
embodiments, the relative concentration of the or:target nucleic acid in the biological
system (e. g. subject’s whole body, , cell, nucleus, mitochondria, or cytoplasm) is n
100:1 to 1:100. In some embodiments, the ve concentration is 50:1 to 1:50, 25:1 to 1:25,
:1 to 1:10, 5:1 to 1:5, 3:1 to 1:3, 2:1 to 1:2 or approximately 1:1. In some embodiments, the
effector:target concentration is at least about 10:1. In some embodiments, the effector:target
concentration is at least about 25:1. In on, in certain embodiments both RNAs are
localized to the same compartment of the cell such that an interaction is more likely. While the
subcellular distribution need not match precisely, it should not be exclusive. For example, the
effector should not be exclusively nuclear while the target c acid is exclusively
cytoplasmic.
Small les can be used to modulate splicing of pre-mRNA for therapeutic
benefit in a variety of settings. One example is spinal muscular atrophy (SMA). SMA is a
consequence of insufficient amounts of the survival of motor neuron (SMN) protein. Humans
have two versions of the SMN gene, SMNl and SMN2. SMA patients have a mutated SMNl
gene and thus rely solely on SMN2 for their SMN protein. The SMN2 gene has a silent mutation
in exon 7 that causes inefficient splicing such that exon 7 is d in the majority of SMN2
transc1ipts, leading to the generation of a defective n that is rapidly degraded in cells, thus
limiting the amount of SMN protein produced from this locus. A small molecule that promotes
the efficient inclusion of exon 7 during the splicing of SMN2 ripts would be an effective
treatment for SMA (Palacino et al., Nature Chem. Biol, 2015, 11, 511-517). Accordingly, in
one , the t invention provides a method of identifying a small molecule that
modulates the splicing, transcription, or cellular half-life of a target pre-mRNA to treat a disease
or disorder, sing the steps of: screening one or more small molecules disclosed herein for
binding to a 3WJ in the target pre-mRNA, and identifying which small molecule(s) bind to the
3WJ and te the splicing, transcription, or cellular half-life of the target pre—mRNA to treat
the disease or disorder. In some embodiments, the method further comprises fying the
target pre-mRNA according to a disclosed ational survey. In some embodiments, the
target pre-mRNA is capable of forming a 3WJ in the presence of an effector RNA such as an
effector miRNA. In some embodiments, the small molecule binds to a trans 3WJ formed
n the effector RNA and the target NA. In some embodiments, the small molecule
binds to a cis 3WJ formed between ns of the target pre-mRNA. In some embodiments, the
pre—mRNA is an SMN2 transcript. In some embodiments, the disease or disorder is spinal
muscular atrophy (SMA).
Even in cases in which defective splicing does not cause the disease, alteration of
splicing patterns can be used to correct the disease. Nonsense mutations leading to premature
translational termination can be eliminated by exon skipping if the exon sequences are in—frame.
This can create a protein that is at least partially onal. One example of the use of exon
skipping is the dystrophin gene in Duchenne ar dystrophy (DMD). A variety of different
mutations leading to premature termination codons in DMD patients can be eliminated by exon
skipping promoted by oligonucleotides (reviewed in Fairclough er al., Nature Rev. Gen, 2013,
14, 373-3 78). Small molecules that bind RNA structures and affect splicing are expected to have
a similar effect. Accordingly, in one aspect, the present invention es a method of
identifying a small le that modulates the splicing pattern of a target pre-mRNA to treat a
disease or disorder, comprising the steps of: ingly, in one aspect, the present invention
provides a method of identifying a small le that modulates the splicing pattern of a target
pre—mRNA to treat a disease or disorder, sing the steps of: screening one or more small
molecules disclosed herein for binding to a 3WJ in the target pre—mRNA; and identifying which
small molecule(s) bind to the 3WJ and modulate the splicing pattern of the target pre-mRNA to
treat the disease or disorder. In some embodiments, the method further comprises identifying the
target pre-mRNA according to a disclosed ational . In some embodiments, the
target pre—mRNA is capable of forming a 3WJ in the presence of an effector RNA such as an
effector miRNA. In some ments, the small molecule binds to a trans 3WJ formed
n the effector RNA and the target pre-mRNA. In some embodiments, the small molecule
binds to a cis 3WJ formed between ns of the target pre-mRNA. In some embodiments, the
target pre-mRNA is a dystrophin gene transcript. In some embodiments, the small molecule
promotes exon skipping to eliminate premature translational termination. In some embodiments,
the disease or disorder is Duchenne muscular dystrophy (DMD).
The present invention contemplates the use of small molecules to up- or down-
regulate the expression of specific proteins based on targeting 3WJs in their cognate mRNAs.
ingly, the present invention provides methods of modulating the downstream protein
expression associated with a target mRNA comprising the step of contacting the target mRNA
with a small molecule disclosed herein that binds to or stabilizes a 3WJ in the target mRNA. In
another aspect, the present invention provides a method of producing a small molecule that
modulates the downstream protein expression ated with a target mRNA, comprising the
steps of: screening one or more small molecules disclosed herein for binding to a 3WJ in the
target mRNA; and identifying which small molecule(s) bind to the 3WJ and modulate the
ream protein expression associated with the target mRNA. In some ments, the
method r comprises identifying the target mRNA ing to a disclosed computational
survey. In some ments, the target mRNA is capable of forming a 3WJ in the presence of
an effector RNA such as an effector miRNA. In some embodiments, the small molecule binds to
a trans 3WJ formed between the effector RNA and the target mRNA. In some embodiments, the
small molecule binds to a cis 3WJ formed between portions of the target mRNA. In some
embodiments, modulation of the downstream protein expression associated with the target
mRNA treats or ameliorates a disclosed disease or condition.
In some embodiments, the t invention provides a method of treating a disease
or disorder mediated by mRNA, comprising the step of administering to a t in need thereof
a small molecule disclosed herein.
Targeting Regulatory RNA
The largest set of RNA targets comprises RNA that is transcribed but not translated
into protein, termed “non-coding RNA”. Non-coding RNA is highly conserved and the many
varieties of non—coding RNA play a wide range of regulatory ons. The term “non—coding
RNA,” as used herein, includes but is not limited to micro-RNA (miRNA), long non-coding
RNA (lncRNA), long intergenic non-coding RNA (lincRNA), Piwi-interacting RNA (piRNA),
competing endogenous RNA ), and -genes. Each of these sub-categories of non-
coding RNA offers a large number of RNA targets with significant therapeutic potential.
Accordingly, in one aspect, the present invention provides a method of producing a small
molecule that modulates a regulatory function of a target ding RNA, comprising the steps
of: screening one or more small molecules disclosed herein for binding to a 3WJ in the target
ding RNA; and identifying which small molecule(s) bind to the 3WJ and modulate the
regulatory function of the target non-coding RNA. In some ments, the method further
comprises identifying the target non-coding RNA according to a sed computational survey.
In some embodiments, the target non-coding RNA is capable of forming a 3WJ in the presence
of an effector RNA such as an effector miRNA. In some embodiments, the small molecule binds
to a trans 3WJ formed between the effector RNA and the target non-coding RNA. In some
embodiments, the small molecule binds to a cis 3WJ formed between portions of the target non-
coding RNA. In some embodiments, modulating the regulatory function of the target non-
coding RNA treats or ameliorates a disease caused by a miRNA, lncRNA, lincRNA, piRNA,
ceRNA, or pseudo-gene.
Targeting miRNA
miRNAs are short double-strand RNAs that regulate gene expression (see Elliott &
Ladomery, lar Biology of RNA, 2nd Ed.). Each miRNA can affect the expression of
many human genes. There are nearly 2,000 miRNAs in humans. These RNAs regulate many
biological processes, including cell entiation, cell fate, motility, survival, and function.
miRNA expression levels vary between different tissues, cell types, and disease settings. They
are ntly aberrantly expressed in tumors versus normal tissue, and their activity may play
significant roles in cancer (for reviews, see Croce, Nature Rev. Genet. -714, 2009;
Dykxhoom Cancer Res. 70:6401-6406, 2010). miRNAs have been shown to regulate oncogenes
and tumor suppressors and themselves can act as oncogenes or tumor suppressors. Some have
been shown to promote epithelial-mesenchymal transition (EMT) and cancer cell invasiveness
and metastasis. In the case of oncogenic miRNAs, their inhibition could be an ive anti-
cancer treatment. Accordingly, in one aspect, the present invention provides a method of
producing a small molecule that modulates the activity of a target miRNA to treat a disease or
disorder, comprising the steps of: screening one or more small molecules disclosed herein for
binding to a 3WJ comprising at least a portion of the target miRNA, and identifying which small
molecule(s) bind to the 3WJ and modulate ty of the target miRNA to treat the disease or
disorder. In some embodiments, the method further comprises identifying the target miRNA
ing to a disclosed computational survey. In some embodiments, the target miRNA is
capable of forming a 3WJ in the presence of an RNA to which it binds, such as an mRNA. In
some embodiments, the small molecule binds to a trans 3WJ formed between the target miRNA
and an RNA to which it binds. In some embodiments, the miRNA regulates an oncogene or
tumor suppressor, or acts as an oncogene or tumor suppressor. In some embodiments, the
disease is cancer. In some embodiments, the cancer is a solid tumor.
In some embodiments, the target miRNA is an oncogenic miRNA such as 5,
~92, miR—l9, miR-Zl, or b (see Stahlhut & Slack, Genome Med. 2013, 5, 111).
miR—155 plays pathological roles in inflammation, hypertension, heart failure, and cancer. In
cancer, miR-155 triggers oncogenic cascades and sis resistance, as well as increasing
cancer cell invasiveness. Altered expression of miR—lSS has been described in le cancers,
ing staging, progress and treatment outcomes. Cancers in which miR-lSS over-expression
has been reported include breast cancer, thyroid carcinoma, colon cancer, cervical , and
lung cancer. It is reported to play a role in drug resistance in breast cancer. miR-17~92 (also
called Oncomir-l) is a polycistronic 1 kb primary ript comprising miR—l7, 20a, 18a, 19a,
92-1 and l9b-1. It is activated by MYC. miR-19 alters the gene expression and signal
transduction pathways in multiple hematopoietic cells, and it triggers leukemogenesis and
lymphomagenesis. It is ated in a wide variety of human solid tumors and hematological
cancers. miR—21 is an oncogenic miRNA that reduces the expression of multiple tumor
suppressors. It stimulates cancer cell invasion and is associated with a wide variety of human
cancers including breast, ovarian, cervix, colon, lung, liver, brain, gus, te, pancreas,
and thyroid s. Accordingly, in some embodiments of the s described above, the
target miRNA is selected from miR-155, miR-l7~92, miR—l9, miR-Zl, or miR—lOb. In some
embodiments, the disease or disorder is a cancer selected from breast cancer, ovarian cancer,
cervical cancer, thyroid carcinoma, colon cancer, liver cancer, brain , esophageal cancer,
prostate cancer, lung cancer, leukemia, or lymph node cancer. In some embodiments, the cancer
is a solid tumor.
Beyond oncology, miRNAs play roles in many other diseases including
cardiovascular and metabolic diseases (Quiant and Olson, J. Clin. Invest. 123111-18, 2013;
Olson, Science Trans. Med. 6: 239ps3, 2014, Baffy, J. Clin. Med. 4:1977-1988, 2015).
Many mature miRNAs are relatively short in length and thus may lack sufficient
folded, three-dimensional structure to be targeted by small les. However, it is believed
that the levels of such miRNA could be reduced by small molecules that bind a 3W1 comprising
at least a portion of the primary transcript or the pre-miRNA to block the biogenesis of the
mature miRNA. ingly, in some embodiments of the methods described above, the target
miRNA is a primary transcript or RNA.
lncRNA are RNAs of over 200 nucleotides (nt) that do not encode proteins (see Rinn
& Chang, Ann. Rev. Biochem. 2012, 81, 145-166; (for reviews, see Morris and Mattick, Nature
s Genetics 15:423-437, 2014, k and Rinn, Nature Structural & Mol. Biol. 22:5-7,
2015; Iyer et al., Nature Genetics 47(:199-208, 2015)). They can affect the expression of the
n-encoding mRNAs at the level of ription, splicing and mRNA decay. Considerable
research has shown that lncRNA can regulate transcription by recruiting epigenetic regulators
that increase or decrease transcription by altering chromatin structure (e.g., Holoch and Moazed,
Nature Reviews Genetics 16:71-84, 2015). lncRNAs are ated with human diseases
including cancer, inflammatory diseases, neurological diseases and cardiovascular disease (for
instance, Presner and Chinnaiyan, Cancer ery 1:391-407, 2011; Johnson, Neurobiology of
Disease 46:245—254, 2012, Gutscher and Diederichs, RNA Biology 9:703-719, 2012, Kumar et
al., PLOS Genetics 9:e1003201, 2013; van de voort et al., Frontiers in Molecular
Neuroscience, 2013, Li et al., Int. J. Mol. Sci. 14118790-18808, 2013). The targeting oflncRNA
could be done to up-regulate or down-regulate the sion of specific genes and ns for
therapeutic benefit (e.g., Wahlestedt, Nature Reviews Drug Discovery 12:433-446, 2013; Guil
and Esteller, Nature Structural & Mol. Biol. 19:1068—1075, 2012). In l, lncRNAs are
expressed at lower levels relative to mRNAs. Many lncRNAs are physically associated with
chromatin (Werner et al., Cell Reports 12, l-lO, 2015) and are ribed in close proximity to
protein—encoding genes. They often remain physically associated at their site of transcription and
act locally, in cis, to regulate the expression of a neighboring mRNA. lncRNAs regulate the
expression of protein-encoding genes, acting at multiple ent levels to affect transcription,
alternative splicing and mRNA decay. For example, lncRNA has been shown to bind to the
epigenetic tor PRC2 to promote its recruitment to genes whose transcription is then
sed via chromatin modification. lncRNA may form complex structures such as 3WJ that
mediate their association with various regulatory ns. A small molecule that binds to these
lncRNA structures could be used to modulate the sion of genes that are normally regulated
by an individual lncRNA. The mutation and dysregulation of lncRNA is associated with human
diseases, therefore, there are a multitude of lncRNAs that could be therapeutic targets.
Accordingly, in some embodiments of the methods described above, the target non-coding RNA
is a lncRNA. In some embodiments, the lncRNA is associated with a cancer, inflammatory
disease, neurological disease, or cardiovascular disease. In some embodiments, the method
further comprises identifying the target lncRNA according to a disclosed computational survey.
In some embodiments, the target lncRNA is capable of forming a 3W] in the ce of an
effector RNA such as an or miRNA. In some embodiments, the small molecule binds to a
trans 3WJ formed between the effector RNA and the target lncRNA. In some embodiments, the
small molecule binds to a cis 3WJ formed n ns of the target lncRNA.
One exemplary target lncRNA is HOTAIR, a lncRNA expressed from the HoxC
locus on human chromosome 12. Its sion level is low (~100 RNA copies per cell). Unlike
many lncRNAs, HOTAIR can act in trans to affect the expression of distant genes. It binds the
epigenetic repressor PRC2 as well as the LSDl/CoREST/REST complex, another repressive
epigenetic regulator (Tsai el al., Science 329, 689-693, 2010). HOTAIR is a highly structured
RNA with over 50% of its nucleotides being involved in base pairing. It is frequently
dysregulated (often up—regulated) in various types of cancer (Yao er all, Int. J. M0]. Sci.
:18985-18999, 2014; Deng et al., PLOS One 9:e110059, 2014). Cancer patients with high
expression levels of HOTAIR have a significantly poorer sis, compared with those with
low sion levels. HOTAIR has been reported to be involved in the control of sis,
proliferation, metastasis, angiogenesis, DNA repair, chemoresistance and tumor cell metabolism.
It is highly expressed in metastatic breast cancers. High levels of expression in primary breast
tumors are a significant predictor of subsequent metastasis and death. HOTAIR also has been
reported to be associated with esophageal squamous cell carcinoma, and it is a prognostic factor
in colorectal , cervical cancer, gastric cancer and endometrial carcinoma. Therefore,
HOTAIR-binding small molecules are novel anti-cancer drug candidates. Accordingly, in some
embodiments of the methods bed above, the target non-coding RNA is HOTAIR. In some
ments, the small molecule binds to a 3WJ in the HOTAIR structure. In some
embodiments, the disease or disorder is breast cancer, esophageal squamous cell carcinoma,
colorectal cancer, cervical cancer, c cancer, or endometrial carcinoma.
] Another potential cancer target among lncRNA is MALAT-l tasis-associated
lung adenocarcinoma transcript 1), also known as NEAT2 (nuclear-enriched abundant transcript
2) hner et 61]., Cancer Res. 73:1180-1189, 2013; Brown et 61]., Nat. ural &Mol. Biol.
21:633-640, 2014). It is a highly conserved 7 kb nuclear lncRNA that is localized in nuclear
speckles. It is ubiquitously expressed in normal tissues, but is up-regulated in many cancers.
MALAT-l is a predictive marker for metastasis development in multiple cancers including lung
cancer. It appears to function as a regulator of gene expression, potentially affecting
transcfiption and/or splicing. MALAT—l knockout mice have no phenotype, indicating that it
has d normal function. However, MALAT-l-deficient cancer cells are impaired in
migration and form fewer tumors in a mouse aft tumor models. Antisense
oligonucleotides (ASO) blocking MALAT-l prevent metastasis formation after tumor
implantation in mice. Some mouse xenograft tumor model data indicates that MALAT-l
knockdown by ASOs may inhibit both primary tumor growth and metastasis. Thus, a small
molecule targeting MALAT-l is ed to be ive in inhibiting tumor growth and
metastasis. Accordingly, in some embodiments of the methods described above, the target non-
coding RNA is MALAT-l. In some ments, the small molecule binds to a 3W] in the
MALAT-l structure. In some ments, the disease or disorder is a cancer in which
MALAT-l is up-regulated, such as lung cancer.
In some embodiments, the present invention provides a method of treating a disease
or disorder mediated by non-coding RNA (such as HOTAIR or MALAT-l), comprising the step
of administering to a patient in need thereof a compound of the present invention. Such
compounds are described in detail herein.
Targeting Toxic RNA [Repeat RNA!
] Simple s in mRNA often are associated with human disease. These are often,
but not exclusively, repeats of three nucleotides such as CAG (“triplet repeats”) (for reviews, see
Gatchel and Zoghbi, Nature Reviews Genetics 6:743-755, 2005; Krzyzosiak et al., Nucleic Acids
Res. 26, 2012, Budworth and McMurray, Methods M01. Biol. 101013-17, 2013). Triplet
repeats are abundant in the human genome, and they tend to undergo expansion over generations.
Approximately 40 human diseases are associated with the expansion of repeat sequences.
Diseases caused by triplet expansions are known as Triplet Repeat Expansion Diseases (TRED).
Healthy individuals have a le number of triplet repeats, but there is a threshold beyond
which a higher repeat number causes disease. The threshold varies in different ers. The
triplet repeat can be unstable. As the gene is inherited, the number of repeats may increase, and
the condition may be more severe or have an earlier onset from generation to generation. When
an individual has a number of repeats in the normal range, it is not expected to expand when
passed to the next generation. When the repeat number is in the premutation range (a normal,
but unstable repeat number), then the repeats may or may not expand upon ission to the
next tion. Normal individuals who carry a premutation do not have the condition, but are
at risk of having a child who has inherited a t repeat in the full mutation range and who will
be affected. TREDs can be autosomal dominant, autosomal recessive or X-linked. The more
common t repeat disorders are autosomal dominant.
The repeats can be in the coding or noncoding portions of the mRNA. In the case of
repeats within noncoding regions, the repeats may lie in the 5' UTR, introns, or 3’ UTR
ces. Some examples of diseases caused by repeat sequences within coding regions are
shown in Table 1.
Table 1: Repeat Expansion Diseases in Which the Repeat Resides in the Coding Regions of
mRNA
Normal repeat e repeat
Disease Gene Repeat
number number
“———36-250
DRPLA ATNl 49-88
SBMA
SCAI —49-88
SCA2 —33-77
SCA3 —55-86
SCA6 —21-30
. Normal repeat se repeat
Disease Gene Repeat
number number
SCA7 ATXN7 38-120
SCA17 m— 25-42 47-63
Some examples of diseases caused by repeat sequences within noncoding regions of
mRNA are shown in Table 2.
Table 2: Repeat Expansion Diseases in Which the Repeat Resides in the ing
Regions of mRNA
location number number
antlsense
SCAlO ATXNlO ATTCT 800-4500
SCA12 PPPZRZB 5’UTR 66-78
C9FTD/ALS C9orf72 GGGGCC
The toxicity that results from the repeat sequence can be direct consequence of the
action of the toxic RNA itself, or, in cases in which the repeat expansion is in the coding
ce, due to the toxicity of the RNA and/or the nt protein. The repeat ion RNA
can act by sequestering al RNA—binding proteins (RBP) into foci, One example of a
sequestered RBP is the Muscleblind family protein MBNLl. Sequestration of RBPs leads to
defects in splicing as well as s in nuclear-cytoplasmic transport of RNA and ns.
Sequestration of RBPs also can affect miRNA biogenesis. These perturbations in RNA biology
can profoundly affect neuronal function and survival, leading to a variety of neurological
diseases.
Repeat sequences in RNA form secondary and tertiary structures that bind RBPs and
affect normal RNA biology. One specific example disease is myotonic dystrophy (DMl;
dystrophia myotonica), a common inherited form of muscle disease characterized by muscle
weakness and slow relaxation of the muscles after contraction (Machuca-Tzili el 61]., Muscle
Nerve 32:1-18, 2005). It is caused by a CUG expansion in the 3' UTR of the dystrophia
myotonica protein kinase (DMPK) gene. This repeat-containing RNA causes the misregulation
of ative splicing of several developmentally regulated ripts through effects on the
splicing regulators lVfl3NL1 and the CUG repeat binding protein (CELFl) (Wheeler el al.,
e 325:336-339, 2009). Small molecules that bind the CUG repeat within the DMPK
transcript would alter the RNA structure and prevent focus formation and alleviate the effects on
these spicing tors. Fragile X Syndrome (FXS), the most common inherited form of mental
retardation, is the consequence of a CGG repeat expansion within the 5’ UTR of the FMRl gene
(Lozano el al., Inlraclable Rare Dis. Res. 3:134-146, 2014). FMRP is critical for the regulation
of translation of many mRNAs and for protein trafficking, and it is an essential protein for
synaptic development and neural plasticity. Thus, its deficiency leads to neuropathology. A
small molecule targeting this CGG repeat RNA may alleviate the suppression of FMRl mRNA
and FMRP protein expression. Another TRED having a very high unmet medical need is
Huntington’s disease (HD). HD is a progressive neurological disorder with motor, cognitive,
and psychiatric changes (Zuccato el al., Physiol Rev. -981, 2010). It is characterized as a
poly-glutamine or polyQ disorder since the CAG repeat within the coding sequence of the HTT
gene leads to a protein having a poly-glutamine repeat that appears to have detrimental effects on
transcription, vesicle king, mitochondrial function, and some activity. However, the
HTT CAG repeat RNA itself also demonstrates toxicity, including the sequestration of MBNLl
protein into nuclear ions. One other c e is the GGGGCC repeat expansion in
the C9orf72 (chromosome 9 open reading frame 72) gene that is prevalent in both familial
temporal dementia (FTD) and familial amyotrophic lateral sis (ALS) (Ling el al.,
Neuron 79:416-438, 2013; Haeusler et al., Nature 507:195—200, 2014). The repeat RNA
structures form nuclear foci that sequester critical RNA binding proteins. The GGGGCC repeat
RNA also binds and sequesters RanGAPl to impair nucleocytoplasmic transport of RNA and
proteins (Zhang el al., Nature 525:56-61, 2015). ively targeting any of these repeat
expansion RNAs could add therapeutic benefit in these neurological diseases.
There is some evidence that triplet repeats adopt 3WJ structures, and as such are
suitable targets for disclosed small les. Without wishing to be bound by , it is
believed that the repeat nucleotide sequences in certain repeat diseases organize themselves into
transient 3WJ such as slipped 3WJ formed by (CAG)-(CTG) repeats. Such 3WJ could be
stabilized by small molecules of the present invention and their toxic effects decreased in order
to treat a TRED such as those described above or in Tables 1 and 2. (See, e.g., Barros ez‘ al.,
Chem. Sci. 2015, 6, 4752-4755.)
The present invention contemplates a method of treating a disease or disorder wherein
aberrant RNAs themselves cause pathogenic s, rather than acting through the agency of
protein expression or regulation of protein expression. In some embodiments, the disease or
disorder is mediated by repeat RNA, such as those described above or in Tables 1 and 2. In
some embodiments, the e or disorder is a repeat expansion disease in which the repeat
resides in the coding regions of mRNA. In some ments, the disease or disorder is a
repeat expansion e in which the repeat resides in the noncoding regions of mRNA. In
some embodiments, the disease or disorder is selected from Huntington’s e (HD),
dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), or a
spinocerebellar ataxia (SCA) ed from SCAl, SCA2, SCA3, SCA6, SCA7, or SCA17. In
some embodiments, the disease or disorder is selected from Fragile X Syndrome, myotonic
dystrophy (DMl or dystrophia myotonica), Friedreich’s Ataxia (FRDA), a spinocerebellar ataxia
(SCA) selected from SCA8, SCAlO, or SCA12, or C9FTD (amyotrophic lateral sclerosis or
ALS).
In some embodiments, the disease is amyotrophic lateral sclerosis (ALS),
Huntington’s e (HD), frontotemporal dementia (FTD), ic dystrophy (DMl or
phia myotonica), or Fragile X Syndrome.
In some embodiments, the present invention provides a method of treating a e
or disorder mediated by repeat RNA, comprising the step of administering to a patient in need
thereof a compound of the present invention. Such compounds are described in detail herein.
Also provided is a method of producing a small molecule that modulates the activity
of a target repeat expansion RNA, comprising the steps of: screening one or more small
molecules disclosed herein for binding to a 3WJ in the target repeat expansion RNA; and
identifying which small molecule(s) bind to the 3WJ and te the activity of the target
repeat ion RNA. In some embodiments, the method further comprises identifying the
target repeat expansion RNA according to a disclosed computational survey. In some
embodiments, the target repeat expansion RNA is e of forming a 3WJ in the presence of
an effector RNA such as an effector miRNA. In some embodiments, the small molecule binds to
a trans 3WJ formed between the effector RNA and the target repeat expansion RNA. In some
embodiments, the small molecule binds to a cis 3WJ formed between portions of the target
repeat expansion RNA. In some embodiments, the repeat expansion RNA causes a e or
disorder selected from HD, DRPLA, SBMA, SCAl, SCA2, SCA3, SCA6, SCA7, or SCA17. In
some embodiments, the disease or disorder is selected from Fragile X Syndrome, DMl, FRDA,
SCA8, SCAIO, SCA12, or C9FTD. In some ments, the small molecule is effective to
treat or ameliorate the disease or disorder.
Other Target RNAs and Diseases/Conditions
An association exists between a large number of onal RNAs and diseases or
conditions, some of which are shown below in Table 3. Accordingly, in some embodiments of
the methods described above, the target RNA is selected from those in Table 3. In some
embodiments, the disease or disorder is selected from those in Table 3.
Table 3: Target RNAs and Associated Diseases/Conditions
RNA Tar_et Indication
A20 inflammatory diseases; liver failure; liver transplant
ABCAl coronary artery disease
ABCBl 1 Primary Biliary Sclerosis
ABCB4 Primary Biliary Sclerosis
ABCGS/8 coronary artery disease
Adiponectin diabetes; y; metabolic syndrome
AMPK diabetes
ApoAl hypercholesterolemia
ApoAS hypercholesterolemia
ApoC3 chylomicronemia syndrome
EEIIIIIIII-IIIIIIIIIIIIIIIIIIIIprostate cancer
ARlnc-l prostate cancer
ATXNl spinocerebellar ataxia 1
ATXN 1 O spinocereb ellar ataxia 10
ATXN2 ereb ellar ataxia 2
ATXN3 Soinocereb ellar ataxia 3
|||i||ATXN7 spinocereb ellar ataxia 7
ATXN8 spinocereb ellar ataxia 8
BACE]
BCL2 cancer
L CML
BDNF gton's Disease
|i|lRNA Tar_et tion
Beta-catenin cancer
cancer
BRCAl cancer
% cancer
BTK cancer
C9orl72 (ALS, FTD) ALS, FTD
A spinocereb ellar ataxia 6
CD2 74 tumor immunology
CD2 79 tumor immunology
CD3zeta inflammation and autoimmune diseases
CD4OLG inflammation
CFTR Cystic Fibrosis
cKIT GIST; mastocytoma
CNTF macular ration
Complement Factor H macular degeneration
CRACMI inflammatory diseases; autoimmune disease; organ transplant
CTLA4 cancer; inflammatory diseases
DGAT2 NASH
D102 dyslipidemia
Dystrophin Duchenne Muscular Dystrophy; Becker's Muscular Dystrophy
EGFR cancer
EIF4E cancer
EZH2 cancer
Factor 7 hemophilia
Factor 8 ilia
Factor 9 hemophilia
Fetal Hemoglobin sickle cell anemia; halassemia
FLT3
FMR1 Fragile X Syndrome
Foxp3 inflammation & autoimmune diseases
in Friedreich's Ataxia
HAlVIP/Hepcidin thalassemia; hereditary hemochromatosis
mERZ cancer
HIF- 1 a cancer
HOTAIR cancer
H Huntington’s Disease
l—lVaHa rheumatoid arthritis
IL- 1 7 inflammatory & autoimmune diseases
IL-23 inflammatory & autoimmune diseases
RNA Tar_et Indication
IL-6 rheumatoid arthritis
Ipfl/del diabetes
KRAS cancer
Laminin— l a Merosin-deficient congenital muscular dystrophy MDCAl
LARGE Muscular Dystroglycanopathy Type B,6
LDLR hypercholesterolemia
LINGOI neurodegeneration
MALATl cancer
MAX cancer
MBNLl Myotonic phy
MCLI cancer
MECP2 Rett Syndrome
Mertk Lupus
miR-103 NASH
miR-107 NASH
miR- 1 0b GBM
miR-155 ALS and others
miR-21 solid tumors
miR-221 ECC
mTOR cancer
MYO cancer
Nanog neurological diseases
neurofibromatosis
z33 multiple sclerosis
phenylketonuria
PCSK6 hyoertension
PCSK9 hypercholesterolemia
PD-l cancer; inflammation
PD-Ll cancer; inflammation
PDKl/2 Polycystic kidney e
PGC l -a/FNDC5 PGCl—a/FNDCS
Progranulin ogical diseases
PTB-lB diabetes
PTEN cancer
PTPN1 Type II diabetes
r(AUUCU)‘:X13 SCAlO
r(CAG “13 Huntington’s Disease
r(CCUG)‘:Xp DM2
r(CGG exp FXTAS
RNA Tar_et Indication
r(CUG)‘”‘p DMl
r(GGGGCC)exP c9ALS (familial)
r(GGGGCC)eXp C9FTD
Ras Cancer
RORC autoimmune e
RTN4 Neurodegeneration
RTN4R Neurodegeneration
Sarcospan ne ar phy
Serca2a congestive heart failure
SirT6 Cancer
SMAD7 6U
SMN2 Spinal Muscular Atrophy
SNCA
SORTl coronary artery disease
U) D—t ry artery disease
STAT3 Cancer
STATS Cancer
T-bet Cancer
Thyroid Hormone Receptor
beta dyslipidemia; NASH; NAFLD
THVI-3 inflammatory diseases; cancer
inflammatory disease
TNFRSF1[A Osteoporosis
TNFSF11 Osteoporosis
TRIBI coronary artery disease
TTpd Amyloidosis
TWISTI Cancer
Utrophin Duchenne Muscular Dystrophy
Wnt Cancer
Targeting Viral RNAs
In an aspect of the invention, the compounds and disclosed methods are used to target
a viral nucleic acid or a transcript thereof. In some ments, the virus has an RNA genome.
Both single-strand RNA and double-strand RNA s have double-stranded RNA sequences
that may be selected as targets for modulation. For viruses such as HCV (Pirakitikulr et al., Mol.
Cell 2016, 61, 1-10) and HIV—1 (Lavender et al, PLOS Comp. Bio. 2015, 11), a substantial
amount of RNA structural information exists, and 3WJs are present in the structures of the viral
RNA genomes. Many viruses have RNA structures or c elements that are rarely found or
not found in ian genomes. Thus, targeting these elements provides selective antiviral
agents with minimal effect on host processes. These genetic elements include RNA sequences
that mediate translation, such as IRES elements that are far more common and functionally
significant in s than in the mammalian genome. Unique RNA sequences also can be
essential for the packaging of the RNA into a virus particle. Accordingly, in some embodiments,
the target nucleic acid is a viral RNA structure or genetic element rarely found in mammalian
genomes or exclusively found in viral genomes. In some ments, the viral RNA structure
or genetic element is an IRES element.
The disclosed compounds and s may be used to target virtually any virus,
because every virus must produce RNA and thence proteins at some point in their life cycles. In
some embodiments the virus is selected from a Group I (dsDNA viruses), Group II (ssDNA
viruses), Group III (dsRNA viruses), Group IV ((+)sense RNA s), Group V ((-) sense RNA
viruses), Group VI (RNA reverse transcribing viruses), Group VII (DNA reverse transcribing
viruses) virus, or a subviral agent such as a satellite or viroid.
For additional target viruses, see, e. g.
http://www.virology.net/Big_Virology/BVFamilyGroup.html. In addition to targeting viral
RNA transcripts ning a 3WJ, mixed RNA/DNA s comprising viral nucleic acids
may be ed. For example, viral genomic DNA interacts with endogenous RNA effectors to
produce 3WJs from mixed RNA/DNA hybridization events. Accordingly, in some embodiments
the target nucleic acid is a mixed RNA/DNA hybrid comprising genetic information from a virus
such as those disclosed above, wherein the hybrid is e of forming or contains one or more
3W1s.
In some embodiments, the virus is an RNA virus (i.e., a virus having an RNA
) such as a flavivirus, for example Zika virus, West Nile virus, Dengue virus, Yellow
Fever virus, or Japanese encephalitis (Shi (editor), Molecular Virology and Control of
Flaviviruses, 2012, Caister ic Press). In some embodiments the virus is a coronavirus.
Coronaviruses are a family of single-strand (ss) RNA viruses that includes human pathogens
such as SARS-causing virus SARS-CoV (Thiel, r), Coronaviruses: Molecular and Cellular
Biology ([31 ed), 2007, r Academic Press). Other RNA viruses that may be treated by the
present invention include Ebola virus. In some embodiments, one or more of the foregoing RNA
viruses is targeted by small molecule-induced 3WJ formation and/or small molecule-mediated
stabilization of the 3W] in order to block replication, translation or packaging of the viral RNA.
In some embodiments, the target RNA is the RNA viral genome of the viral pathogen and the
effector RNA is an endogenous small RNA in the infected cell.
] In some embodiments, the virus is selected from a Group IV —stranded RNA
virus such as Coxsackie virus, Norovirus, Measles virus, Hepatitis C virus, Zika virus, Ebola
virus, Rabies virus, West Nile virus, Dengue virus, SARS coronavirus, or Yellow fever virus. In
some ments, the virus is selected from a Coronavirus such as Avian infectious bronchitis
virus, Bovine virus, Canine coronavirus, Feline infectious peritonitis virus, Human
coronavirus 299E, Human coronavirus OC43, Murine hepatitis virus, Porcine epidemic diarrhea
virus, Porcine hemagglutinating encephalomyelitis virus, e transmissible gastroenteritis
virus, Rat coronavirus, Turkey coronavirus, Rabbit coronavirus, Torovirus, Beme virus, or Breda
virus. In some embodiments, the virus is ed from a frlovirus, Marburg virus, or Ebola
virus.
Microbial Nucleic Acid Targets
Various other infectious agents provide suitable target nucleic acids. In some
embodiments, the target nucleic acid is a bacterial nucleic acid. Nucleic acids such as RNA in
pathogenic bacteria provide target sequences that are critical for the life cycle of the ens or
affect the bacterial life cycle, nce, or pathogenicity and in some cases differ y in
sequence and function from human RNAs. For e, the target bacteria may be a
tuberculosis-causing bacteria.
Fungal and Parasitic s
In some embodiments, the target nucleic acid is a fungal or parasitic nucleic acid,
such as a malarial nucleic acid. Nucleic acids such as RNA in pathogenic fungi and parasites
provide target sequences that are critical for the life cycle of the pathogens or affect the life
cycle, virulence, or pathogenicity and in some cases differ greatly in sequence and function from
human RNAs.
miRNA as Effectors
MicroRNA (miRNA) is a class of single-strand noncoding RNAs that are
approximately 22 nucleotides (nt) in length (Friedman et al., Genome Res. 2009, 19, 92-105,
Ghildiyal & , Nat. Rev. Genet. 2009, 10, 94-108; Ipsaro & Joshua-Tor, Nat. Struct. M0].
Biol. 2015, 22, 20-28; alde, Science 2015, 349, 380—3 82). They play important regulatory
roles in repressing gene expression by functioning at the post-transcriptional level. They are
well-conserved and are believed to be critical components in gene regulation across many
species in plants and animals. Their “seed” sequence can interact with sequences in the 3’
slated region (UTR) of target mRNA, and less frequently, the 5’ end of mRNA. This
base-pairing event leads to a se in the expression of the target gene by inducing
degradation of the mRNA or by inhibiting translation of the mRNA into protein. miRNAs
influence a wide range of biological processes including cell entiation, cell fate, motility,
survival, and cell on. miRNAs can be associated with a variety of human diseases
including cancer as well as metabolic, cardiovascular, neurological and inflammatory diseases
(Croce, Nature Rev. Genet. 2009, 10, 704-714; Dykxhoorn Cancer Res. 2010, 70, 6401-6406;
Quiant & Olson, J. Clin. Invest. 2013, 123, 11-18, Olson, Science Trans. Med. 2014, 6, 239ps3;
Baffy, J. Clin. Med. 2015, 4, 988). In many settings, the alteration in miRNA expression
is associated with human diseases.
Accordingly, in some embodiments the present invention provides a method of
modulating the activity of a target miRNA or precursor or protein-bound complex or mRNA- or
ncRNA-bound complex thereof, comprising contacting the target miRNA or precursor or
protein-bound complex or mRNA- or ncRNA-bound complex thereof with a disclosed
compound.
miRNAs are expressed from their own distinct genes or are expressed within introns,
or in some cases exons, of other genes. Most miRNAs are transcribed by RNA rase II,
The y transcript containing the miRNA ce is known as a pri-miRNA. Pri-miRNAs
have unique structural features that differ from other types of RNA, including a segment that
folds to form a “hairpin” or stem-loop structure that is flanked by segments of a single-strand
RNA. This double-stranded RNA structure can be recognized by the microprocessor complex
that contains the RNA-binding n DGCR8 (DiGeorge Syndrome Critical Region 8) and the
RNase III enzyme Drosha. DGCR8 orients the catalytic RNase 111 domain of Drosha to produce
r hairpins from pri-miRNAs by cleaving RNA at approximately eleven nucleotides from
the hairpin base (one helical double-strand RNA turn into the stem). The microprocessor
complex cuts the RNA to generate a pre-miRNA of 60 to 70 nt. A single pri-miRNA can
n one to six pre-miRNAs. The RNA itself has a distinct stem-loop structure. It
interacts with Exportin-S and Ran GTPase, leading to its transport from the nucleus into the
cytoplasm. In the cytosol, the miRNA-protein complex is ized by the RNase III enzyme
Dicer that cleaves the pre-miRNA into a mature miRNA. The Dicer endoribonuclease interacts
with 5’ and 3’ ends of the hairpin and cleaves the loop joining the 3’ and 5’ arms, ng a
miRNA duplex. The final miRNA is an approximately 22 base pair duplex having 2 nt 3’
overhangs and 5’ phosphate groups. One strand of this mature miRNA is then loaded onto
Argonaute (Ago) protein to form the RNA-Induced Silencing Complex (RISC). Members of the
Ago protein family are central to RISC function. Ago proteins are needed for miRNA-induced
silencing and contain two conserved RNA binding s — a PAZ domain that can bind the
single stranded 3’ end of the mature miRNA and a PIWI domain that structurally resembles
ribonuclease H and functions to ct with the 5’ end of the guide strand of the miRNA.
These proteins bind the mature miRNA and orient it for interaction with a target mRNA. Some
Ago family members such as the human AgoZ cleave the RNA targets directly. Ago proteins
also can recruit additional proteins to inhibit translation. Although either strand of the duplex
may potentially act as a functional miRNA, only one strand is incorporated into the RISC
complex with which the miRNA and its mRNA target ultimately ct. miRNA base pairs
with its target mRNA, leading to decreased target mRNA levels or tion of its translation.
Induction of mRNA cleavage leading to mRNA decay appears to be the more common
mechanism of inhibition of expression. Translational repression is less well understood than the
induction of mRNA degradation. As noted above, a single miRNA can have many target
mRNAs. Besides targeting mRNA, miRNAs also can target noncoding RNAs.
Thus, in some embodiments, the target miRNA is a precursor to a corresponding
mature miRNA. In some embodiments, the target miRNA is a pri-miRNA. In some
embodiments, the target miRNA is a pre—miRNA. In some ments, the target miRNA is
an mRNA- or ncRNA-bound complex such as a mature miRNA bound to an mRNA or ncRNA
whose activity it tes. In some embodiments, the target miRNA is associated with a disease
or disorder such as cancer, a metabolic disorder, a cardiovascular er, a neurological
disorder, or an inflammatory disease, In some embodiments, the compound up-regulates the
activity of the target miRNA. In some embodiments, the compound down-regulates the activity
of the target miRNA, In some ments, the down-regulation is via binding to a 3WJ in the
target miRNA-mRNA or miRNA-ncRNA complex. In some embodiments, binding to the 3WJ
inhibits translation of the mRNA. In some embodiments, binding to the 3WJ induces
degradation of the mRNA or ncRNA.
] Also provided is a method of producing a small molecule that modulates the ty
of a target miRNA or precursor or protein-bound complex or mRNA- or ncRNA-bound complex
thereof, comprising the steps of: screening one or more small les disclosed herein for
binding to a 3WJ in the target miRNA or sor or n—bound complex or mRNA- or
ncRNA-bound x thereof; and identifying which small molecule(s) bind to the 3WJ and
modulate the activity of the target miRNA or precursor or protein-bound x or mRNA- or
ncRNA-bound complex thereof. In some embodiments, the method further comprises
identifying the target miRNA or precursor or protein-bound complex or mRNA— or ncRNA-
bound complex thereof according to a disclosed computational survey. In some embodiments,
the target miRNA is an effector capable of forming a 3WJ with an mRNA or ncRNA which it
regulates. In some embodiments, the small molecule binds to a trans 3WJ formed between the
effector and the mRNA or ncRNA. In some embodiments, the small molecule binds to a cis
3WJ formed by a portion of the mRNA or ncRNA.
There are roughly 2000 miRNAs in the human genome. miRNAs can be expressed in
a tissue-specific and cell-specific manner. Their expression can be affected by a variety of
stimuli including hormones, cell stress, oncoproteins, cytokines and hypoxia. As stated above,
aberrant miRNA expression can be associated with a wide variety of human es, and this
aberrant miRNA expression is correlated with the dysregulation of the sion of their target
genes.
Perfect base-pairing of the entire miRNA with target mRNA is rare in mammalian
cells. It is believed that perfect or near perfect base pairing with the target mRNA promotes
cleavage of the target RNA. A short stretch of nt in the 5’ end of the miRNA (“the seed”)
s to be sufficient for airing to the target sequence in the mRNA. This base-pairing
can comprise as few as 6 nt. The limited sequence complementarity needed for miRNA-mRNA
interaction enables single miRNAs to regulate a large number of ripts. It has been
documented that individual miRNA can affect the expression of well over 100 different mRNA,
with estimates as high as 400 mRNA targets. Although the requirements for miRNA recognition
of mRNA do not appear to be stringent, research has led to a description of rules that predict
canonical miRNA targeting. Seed nucleotides 2-8 at the 5’ end of the miRNA are important for
target recognition. Crystal structures of the Ago-miRNA complex (Schirle et al., Science 2014,
346, 608-613) suggest that the seed ce is positioned to initiate the interaction between the
miRNA and its target mRNA. A t seed complementarity is considered to be canonical
targeting, but ect or ed interactions are ed (reviewed in Seok et al., M0]. Cell,
2016, 39, 375-381). Thus, it appears that a variety of airing can be used to achieve
miRNA-mRNA recognition. Non-canonical pairing can take place and may have biological
consequences, It has been esized that these weaker, non-canonical, binding events could
lead to less effective repression of expression and could, therefore, provide for a range of
effectiveness in the repressive activity of miRNA. It even has been reported that human
microRNA miR369-3 directs the association of Ago and FXRl ns with sequences in the 3’
UTRs of mRNA to increase translation, while another miRNA, Let—7, also can increase
translation of target mRNAs upon cell cycle arrest (Vasudevan et al., Science 2007, 318, 1931-
1934). Therefore, it is demonstrated that miRNAs can have a wide range of effects in
mammalian cells. Accordingly, in some embodiments, a sed compound binds to a 3WJ
formed between a target miRNA and an mRNA or ncRNA that the miRNA regulates. In some
embodiments, a sequence of nt in the 5’ end of the miRNA (the seed) is capable of base—pairing
to a target sequence in the mRNA. In some embodiments, a sequence of nt in the 3’ end of the
miRNA (the seed) is capable of base—pairing to a target sequence in the mRNA. In some
embodiments the base—pairing comprises cal (Watson-Crick) base-pairing, for example
between 75%, 80%; 85%; 90%; 95%, 98%; 99%; or all of the seed and its target sequence. In
some embodiments, the airing comprises at least 6 nt. In some embodiments, the base-
pairing comprises at least 7, 8, 9, 10, ll, 12, l3, l4, 15, or 20 nt. In some embodiments, the
base-pairing comprises at least 25, 30, 35, 40, 45, 50, 75, or 100 nt.
The rapid and controlled turnover of mature miRNA is needed for rapid changes in
miRNA expression s and resulting gene regulation. For example, the binding of miRNA
to Ago protein in the cytoplasm is believed to ize the miRNA guide strand, while the
opposite ger strand is degraded. Target ment also may stabilize the miRNA. Post-
transcriptional modifications of the miRNA also can affect their half-lives in cells.
In some embodiments, the miRNA is an effector such that the miRNA recognizes a
target sequence in an RNA such as an mRNA and bind in a manner which leads to the formation
of a 3WJ. A described above, in some embodiments this 3WJ ses a stem-loop structure
flanked on each side by sequences of base pairing between the miRNA and mRNA. The stem-
loop can be formed by either the miRNA or mRNA. In some embodiments, a stable 3WJ is
formed and degradation of the mRNA is not promoted. In some embodiments, there is not
perfect complementarity (canonical base-pairing) between the miRNA seed (miRNA positions 2-
7 or 2-8 from the 5’ end) and the target mRNA. In some embodiments, the base pairing is just
downstream of the seed or can overlap with the seed, but does not include the entire seed. For
example, nt 5-10 of the miRNA could have perfect base pairing with the mRNA target,
immediately followed by a formation of a stem-loop by the mRNA sequence, and then the
miRNA could base pair perfectly with the mRNA over a stretch of 4-12 nt starting at miRNA nt
11,12,13 or 14.
piRNA as Effectors
Piwi-interacting RNA (piRNA) is the largest class of small ing RNA
expressed in animal cells wed in Seto et al., Molecular Cell, 2007, 26, 603-609;
Klattenhoff and Theurkauf, Development 2008, 135, 3-9, 2008). There may be 0 or more
piRNAs in animal cells. These RNAs are 26-31 nt in length. piRNAs derive their name due to
their interaction with piwi proteins. They are ed to be involved in gene silencing, but far
less is known about the generation of piRNA and their mechanism of gene silencing relative to
miRNA. Many piRNAs are antisense to transposons, therefore, the silencing of transposons may
be a critical role of piRNAs. They are found to localize to both nuclear and cytosolic
compartments of the cell. piRNAs are abundant in germ cells where they may play critical roles
in germ cell pment and function. However, they also are abundant in a wide variety of
mammalian somatic cells and tissues.
Accordingly, in some embodiments the present invention provides a method of
modulating the activity of a target piRNA or precursor or protein-bound complex thereof,
comprising contacting the target piRNA or sor or protein-bound complex thereof with a
disclosed compound.
In some embodiments, the target piRNA is associated with a disease or disorder such
as a disease involving aberrant germ cell development or function. In some embodiments, the
nd up-regulates the activity of the target piRNA. In some embodiments, the compound
down—regulates the activity of the target piRNA. In some embodiments, the egulation is
via binding to a 3WJ in the target piRNA or precursor or protein-bound complex thereof. In
some embodiments, binding to the 3W] induces degradation of the piRNA.
Also provided is a method of producing a small molecule that modulates the activity
of a target piRNA or sor or protein-bound complex thereof, comprising the steps of:
screening one or more small molecules disclosed herein for binding to a 3W1 in the target
piRNA or precursor or protein-bound complex thereof; and identifying which small le(s)
bind to the 3WJ and modulate the activity of the target piRNA or precursor or protein—bound
complex thereof. In some embodiments, the method further comprises identifying the target
piRNA or precursor or protein—bound complex thereof according to a disclosed computational
survey. In some embodiments, the target piRNA is an effector capable of g a 3WJ with an
RNA which it regulates. In some embodiments, the small molecule binds to a trans 3W] formed
n the effector and the RNA which it tes. In some embodiments, the small molecule
binds to a cis 3W1 formed by a portion of the piRNA.
snoRNA as Effectors
Small nucleolar RNAs (snoRNAs) are small RNAs that direct chemical modifications
such as methylation or pseudouridylation of other RNAs such as mal RNAs, tRNAs, and
small nuclear RNAs (snRNA) (reviewed in Bachellerie et al., Biochimie 2002, 84, 775-790;
Jorjani et al., c Acids Res. 2016, May 12 [Epub ahead of print]). They are lly 70-90
nt in . 1740 snoRNAs have been identified. They are fairly abundant in mammalian cells.
To carry out RNA modifications, snoRNAs associate with proteins in a small nucleolar
cleoprotein (snoRNP) complex. The snoRNA contains an antisense sequence of 10-20 nt
complementary to the sequence surrounding the base targeted for modification. Most snoRNAs
are encoded in the s of genes encoding proteins involved in ribosome synthesis or
translation. They also can be located in intergenic regions, ORFs of protein coding genes, and in
UTRs. They can be transcribed by either RNA polymerase II or III. The sequences of snoRNA
are ved, but their expression levels differ dramatically between s.
Accordingly, in some ments the present invention provides a method of
modulating the ty of a target snoRNA or precursor or protein-bound (such as a snoRNP)
complex thereof, comprising contacting the target snoRNA or precursor or protein-bound
complex thereof with a disclosed nd.
In some embodiments, the target snoRNA is associated with a disease or disorder
such as a disease involving aberrant nucleic acid modification such as methylation or
pseudouridylation. In some embodiments, the aberrantly modified nucleic acid is a mal
RNA, a tRNA, or an snRNA. In some embodiments, the compound up-regulates the activity of
the target snoRNA. In some embodiments, the compound down-regulates the activity of the
target snoRNA. In some embodiments, the down-regulation is via binding to a 3WJ in the target
snoRNA or precursor or protein—bound x thereof.
Also provided is a method of ing a small molecule that modulates the activity
of a target snoRNA or precursor or protein—bound (such as a snoRNP) complex thereof,
comprising the steps of: screening one or more small molecules disclosed herein for binding to a
3WJ in the target snoRNA or precursor or protein-bound (such as a snoRNP) complex thereof;
and identifying which small molecule(s) bind to the 3WJ and modulate the activity of the target
snoRNA or precursor or protein-bound complex thereof. In some embodiments, the method
further comprises identifying the target snoRNA or precursor or protein—bound complex thereof
according to a disclosed computational survey. In some ments, the target snoRNA is an
effector capable of g a 3WJ with an RNA which it regulates. In some embodiments, the
small le binds to a trans 3WJ formed between the effector and the RNA which it
regulates. In some embodiments, the small molecule binds to a cis 3WJ formed by a portion of
the snoRNA.
3. Methods ofIdentifying Target 3 WJs
Computational Survey of Potential Effector/Target c Acid Interactions
The present ion provides methods of identifying a potential target 3WJ in a
nucleic acid. ation about nucleic acid three-dimensional structure is more challenging to
obtain than proteins, and there exists a need for better methods for interrogating nucleic acid
structure, particularly in vivo. Accordingly, in one aspect the present invention provides a
method of identifying a target nucleic acid capable of g a cis or trans 3WJ, wherein
g by a disclosed nd stabilizes the 3WJ, thus modulating the activity of the nucleic
acid, for example to treat a disclosed disease or condition. In some embodiments, a provided
method comprises as a first step g an in silico search that identifies potential ctions
between effector RNAs and candidate target RNAs. In some embodiments, the search focuses
on a target RNA selected based on prior knowledge of the value of targeting either that RNA
itself or the protein that it expresses upon translation. The output of the search is a list of effector
RNAs (such as small effector RNAs of, for example, 20-100 nt) that can hybridize with the
target RNA such that a 3WJ can form. In other embodiments, a broader or even comprehensive,
in silico search of all potential interactions between RNAs (as effectors) and all target RNAs in
the cell is run. In some embodiments, the search is conducted initially without reference to any
extant body of knowledge of the actual, known ical ons of the effector RNAs.
Rather, each effector RNA is treated essentially as a chemical without reference to its biological
role.
In one aspect, the present invention provides a method of identifying a target nucleic
acid capable of forming a 3WJ comprising the following steps:
(a) providing one or more effector c acids (such as a human small RNA)
comprising one or more energetically accessible stem-loop ures,
(b) providing one or more target nucleic acids (such as a human mRNA) comprising one
or more energetically accessible stem-loop structures,
(c) screening for one or more effector/target nucleic acid hybridization ctions that
accommodate a stem-loop ure such as a 3WJ in either the effector nucleic acid or the target
nucleic acid,
((1) optionally, categorizing the resulting database of 3WJS comprising an effector nucleic
acid and target nucleic acid by one or more of: (1) loop topology, (2) loop sequence, (3) stem-
loop stability, and (4) identity of the nucleobases that impinge ly on the 3WJ cavity; and
(e) optionally, cross-referencing the resulting database of possible 3WJs comprising an
effector nucleic acid and target nucleic acid with available dge about the biological role
and therapeutic cance of the target nucleic acids as well as available knowledge about the
relative abundance and cellular location of effector nucleic acids such as small effector RNAs.
In step (a) above, in the case of effector miRNAs, in some embodiments a publicly
ble database is used as a source of potential or miRNAs. In some embodiments, the
database is selected from: (hit3:1","wwwmirbaseora, hit :x’x’mi rdb " "miRIifiBf
. or
htt 3 :.//www .miaroma. org/miminim/home , d o"7
hill}.SIZE}.itfiXaIlQQLEEZQswig; 12Iii:3111’5ziammnit;fiimsmtg; blil:?§.c’ii’félfl£.QEQIEEJAEEE
lieideiherg(fie/apps/sz/minvalkZI"; or http:.I’z‘immvtargetscanorg). In some ments, the
target c acid is human. In some embodiments, the target c acid is viral. In some
embodiments, a database of viral genomes is used, such as that available at
htt):f/wwwnchinlnrnih.gov/Genomex’viFuses/C In some embodiments, the target nucleic acid is
ial. In some embodiments, the target nucleic acid is fungal. In some embodiments, the
target nucleic acid is parasitic.
In step (a) above, in some embodiments, “energetically accessible” means that at least
0.1% of that miRNA in a living cell adopts (or is predicted to adopt) a given stem—loop structure.
In other embodiments, the threshold of “accessible” might be set at 0.1% to 1%, 1% to 10%, or
about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some embodiments, to be included in
the method above, such stem-loop needs to leave at least 4 tides upstream (5’) and at least
4 nucleotides downstream (3’) of the oop that can participate in hybridization with the
target nucleic acid.
In step (b) above, in some ments, “energetically accessible” means that at least
0.1% of that miRNA in a living cell adopts (or is predicted to adopt) a given stem—loop structure,
In other embodiments, the threshold of “accessible” might be set at 0.1% to 1%, 1% to 10%, or
about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In some embodiments, to be included in
the method above, such stem-loop needs to leave at least 4 nucleotides upstream (5’) and at least
4 nucleotides downstream (3’) of the stem-loop that can participate in hybridization with the
target nucleic acid.
In step (c) above, in some embodiments, the method es a compilation of all
effector/target c acid pairs that create a 3W1 ying the constraints described above. In
all three of the possible unhybridized inter-stem loops in a 3W], the lengths of those loops can
vary ndently from O to 4 (or, in some embodiments, 0 to 2 or 0 to l), with any of the
standard or post-transcriptionally modified nucleobases occupying those loop positions.
In some embodiments, pairings that engage therapeutically high-value target nucleic
acids with relatively high-abundance small effector RNAs will be the focus of further study. The
structure, dynamics, and ical significance of the selected key 3WJs can be characterized
extensively using a wide range of established s described below.
In some embodiments, a 3WJ such as a 3WJ identified in the above computational
screening methods, may be characterized by synthesizing the or nucleic acid (such as a
small effector RNA) and its cognate target c acid using standard, commercially ble,
machine-enabled synthetic s. In addition, in many ments, a single-stranded
(“cis”) analog of a trans-3WJ is synthesized, in such embodiments, the two components can be
linked either 5’—to-3’ or 3’-to-5’. The advantages of a single-stranded model include simplified
assay formats and controlled stoichiometry in structural characterization.
Whether in trans or in cis, the model effector/target nucleic acid interacting pairs can
be structurally characterized in detail using X-ray llography, cryo-EM (Binshtein & Ohi,
Biochemistry 2015, 54, 3133-3141), NMR, circular dichroism, UV-vis spectroscopy, and
MaP (Siegfried et al., Nature Methods 2014, 11, 959-973), and related methods that help
to establish the detailed 3D structure and dynamics of the construct and thus, by implication,
allow inference of the structure and dynamics of the congeneric trans pair in cells.
Building Binding Assays to Measure SM Binding to 3WJs
One of the principal purposes in building the above cis and trans models is to screen
for small molecules that will bind selectively and with high affinity into the cavities defined by
the 3W1s. Many methods that are currently deployed for analogous screening of small
molecules against n targets can be adapted to similar effect in 3WJs. In some
embodiments, one of the following exemplary assays is used to measure small molecule binding
to an effector/target nucleic acid 3WJ:
NMR — NlVIR can be used to assess the binding of small molecules to biomolecules
such as RNA. One key advantage of this method is that, if the ure of the target biomolecule
can be observed during the screen, then the screen will yield not just the fact of molecular
association between SM and RNA, but also the subsite and binding mode. Some of the
disadvantages of NlVIR are that relatively high trations of ligand are required (uM to
mM), meaning that high—affinity interactions are difficult to measure, and that the method is
slow, thus permitting only limited hput. But these latter ms actually make NMR an
ideal read-out in carrying out fragment screening, where high-concentration/low-affinity
interactions are the focus and relatively few fragments (typically <5000) are usually ed in
nt-based lead discovery. In some cases the atomic nucleus that is observed is 19F rather
than s.
SPR — Surface plasmon resonance is a widely implemented method for assessing
molecular associations. SPR requires that one component (the target RNA or the small molecule
ligand) be covalently affixed to the surface and the plasmon resonance is measured in response
to partner molecules added to the solution above the lized partner. On rates and off rates
can be measured directly, affording equilibrium dissociation constants.
DELs — DNA—encoded libraries serve here as an example of a broader category of
pull-down s. In DELs, a cis model of a key 3WJ would be modified with functional
groups (e.g., biotin) that allowed facile pull-down (e.g., with streptavidin) after having been
exposed to a homogeneous DNA-encoded library. Those small molecule ligands in the DEL that
ate tightly with the 3WJ will also be pulled down when the 3WJ is pulled down and the
identity of the ligands exposed using PCR and sequencing.
Chromophoric 3WJs — There are multiple reports of derivatizing RNAs such that
two chromophores interact when proximal, leading to either activation or suppression of
emission, which in turn relies upon the RNA assuming the correct conformation (in this case the
anticipated 3WJ). Loop sequence, loop length, temperature, and other parameters can be
optimized so that achieving the 3WJ conformation depends on the stabilizing ce of an
added small molecule that binds into the 3WJ cavity. These assays are straightforward and can
be run in geneous or homogeneous formats and allow screening of standard, commercially
available ies of small molecules.
WO 06074
RNA Microarrays — One can immobilize a wide range of 3WJs onto a microarray —
in effect an immobilized library of targets. The chromophore functionality described above can
be incorporated so that when the surface-immobilized y of RNA (or other nucleic acid)
targets is exposed to small molecules (singly or as mixtures), chromophoric change at c
sites on the microarray indicate that that RNA (or other nucleic acid) is ligated and, more
importantly, stabilized in the 3WJ conformation.
SM Microarrays — One can immobilize a wide range of small les onto a
microarray. There is a broad literature on this topic and a commensurately broad range of
immobilization techniques. In such microarrays, the identity of the SM is typically associated
with its position in the microarray. Chromophoric 3W] models can then be exposed in to the
rray and specific SM/3WJ interactions identified by the observation of the chromophore at
specific sites on the microarray.
MS — Nucleic acids can be analyzed by a variety of liquid chromatography
techniques. Chromatography of RNA in the presence of potential small molecule ligands would
lead to bound small le ligands being d with the RNA as it passes through the
chromatography media. Post-chromatographic separation of the ligand from the RNA will allow
identification of the g ligands by mass spectrometry. If two ligands share the same mass
(are isomers), there are numerous pathways to disambiguation.
Initial Screens of 3WJs — Initial screening of small molecules against the 3W]
models will focus on three types of ies: (1) Compounds that are C3-symmetric or are
variations on C3-symmetric scaffolds; (2) Compounds that are variations on reduced-symmetry
scaffolds that still approximate the C3-symmetric congeners, and (3) A structurally diverse
library. These scaffolds and exemplary ments thereof are described in detail below.
Structural Characterization of 3WJ/SM Complexes — Many therapeutically
interesting RNA targets are large and only modestly structured, so detailed structural
characterization is either not possible or not practical. However, targeting of 3WJs as in the
present invention means that the target ucture modeled by the synthetic 3WJ is relatively
small and its complexes with SMs may be characterized in detail. Methods of structural
characterization e NMR, x-ray crystallography, cryo-EM, and CD. Such a detailed
terization of a small molecule complexed with its target RNA provides nearly
unprecedented opportunities for structure—based drug design against an RNA target.
Confirmation of Trans Effect
In much of the foregoing the focus is on the cis (single-stranded) models. When the
therapeutic targets are composed of a trans interaction n an effector RNA and a target
RNA, then the single—stranded cis construct is studied out of convenience and any small
molecule ligand needs to be demonstrated to bind to and stabilize the congeneric trans complex
upon which the cis model is based. Put differently, it is essential that the selected small
molecules identified via design or ing induce the formation of the intended
effector/SM/target ternary complex.
Building Competitive Displacement Assays
The screens bed above will identify small le leads against trans
effector/target c acid 3WJs of interest. While exploration of the SAR from those leads to
development candidates may be possible using the methods and techniques bed above, it is
often important to develop assays where new molecules compete with a reference le for
the same biologically essential subsite, in this case the cavity circumscribed by the 3W]. This
assay format is often very high—throughput and focuses the screen to find molecules that share
the same binding mode as the reference molecule. In some embodiments, such an assay
complises the steps of: (l) identification of the initial small molecule for testing, described above
and below, (2) modification of that small molecule, where necessary, to include a chromophoric
read-out where displacement either activates or suppresses on and (3) concomitant
modification, where necessary, of the nucleic acid target proximal to the binding site to
orate a mentary chromophoric read-out.
Demonstration of Cellular Activity
All of the foregoing takes place in relatively artificial biochemical settings. But the
selected small molecules need to induce the formation of the ternary complex inside cells.
rmore, the formation of that ternary complex needs to impact the stability, function, and/or
translation of the targeted nucleic acid, e.g. RNA, which will in some embodiments is a pre-
mRNA or mRNA. The demonstration that the required y complex as formed inside the cell
can be readily achieved by making a tethered reagent, based on the ted , and
performing seq or other affinity—based techniques on that ternary x.
] Impacting the functional career of a targeted RNA can be demonstrating using
standard reporter gene assay methods. In some embodiments, the target nucleic acid, e.g. RNA
sequence, is introduced into a Luciferase er vector or other standard reporter uct and
the impact of the 3WJ and small molecule on the reporter expression is measured in cells. In
cases in which the 3WJ and small molecule will affect the expression levels of the target, the
levels of the RNA can be measured by quantitative RT-PCR and the level of protein measured by
ELISA, Western blot or FRET assay.
2. Compounds and Embodiments Thereof
In one aspect, the present invention provides compounds, and pharmaceutically
acceptable compositions thereof, for use in modulating the activity of a target nucleic acid by
binding to a 3WJ in the target nucleic acid or an effector/nucleic acid complex. Such compounds
are effective for treating, preventing, or ameliorating a disease or condition associated with a
target RNA, and for use in methods described herein.
Compounds that may be used in the present invention and methods of discovering
such compounds (such as SHAPE-MaP, RING-MaP, and PEARL-seqTM (also known as Hook
the Worm and Hook and Click methods) are described in US. Provisional Patent Application
USSN 62/289,671, which is hereby incorporated by reference in its entirety.
Compounds of the present invention include those described generally herein, and are
r illustrated by the classes, subclasses, and species disclosed herein. As used herein, the
following definitions shall apply unless otherwise ted. For purposes of this invention, the
al elements are identified in accordance with the ic Table of the Elements, CAS
version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of
organic chemistry are described in “Organic Chemistry”, Thomas l, University Science
Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, MB.
and March, J John Wiley & Sons, New York:
., 2001, the entire contents of which are hereby
incorporated by reference.
Small Molecules
The design and synthesis of novel, small molecule ligands capable of g RNA
represents largely untapped therapeutic potential. Certain small molecule ligands including
ides (e.g., omycin, azithromycin), ids (e.g., berberine, palmatine),
aminoglycosides (e,g., paromomycin, in B, kanamycin A), tetracyclines (e. g.,
doxycycline, oxytetracycline), theophyllines, and oxazolidinones (e.g., linezolid, tedizolid) are
known to bind to RNA, paving the way for the search for small molecules as RNA targeting
drugs. c dyes, amino acids, biological cofactors, metal xes as well as peptides also
show RNA binding ability. It is possible to modulate RNAs such as riboswitches, RNA
molecules with expanded nucleotide repeats, and viral RNA elements.
The terms “small molecule that binds a target RNA,” “small molecule RNA binder,”
“affinity moiety,” or d moiety,” as used herein, include all compounds generally classified
as small molecules that are capable of binding to a target RNA with sufficient affinity and
specificity for use in a disclosed method, or to treat, prevent, or ameliorate a disease associated
with the target RNA. Small molecules that bind RNA for use in the present invention may bind
to one or more secondary or tertiary structure ts of a target RNA. These sites include
RNA triplexes, 3WJs, 4WJs, parallel-Y ons, hairpins, bulge loops, pseudoknots, internal
loops, and other higher-order RNA structural motifs described or referred to herein.
Accordingly, in some embodiments, the small molecule that binds to a target RNA is
ed from a macrolide, alkaloid, aminoglycoside, a member of the tetracycline family, an
oxazolidinone, an SMN2 ligand, ribocil or a related compound, an anthracene, or a triptycene. In
some embodiments, the small molecule RNA binder is selected from paromomycin, a neomycin
(such as neomycin B), a cin (such as cin A), linezolid, tedizolid, pleuromutilin,
ribocil, NVS-SMl, anthracene, or triptycene. In some embodiments, the small molecule is
selected from those shown in US. Provisional Patent Application USSN 62/289,671, which is
hereby incorporated by reference in its entirety; or a pharmaceutically acceptable salt,
isomer, or tautomer thereof. Further exemplary small molecules are described in detail
below.
In some embodiments, the present invention provides a compound of Formula I:
or a pharmaceutically acceptable salt thereof, wherein:
Rings A, B, and C are each, independently, a 3-8 membered saturated or partially unsaturated
monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring,
a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5-6 membered
monocyclic aromatic ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur, or an 8—10 ed bicyclic heteroaromatic ring having 1-5
heteroatoms independently selected from en, oxygen, or ;
each Y is independently CR or N,
each R1 is independently -R, n, -CN, -OR, -N(R)2, -N02, -N3, -SR, or -L1-R6,
each R2 is independently -R, halogen, -CN, -OR, -N(R)2, -N02, -N3, -SR, —L2-R6, or two R2
groups on the same carbon are optionally taken together to form =NR6, =NOR6, =0, or :8,
each R3 is independently -R, halogen, -CN, -OR, -N(R)2, -NOz, —N3, -SR, or —L3-R6,
each R6 is independently hydrogen or C1-6 alkyl optionally substituted with l, 2, 3, 4, 5, or 6
each R is independently hydrogen or an ally substituted group selected from C1-6 tic,
a 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an
8-10 membered bicyclic aromatic yclic ring, a 4-8 membered saturated or partially
unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected
from nitrogen, oxygen, or sulfur, a 5—6 membered monocyclic heteroaromatic ring having 1-
4 heteroatoms independently selected from nitrogen, , or sulfur, or an 8-10
membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from
nitrogen, , or sulfur,
each L1, L2, and L3 is independently a covalent bond or a C1-8 nt straight or branched
hydrocarbon chain wherein 1, 2, or 3 methylene units of the chain are independently and
optionally replaced with -O-, -C(O)—, -C(O)O-, -OC(O)—, , -C(O)N(R)—, -(R)NC(O)-, -
OC(O)N(R)—, -(R)NC(O)O—, (O)N(R)—, -S-, —SO-, -SOz-, -S02N(R)-, -(R)NSOz-, -
C(S)—, -C(S)O-, -OC(S)-, -C(S)N(R)—, -(R)NC(S)-, -(R)NC(S)N(R)-, or -Cy-;
m is 0,1, 2, 3, or 4;
n is 0,1, 2, 3, or 4, and
p is 0, 1, 2, 3, or4.
As defined generally above, Rings A, B, and C are each, independently, a 3-8
membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10
membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated
clic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, a 5—6 membered monocyclic heteroaromatic ring having 1—4 heteroatoms
ndently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered bicyclic
heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
In some embodiments, Ring A is a 3—8 membered saturated or partially unsaturated
monocyclic carbocyclic ring. In some ments, Ring A is phenyl. In some embodiments,
Ring A is an 8-10 membered ic aromatic carbocyclic ring. In some embodiments, Ring A
is a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2
heteroatoms independently ed from en, oxygen, or sulfur. In some embodiments,
Ring A is a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some embodiments, Ring A is an 8-
membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from
nitrogen, , or sulfur.
] In some embodiments, Ring A is a 5-6 membered monocyclic heteroaromatic ring
having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
In some embodiments, Ring B is a 3—8 membered saturated or partially rated
monocyclic carbocyclic ring. In some embodiments, Ring B is phenyl. In some embodiments,
Ring B is an 8-10 membered bicyclic aromatic yclic ring. In some embodiments, Ring B
2017/040514
is a 4—8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2
atoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments,
Ring B is a 5-6 ed monocyclic heteroaromatic ring having 1—4 heteroatoms
independently selected from nitrogen, oxygen, or . In some embodiments, Ring B is an 8-
membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
In some embodiments, Ring B is a 5-6 membered monocyclic heteroaromatic ring
having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
In some ments, Ring B is absent.
In some embodiments, Ring C is a 3-8 membered saturated or partially unsaturated
monocyclic carbocyclic ring. In some ments, Ring C is phenyl. In some embodiments,
Ring C is an 8-10 membered bicyclic aromatic carbocyclic ring. In some embodiments, Ring C
is a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2
heteroatoms independently ed from nitrogen, oxygen, or sulfur. In some embodiments,
Ring C is a 5-6 membered clic aromatic ring having 1—4 heteroatoms
independently selected from nitrogen, oxygen, or sulfur. In some embodiments, Ring C is an 8-
membered bicyclic heteroaromatic ling having 1-5 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
] In some embodiments, Ring C is a 5-6 membered monocyclic heteroaromatic ring
having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
;In some embodiments, Rings A, B, and C are each, ndently, selected from:
NR )3) 511333350
phenym 3:3 :11]
:n m I? if? hm
In some embodiments, Rings A, B, and C are each, independently, ed from:
3 x
“130 5/5/ 3/ f/N
\| 1,293,191
7 7 7 7 7 7 7
3 N f
‘2le “all? 5 o 0—\o
/ / N / g‘
a \NI 1 \NI
In some embodiments, Ring A is selected from:
phenyl, 11:13
In some embodiments, Ring B is selected from:
phenyl, R13 1113
In some embodiments, Ring C selected from:
phenyl, 13:13
‘95
I I
\ \ N
In some embodiments, at least one of Ring A, B, or C is ‘1 N
or at
] As defined generally above, each R1 is independently R, halogen, -CN, —OR, -N(R)2, -
N02, -N3, -SR, or -L1-R6.
] In some ments, R1 is R. In some embodiments, R1 is halogen. In some
embodiments, R1 is -CN. In some embodiments, R1 is -OR. In some embodiments, R1 is -N(R)2.
In some embodiments, R1 is -N02. In some embodiments, R1 is -N3. In some embodiments, R1
is -SR. In some ments, R1 is -L1-R6.
In some embodiments, R1 is hydrogen. In some embodiments, R1 is an optionally
substituted C1.6 aliphatic group. In some embodiments, R1 is an optionally substituted 3-8
membered ted or partially unsaturated monocyclic carbocyclic ring. In some
embodiments, R1 is an ally substituted phenyl. In some embodiments, R1 is an optionally
substituted 8-10 membered bicyclic aromatic carbocyclic ring. In some embodiments, R1 is an
optionally substituted 4—8 membered saturated or partially unsaturated monocyclic cyclic
ring having 1-2 heteroatoms independently selected from en, oxygen, or . In some
embodiments, R1 is an optionally substituted 5—6 membered clic heteroaromatic ring
having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, R1 is an optionally substituted 8-10 membered bicyclic heteroaromatic ring having
1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
As defined generally above, each R2 is independently R, halogen, -CN, -OR, -N(R)2, -
N02, -N3, -SR, or —L2-R6.
In some embodiments, R2 is R. In some embodiments, R2 is halogen. In some
embodiments, R2 is -CN. In some embodiments, R2 is -OR. In some embodiments, R2 is —N(R)2.
In some embodiments, R2 is -N02. In some embodiments, R2 is -N3. In some embodiments, R2
is -SR. In some embodiments, R2 is —L2—R6.
In some embodiments, R2 is hydrogen. In some embodiments, R2 is an optionally
substituted C1.6 aliphatic group. In some ments, R2 is an optionally substituted 3-8
membered saturated or partially unsaturated clic carbocyclic ring. In some
embodiments, R2 is an optionally substituted . In some embodiments, R2 is an optionally
substituted 8-10 membered bicyclic aromatic carbocyclic ring. In some embodiments, R2 is an
2017/040514
ally substituted 4-8 membered saturated or partially unsaturated monocyclic heterocyclic
ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, R1 is an optionally substituted 5-6 membered monocyclic heteroaromatic ring
having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, R2 is an optionally substituted 8-10 membered bicyclic heteroaromatic ring having
1—5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
As defined generally above, each R3 is independently R, halogen, -CN, -OR, -N(R)2, -
N02, —N3, -SR, or -L3-R6.
In some embodiments, R3 is R. In some embodiments, R3 is halogen. In some
embodiments, R3 is -CN. In some embodiments, R3 is -OR. In some embodiments, R3 is -N(R)2.
In some embodiments, R3 is -N02. In some embodiments, R3 is -N3. In some embodiments, R3
is -SR. In some embodiments, R3 is —L3-R6.
In some embodiments, R3 is hydrogen. In some embodiments, R3 is an optionally
substituted C14, aliphatic group. In some embodiments, R3 is an optionally substituted 3-8
membered saturated or partially rated monocyclic carbocyclic ring. In some
embodiments, R3 is an optionally substituted phenyl. In some embodiments, R3 is an ally
substituted 8-10 membered bicyclic aromatic carbocyclic ring. In some embodiments, R3 is an
optionally substituted 4-8 membered saturated or partially rated monocyclic heterocyclic
ring having 1-2 atoms independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, R3 is an optionally tuted 5-6 membered clic heteroaromatic ring
having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, R3 is an ally substituted 8-10 membered bicyclic heteroaromatic ring having
1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
As defined generally above, each L1, L2, and L3 is ndently a nt bond or a
C1-s bivalent straight or branched hydrocarbon chain wherein l, 2, or 3 methylene units of the
chain are independently and optionally ed with -O—, -C(O)—, -C(O)O-, -, -N(R)-, -
C(O)N(R)-, -(R)NC(O)—, N(R)-, -(R)NC(O)O-, (O)N(R)-, -S-, -SO-, —SOz-, -
SOzN(R)—, -(R)NSOz-, —C(S)—, -C(S)O-, —OC(S)—, —C(S)N(R)—, -(R)NC(S)—, -(R)NC(S)N(R)-, or -
Cy-.
In some embodiments, L1 is a covalent bond. In some embodiments, L1 is a C1-s
bivalent straight or branched hydrocarbon chain. In some embodiments, L1 is a C1-8 bivalent
straight or ed hydrocarbon chain wherein l, 2, or 3 methylene units of the chain are
independently and ally replaced with -O-, —C(O)-, —C(O)O—, —OC(O)—, -N(R)—, -C(O)N(R)-,
-(R)NC(O)-, N(R)—, -(R)NC(O)O-, -N(R)C(O)N(R)-, -S-, -SO-, -SOz—, -SOzN(R)-, -
(R)NSOz-, -C(S)-, -C(S)O-, -, -C(S)N(R)-, -(R)NC(S)-, -(R)NC(S)N(R)-, or -Cy-.
In some embodiments, L1 is a C1-6 bivalent straight or branched hydrocarbon chain
wherein 1, 2, or 3 methylene units of the chain are independently and optionally replaced with -
O-, -C(O)-, -N(R)-, -S—, -SO-, -SOz-, —SOzN(R)-, -(R)NSOz-, —C(S)—, or -Cy-, and each R is
independently hydrogen, -CH2-phenyl, phenyl, C1-6 alkyl, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, -CH2F, -CHF2, -CF3, -CH2CHF2, or -CH2CF3; or each R is independently hydrogen
or methyl, or R is hydrogen.
In some embodiments, L2 is a covalent bond. In some embodiments, L2 is a C14;
bivalent straight or branched hydrocarbon chain. In some ments, L2 is a C14; nt
straight or branched hydrocarbon chain wherein 1, 2, or 3 methylene units of the chain are
independently and optionally replaced with -O-, -C(O)-, -C(O)O—, -OC(O)-, -N(R)-, -C(O)N(R)-,
—(R)NC(O)-, -OC(O)N(R)—, -(R)NC(O)O-, -N(R)C(O)N(R)-, -S—, -SO—, -SOz—, —SOzN(R)—, -
(R)NSOz-, -C(S)-, -, -OC(S)—, -C(S)N(R)-, (S)—, -(R)NC(S)N(R)-, or -Cy-.
In some ments, L2 is a C1.6 nt straight or branched hydrocarbon chain
wherein l, 2, or 3 methylene units of the chain are independently and optionally replaced with -
0-, —C(O)-, —N(R)-, -S—, -SO-, -SOz—, —SOzN(R)-, -(R)NSOz-, -C(S)—, or -Cy-, and each R is
ndently hydrogen, -CH2-phenyl, phenyl, C1-6 alkyl, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, -CH2F, -CHF2, -CF3, -CH2CHF2, or -CH2CF3; or each R is independently hydrogen
or methyl, or R is hydrogen.
In some embodiments, L3 is a covalent bond. In some embodiments, L3 is a C1-s
bivalent straight or branched hydrocarbon chain. In some ments, L3 is a C1-8 bivalent
straight or branched hydrocarbon chain wherein l, 2, or 3 methylene units of the chain are
independently and ally replaced with -O-, —C(O)-, —C(O)O—, —, , -C(O)N(R)-,
-(R)NC(O)-, -OC(O)N(R)—, -(R)NC(O)O-, -N(R)C(O)N(R)-, -S-, -SO-, -SOz—, -SOzN(R)-, -
(R)NSOz—, -C(S)—, -C(S)O—, -OC(S)—, -C(S)N(R)—, —(R)NC(S)—, -(R)NC(S)N(R)-, or -Cy-.
In some embodiments, L3 is a C14, bivalent straight or branched hydrocarbon chain
wherein l, 2, or 3 methylene units of the chain are independently and optionally replaced with -
0-, -C(O)-, -N(R)-, -S—, -SO-, -SOz-, -SOzN(R)-, -(R)NSOz-, , or -Cy-, and each R is
independently hydrogen, -CH2-phenyl, phenyl, C1-6 alkyl, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, -CH2F, -CHF2, -CF3, F2, or 3, or each R is independently hydrogen
or ; or R is hydrogen.
As defined generally above, each -Cy- is independently a bivalent optionally
substituted 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring,
optionally substituted phenylene, an optionally substituted 4-8 membered saturated or partially
unsaturated monocyclic cyclic ring having 1-3 heteroatoms ndently selected from
nitrogen, oxygen, or sulfur, an optionally substituted 5-6 membered monocyclic heteroaromatic
ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an
optionally substituted 8-10 membered bicyclic or bridged bicyclic saturated or partially
unsaturated heterocyclic ring having 1-5 heteroatoms independently selected from nitrogen,
oxygen, or sulfur, or an optionally substituted 8-10 membered bicyclic or bridged bicyclic
aromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or
sulfur.
In some embodiments, -Cy- is a nt optionally substituted 3-8 membered
saturated or partially rated monocyclic carbocyclic ring. In some embodiments, -Cy- is an
optionally substituted phenylene. In some embodiments, -Cy- is an optionally tuted 4-8
ed saturated or partially unsaturated monocyclic heterocyclic ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some embodiments, -
Cy- is an optionally tuted 5-6 membered monocyclic heteroaromatic ring having 1-4
heteroatoms independently selected from en, oxygen, or sulfur. In some embodiments, -
Cy- is an optionally substituted 8-10 membered bicyclic or bridged bicyclic saturated or partially
unsaturated cyclic ring having 1—5 heteroatoms independently selected from en,
oxygen, or sulfur. In some embodiments, -Cy- is an optionally substituted 8-10 membered
bicyclic or bridged bicyclic heteroaromatic ring having 1—5 heteroatoms ndently selected
from nitrogen, oxygen, or sulfur.
2017/040514
As defined generally above, each R6 is ndently hydrogen or Cm alkyl
optionally substituted with l, 2, 3, 4, 5, or 6 halogens.
As defined generally above, m is O, 1, 2, 3, or 4. In some embodiments, m is O. In
some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In
some embodiments, m is 4. In some embodiments, m is 0, l, 2, or 3. In some ments, m
is O, 1, or 2. In some embodiments, m is l, 2, or 3.
As defined generally above, 11 is O, 1, 2, 3, or 4. In some embodiments, n is O. In
some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some
embodiments, n is 4. In some embodiments, n is O, l, 2, or 3. In some embodiments, n is O, 1, or
2. In some embodiments, n is 1, 2, or 3.
As defined generally above, p is O, 1, 2, 3, or 4. In some embodiments, p is O. In
some embodiments, p is 1. In some ments, p is 2. In some embodiments, p is 3. In some
embodiments, p is 4. In some embodiments, p is O, l, 2, or 3. In some embodiments, p is 0, 1, or
2. In some embodiments, p is 1, 2, or 3.
In some ments, a compound of Formula I is covalently , either directly
or h a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In another aspect, the present invention provides a compound of Formula 11:
4% / / \
\ —\(R3)p
or a pharmaceutically acceptable salt thereof, wherein each of R, R1, R2, R3, R6, L1, L2, L3, -Cy-,
m, n, and p is as defined above and described in embodiments herein, both singly and in
combination.
In some embodiments, a compound of Formula II is covalently linked, either directly
or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention provides a compound of a III:
or a pharmaceutically acceptable salt thereof, wherein each of R, R1, R2, R3, R6, L1, L2, L3, -Cy-,
m, n, and p is as defined above and described in ments herein, both singly and in
combination.
In some embodiments, a compound of Formula 111 is covalently , either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present ion provides a compound of Formula IV:
or a pharmaceutically acceptable salt thereof, wherein each of R, R1, R2, R3, R6, L1, L2, L3, -Cy-,
m, n, and p is as defined above and described in embodiments herein, both singly and in
combination.
In some ments, a compound of Formula IV is covalently linked, either directly
or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
] In some embodiments, the present invention provides a compound of Formula V:
or a pharmaceutically acceptable salt thereof, wherein each of R, R1, R2, R3, R6, L1, L2, L3, -Cy-,
m, and p is as defined above and descfibed in embodiments herein, both singly and in
combination.
In some ments, a compound of Formula V is covalently linked, either directly
or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some ments, the present invention provides a compound of Formula VI:
or a pharmaceutically able salt thereof, wherein each of R, R1, R2, R3, R6, L1, L2, L3, and -
Cy- is as defined above and described in embodiments herein, both singly and in ation.
In some ments, a compound of Formula V1 is covalently linked, either directly
or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention provides a compound of Formula V:
or a pharmaceutically acceptable salt thereof, wherein each of R, R1, R3, R6, L1, L3, -Cy-, m, and
p is as defined above and described in embodiments herein, both singly and in combination, and
X is -C(R)2-, -NR-, or -O-.
In some embodiments, a compound of Formula VII is covalently linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In r aspect, the present invention provides a nd of Formula VIII:
VIII
or a pharmaceutically acceptable salt thereof, wherein each of X, R, R1, R2, R3, R6, L1, L2, and L3
is as defined above and described in embodiments herein, both singly and in combination; and R4
is independently -R, halogen, -CN, -OR, —N(R)2, —N02, -N3, -SR, or -L3-R6.
In some embodiments, R4 is R. In some embodiments, R4 is halogen. In some
embodiments, R4 is -CN. In some ments, R4 is -OR. In some embodiments, R4 is —N(R)2.
In some ments, R4 is -N02. In some embodiments, R4 is -N3. In some ments, R4
is -SR. In some embodiments, R4 is -L3-R6.
In some ments, R4 is hydrogen. In some ments, R4 is an optionally
substituted C1.6 aliphatic group. In some embodiments, R4 is an optionally substituted 3-8
membered saturated or partially rated monocyclic carbocyclic ring. In some
embodiments, R4 is an ally substituted phenyl. In some embodiments, R4 is an optionally
substituted 8-10 membered bicyclic aromatic carbocyclic ring. In some embodiments, R4 is an
optionally tuted 4-8 membered saturated or partially unsaturated monocyclic heterocyclic
ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In some
embodiments, R4 is an optionally substituted 5-6 membered monocyclic heteroaromatic ring
having 1-4 heteroatoms independently selected from nitrogen, , or sulfur. In some
embodiments, R4 is an optionally substituted 8-10 membered bicyclic heteroaromatic ring having
1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
In some embodiments, R4 is selected from R, halogen, -CN, -OR, -N(R)2, -SR, C1-6
aliphatic, or —L4-R6, n L4 is a C1-6 bivalent straight or branched hydrocarbon chain
wherein l, 2, or 3 methylene units of the chain are independently and optionally replaced with -
0-, -C(O)—, , -S—, -SO-, -SOz-, —C(S)-, or -Cy-; wherein the C1-6 tic group is
optionally substituted with l, 2, or 3 groups independently selected from halogen, -CN, -N(R)2, -
N02, -N3, =NR, =NOR, =0, :8, -OR, -SR, -SOzR, -S(O)R, -R, -Cy-R, -C(O)R, -C(O)OR, -
OC(O)R, -C(O)N(R)2, -(R)NC(O)R, -OC(O)N(R)2, -(R)NC(O)OR, -N(R)C(O)N(R)2, -
SOzN(R)2, -(R)NSOzR, -C(S)R, or -C(S)OR; and each R is independently hydrogen, -CH2-
, phenyl, C1-6 alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, -CH2F, -CHF2, -
CF3, F2, or -CH2CF3; or each R is independently hydrogen or methyl, or R is hydrogen.
In some embodiments, L4 is a covalent bond. In some embodiments, L4 is a C1-8
bivalent straight or branched hydrocarbon chain. In some ments, L4 is a C1.s bivalent
ht or branched hydrocarbon chain wherein l, 2, or 3 methylene units of the chain are
independently and optionally replaced with -O-, -C(O)-, -C(O)O—, -OC(O)-, -N(R)-, -C(O)N(R)-,
—(R)NC(O)-, -OC(O)N(R)—, -(R)NC(O)O-, -N(R)C(O)N(R)-, -S—, -SO—, -SOz—, -SOzN(R)—, -
z-, -C(S)-, -, -OC(S)—, -C(S)N(R)-, -(R)NC(S)—, -(R)NC(S)N(R)-, or -Cy-.
In some embodiments, L4 is a C1.6 bivalent straight or branched hydrocarbon chain
wherein l, 2, or 3 methylene units of the chain are independently and optionally replaced with -
0-, -C(O)-, -N(R)-, -S-, -SO-, -SOz-, -SOzN(R)-, -(R)NSOz-, -C(S)—, or -Cy-, and each R is
independently hydrogen, -CH2-phenyl, phenyl, C1-6 alkyl, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, -CH2F, -CHF2, -CF3, -IF2, or -CH2CF3; or each R is ndently en
or methyl, or R is hydrogen.
In some embodiments, a compound of Formula VIII is covalently linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In r aspect, the present invention provides a compound of Formula IX:
or a pharmaceutically acceptable salt thereof, wherein each of R, R1, R2, R3, R4, R6, L1, L2, L3, L4
and -Cy— is as defined above and described in embodiments herein, both singly and in
combination.
In some embodiments, a compound of Formula IX is covalently linked, either directly
or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some ments, the present invention provides a compound of Formula X:
or a pharmaceutically acceptable salt thereof, wherein each of R, R3, R6, L3, and -Cy- is as
defined above and described in embodiments herein, both singly and in combination.
In some ments, a compound of a X is covalently linked, either directly
or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In another aspect, the present invention provides a nd of Formula XI:
z/ 2
kaRs
or a ceutically acceptable salt thereof, wherein:
each of Y, R, R1, R2, R3, R4, R6, L1, L2, L3, L4 and -Cy- is as defined above and described in
embodiments herein, both singly and in combination, and
Z is -C(R)2- or —O—.
In some embodiments, a nd of Formula X1 is covalently linked, either directly
or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention es a nd of Formula XII:
WO 06074
or a pharmaceutically acceptable salt thereof, wherein:
each of R, R1, R2, R3, R4, R6, L1, L2, L3, L4 and -Cy- is as defined above and described in
embodiments herein, both singly and in combination.
In some embodiments, a compound of Formula XII is covalently linked, either
directly or through a linker such as L1, to at least one ure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention provides a compound of Formula XIII:
XIII
or a pharmaceutically acceptable salt thereof, n:
each of R, R1, R2, R3, R4, R6, L1, L2, L3, L4 and -Cy- is as defined above and described in
embodiments herein, both singly and in combination.
In some embodiments, a compound of a XIII is covalently linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In another , the present invention provides a compound of Formula XIV:
or a pharmaceutically acceptable salt thereof, wherein:
each of R, R1, R2, R3, R6, L1, L2, L3, and —Cy- is as defined above and described in embodiments
herein, both singly and in combination; and
W is -NR—, -O-, or -S-.
In some embodiments, a compound of Formula XIV is covalently linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In another aspect, the present invention provides a compound of Formula XV:
RINJK/NH'
or a pharmaceutically acceptable salt f, wherein:
each of Y, R, R1, R2, R3, R6, L1, L2, L3, and -Cy- is as defined above and described in
ments herein, both singly and in combination, except that one of R1 or R2 may be absent
and replaced by Rs.
In some ments, a compound of Formula XV is covalently linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the t invention provides a compound of Formula XVI:
RIN H
A I N/>_<:N_R3
or a pharmaceutically acceptable salt thereof, wherein:
each of R, R1, R2, R3, R6, L1, L2, L3, and —Cy- is as d above and bed in embodiments
herein, both singly and in combination.
In some embodiments, a compound of Formula XVI is covalently linked, either
directly or h a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention provides a nd of Formula XVII:
R30 0 N 02W)thI /> N
XVII
or a ceutically acceptable salt thereof, wherein:
each of R, R2, R3, R6, L2, L3, and -Cy- is as defined above and described in embodiments herein,
both singly and in combination.
In some embodiments, a compound of Formula XVII is covalently linked, either
directly or h a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention provides a compound of Formula XVIII:
RIN1 N
2\ I/
0 N
XVIII
or a pharmaceutically acceptable salt thereof, wherein:
each of R, R1, R2, R3, R6, L1, L2, L3, and -Cy- is as defined above and described in embodiments
herein, both singly and in ation.
In some embodiments, a compound of Formula XVIII is ntly linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention provides a compound of Formula XIX:
RIN |H>/
02W N
6'};
or a pharmaceutically acceptable salt thereof, wherein:
each of R, R1, R3, R6, L1, L3, and -Cy- is as defined above and described in embodiments ,
both singly and in combination.
] In some embodiments, a compound of Formula XIX is covalently , either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention provides a compound of Formula XX:
RIN H
A | N-R3
O N
R2 R
or a pharmaceutically acceptable salt thereof, wherein:
each of R, R1, R2, R3, R6, L1, L2, L3, and —Cy- is as defined above and described in ments
herein, both singly and in combination.
In some embodiments, a compound of Formula XX is covalently linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In another aspect, the present invention provides a compound of a XXI:
R2 R2
L5J\¥/RK5L5R1I
or a pharmaceutically acceptable salt thereof, wherein:
each R, R1, R2, R6, L1, L2, and -Cy- is as defined above and described in embodiments ,
both singly and in combination; L5 is CH2 or a single or a double bond; and
R5 is absent or is -O'.
In some ments, a compound of Formula XXI is covalently linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention provides a compound of Formula XXII:
XXII
or a pharmaceutically acceptable salt thereof, wherein:
each R, R1, R6, L1, and -Cy- is as defined above and described in embodiments herein, both
singly and in combination.
In some embodiments, a compound of Formula XXII is covalently linked, either
ly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the t invention provides a nd of a XXIII:
R6,Cy Cy\ Re
XXIII
or a pharmaceutically acceptable salt thereof, wherein:
each R, R6, and -Cy- is as defined above and described in embodiments herein, both singly and
in combination.
] In some embodiments, a compound of Formula XXIII is covalently linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention provides a compound of Formula XXIV:
N+/O»
/Cy Cy\
XXIV
or a ceutically acceptable salt thereof, wherein:
each R, R1, R6’ L1, and —Cy- is as defined above and described in embodiments herein, both
singly and in combination.
In some embodiments, a compound of Formula XXIV is covalently linked, either
directly or through a linker such as L1, to at least one ure shown in Figure 24 or 33 by any
chemically feasible means.
In r aspect, the present invention provides a compound of Formula XXV:
or a pharmaceutically acceptable salt thereof, wherein:
each R, R1, R6’ L1, and —Cy- is as defined above and bed in embodiments herein, both
singly and in combination.
In some ments, a compound of Formula XXV is covalently linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention provides a compound of Formulae
, XXVI-b, XXVI-c, XXVI-d, or XXVI-e:
o NH2 NH2 0
N:Ek N
</ N H NH
I </ I
. NANH2 N ,N *0 lN/ILO NA0
H1 ,
H1 ”S” '1 '11
N N N N N
F1 l?1 lRl [Q1 [Q1
XXVI-a XXVI-b XXVI-c XXVI-d XXVI-e
or a diastereomer, or pharrnaceutically acceptable salt thereof, wherein:
each R, R1, R6, L1, and -Cy- is as defined above and described in embodiments herein, both
singly and in combination.
] In some embodiments, a nd of Formulae XXVI-a, XXVI-b, XXVI-c, XXVI-
d, or XXVI-e is covalently linked, either directly or through a linker such as L1, to at least one
structure shown in Figmre 24 or 33 by any chemically feasible means.
In r aspect, the present invention provides a compound of Formula XXVII:
NC“)--<CR2
R10: OH
XXVII
or a pharmaceutically acceptable salt thereof, wherein:
each R, R1, R2, R6, L1, L2, and -Cy- is as defined above and described in embodiments herein,
both singly and in combination.
In some embodiments, a compound of Formula XXVII is ntly linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
ally feasible means.
In r aspect, the present invention provides a compound of Formula XXVIII:
OR10 OHOHO N(R2)2
XXVIII
or a pharmaceutically acceptable salt f, wherein:
each R, R1, R2, R3, R6’ L1, L2, L3 and -Cy- is as defined above and described in embodiments
herein, both singly and in combination.
In some embodiments, a compound of Formula XXVIII is covalently linked, either
directly or through a linker such as L1, to at least one ure shown in Figure 24 or 33 by any
chemically le means.
In another aspect, the present invention provides a compound of Formula XXIX:
N\ l
\ NR
R2 N\ /
N N
XXIX
or a pharmaceutically acceptable salt thereof, wherein:
each R, R1, R2, R3, R6, L1, L2, L3 and -Cy- is as defined above and described in embodiments
herein, both singly and in combination.
In some embodiments, a compound of a XXIX is covalently linked, either
directly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
ally feasible means.
In another aspect, the present invention provides a compound of a XXX:
R2 NJ2
‘\<:
J(R3)p
or a pharmaceutically acceptable salt thereof, wherein:
each R, R1, R2, R3, R6, L1, L2, L3 and -Cy- is as defined above and described in embodiments
herein, both singly and in combination; and
JisN,O orC, andpis 1, 2, or3.
In some embodiments, a compound of Formula XXX is covalently linked, either
ly or through a linker such as L1, to at least one structure shown in Figure 24 or 33 by any
chemically feasible means.
In some embodiments, the present invention provides a compound of Formula XXXI:
KM?»
XXXI
or a pharmaceutically acceptable salt thereof, wherein:
each X, R, R2, R3, R6’ L1, L2, L3 and -Cy— is as defined above and described in embodiments
, both singly and in combination.
In some embodiments, a compound of Formula XXXI is covalently linked, either
directly or h a linker such as L1, to at least one least one structure shown in Figure 24 or
33 by any chemically feasible means.
In some embodiments, the present invention provides a compound of a
XXXII:
XXXII
or a pharmaceutically acceptable salt thereof, n:
each R, R2, R3, R6’ L1, L2, L3 and -Cy- is as defined above and described in embodiments herein,
both singly and in combination.
In some embodiments, a compound of Formula XXXII is covalently linked, either
directly or through a linker such as L1, to at least one least one structure shown in Figure 24 or
33 by any chemically feasible means.
Furthermore, it has now been found that certain compounds comprising a quinoline
core, of which CPNQ is one, are capable of binding RNA. CPNQ has the following ure:
] Accordingly, in some embodiments, the small molecule ligand is ed from
CPNQ or a pharmaceutically acceptable salt thereof. In other ments, the ligand is
selected from a quinoline compound related to CPNQ, such as those provided in any one of
Tables 5 or 6, below, or in any one of Figures 63—71, or a pharmaceutically acceptable salt
thereof.
In some embodiments, CPNQ or a quinoline related to CPNQ is modified at one or
more ble positions to e a hydrogen with a tether (-Tl- and/or -T2-), click-ready group
(-RCG), or warhead (-R‘md), according to embodiments of each as described herein and in USSN
62/289,671, which is hereby incorporated by reference in its entirety. For example, CPNQ or a
quinoline related to CPNQ may have one of the following formulae:
O “/0 /
N/fi / N N
1J\/N CI
CI T T1
XXXIII XXXIV
or a pharmaceutically able salt thereof; n Rm”l is optionally substituted
with -RCG or -T2-RCG, and further optionally substituted with a pull—down group. The compound
of formulae IX or X may further be optionally substituted with one or more optional substituents,
as defined below, such as l or 2 optional substituents,
The term “aliphatic” or atic , as used herein, means a straight-chain (i.e.,
unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely
saturated or that contains one or more units of ration, or a monocyclic hydrocarbon or
bicyclic hydrocarbon that is completely saturated or that contains one or more units of
unsaturation, but which is not aromatic (also referred to herein as “carbocycle, 77 (L cycloaliphatic”
or alkyl”), that has a single point of attachment to the rest of the molecule. Unless
otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some ments,
aliphatic groups contain 1-5 aliphatic carbon atoms. In other embodiments, aliphatic groups
contain 1-4 aliphatic carbon atoms. In still other embodiments, tic groups contain 1-3
aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic
carbon atoms. In some embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refers
to a monocyclic C3-C6 hydrocarbon that is completely ted or that contains one or more
units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest
of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched,
substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as
(cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
As used herein, the term “bridged bicyclic” refers to any bicyclic ring system, i.e.
carbocyclic or heterocyclic, saturated or partially unsaturated, having at least one . As
defined by IUPAC, a “bridge” is an unbranched chain of atoms or an atom or a valence bond
connecting two bridgeheads, where a “bridgehead” is any skeletal atom of the ring system which
is bonded to three or more skeletal atoms (excluding hydrogen). In some ments, a
d bicyclic group has 7-12 ring members and 0-4 heteroatoms independently selected from
en, oxygen, or sulfur. Such bridged ic groups are well known in the art and include
those groups set forth below where each group is attached to the rest of the molecule at any
substitutable carbon or en atom. Unless otherwise specified, a bridged bicyclic group is
optionally substituted with one or more substituents as set forth for aliphatic groups.
Additionally or alternatively, any substitutable nitrogen of a bridged bicyclic group is optionally
substituted. ary bridged bicyclics include:
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The term “lower alkyl” refers to a C1-4 straight or branched alkyl group. ary
lower alkyl groups are , ethyl, propyl, isopropyl, butyl, isobutyl, and tert-butyl.
The term “lower haloalkyl” refers to a C1-4 straight or branched alkyl group that is
substituted with one or more halogen atoms.
The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus,
or silicon (including, any oxidized form of en, sulfur, phosphorus, or silicon; the
quatemized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for
example N (as in 3,4-dihydro-2H—pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N—substituted
pyrrolidinyl)).
The term “unsaturated”, as used herein, means that a moiety has one or more units of
unsaturation.
As used herein, the term ent C14; (or C1-6) saturated or unsaturated, straight or
branched, hydrocarbon chain”, refers to bivalent ne, alkenylene, and alkynylene chains that
are straight or branched as defined herein.
The term “alkylene” refers to a bivalent alkyl group. An “alkylene chain” is a
polymethylene group, i.e., —(CH2)n—, wherein n is a positive r, preferably from 1 to 6, from
1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylene
group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable
substituents include those described below for a substituted tic group.
The term “alkenylene” refers to a bivalent alkenyl group. A substituted alkenylene
chain is a polymethylene group containing at least one double bond in which one or more
hydrogen atoms are replaced with a tuent. Suitable substituents include those described
below for a substituted aliphatic group.
The term “halogen” means F, Cl, Br, or I.
The term “aryl” used alone or as part of a larger moiety as in “aralkyl,” “aralkoxy,” or
xyalkyl,” refers to monocyclic or bicyclic ring systems having a total of five to fourteen
ring members, wherein at least one ring in the system is aromatic and wherein each ring in the
system contains 3 to 7 ring members. The term “aryl” may be used interchangeably with the
term “aryl ring.” In certain embodiments of the present invention, “aryl” refers to an aromatic
ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like,
which may bear one or more substituents. Also included within the scope of the term “aryl,” as
it is used herein, is a group in which an aromatic ring is fused to one or more omatic rings,
such as indanyl, phthalimidyl, imidyl, phenanthridinyl, or tetrahydronaphthyl, and the
like.
The terms oaryl” and “heteroar—,” used alone or as part of a larger moiety, e. g.,
“heteroaralkyl,” or “heteroaralkoxy,” refer to groups having 5 to 10 ring atoms, preferably 5, 6,
or 9 ring atoms; having 6, 10, or 14 7E ons shared in a cyclic array; and having, in addition
to carbon atoms, from one to five atoms. The term “heteroatom” refers to nitrogen,
oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quatemized
form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl,
yl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl,
isothiazolyl, thiadiazolyl, pyridyl, zinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl,
naphthyridinyl, and pteridinyl. The terms oaryl” and “heteroar—” as used herein, also
include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or
heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring.
Nonlimiting es include l, isoindolyl, hienyl, benzofuranyl, dibenzofuranyl,
indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, nolyl, cinnolinyl, phthalazinyl,
quinazolinyl, quinoxalinyl, 4H—quinolizinyl, olyl, acridinyl, phenazinyl, phenothiazinyl,
phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]—l,4-oxazin-
3(4110-one. A heteroaryl group may be mono- or bicyclic. The term “heteroaryl” may be used
interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of
which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an
alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently
are optionally substituted.
As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and
“heterocyclic ring” are used interchangeably and refer to a stable 5— to 7—membered monocyclic
or 7—10—membered bicyclic heterocyclic moiety that is either saturated or partially rated,
and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as
defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen"
includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having
0—3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4—
dihydro—2Hipyrrolyl), NH (as in pyrrolidinyl), or +NR (as in Nisub stituted pyrrolidinyl).
A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon
atom that results in a stable structure and any of the ring atoms can be optionally substituted.
Examples of such saturated or lly unsaturated heterocyclic radicals include, t
limitation, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, dinyl, pyrrolinyl,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, droquinolinyl, oxazolidinyl, piperazinyl,
dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and lidinyl. The
terms “heterocycle,” ocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic
moiety,” and “heterocyclic radical,” are used interchangeably herein, and also include groups in
which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or liphatic rings, such as
indolinyl, 3Hiindolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl
group may be mono— or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group
substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are
optionally substituted.
] As used herein, the term “partially unsaturated” refers to a ring moiety that includes
at least one double or triple bond. The term “partially unsaturated” is ed to encompass
rings having multiple sites of ration, but is not intended to include aryl or heteroaryl
moieties, as herein defined.
As described herein, compounds of the invention may contain “optionally
substituted” moieties. In general, the term “substituted,” whether preceded by the term
“optionally” or not, means that one or more hydrogens of the designated moiety are replaced
with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may
have a suitable substituent at each tutable position of the group, and when more than one
position in any given structure may be substituted with more than one substituent selected from a
specified group, the substituent may be either the same or different at every position.
Combinations of substituents envisioned by this invention are preferably those that result in the
formation of stable or chemically feasible compounds. The term e,” as used , refers
to compounds that are not substantially altered when ted to conditions to allow for their
production, detection, and, in certain embodiments, their recovery, purification, and use for one
or more of the es disclosed herein.
Suitable monovalent substituents on a substitutable carbon atom of an “optionally
substituted” group are independently halogen, —(CH2)0_4R°, —(CH2)0_4OR°, -O(CH2)0-4R°, —O—
(CH2)0_4C(O)OR°; —(CH2)0_4CH(OR°)2; —(CH2)04SR°; —(CH2)0_4Ph, which may be substituted
with R°; —(CH2)0_4O(CH2)0_1Ph which may be substituted with R0, —CH=CHPh, which may be
tuted with R0, 0_4O(CH2)0_1-pyridyl which may be substituted with R0; —N02; —CN;
—N3, -(CH2)o—4N(R°)2; —(CH2)0_4N(R°)C(O)R°; —N(R°)C(S)R°; —(CH2)o—
4N(R°)C(O)NR02; -N(R°)C(S)NR°2; —(CH2)o—4N(R°)C(O)OR°, —
N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; —N(R°)N(R°)C(O)OR°; —(CH2)0—4C(O)R°; —
; —(CH2)0_4C(O)OR°, —(CH2)0_4C(O)SR°, -(CH2)0_4C(O)OSiR°3; 0—4OC(O)R°, —
OC(O)(CH2)0_4SR—, SC(S)SR°; —(CH2)o_4SC(O)R°; 04C(O)NR°2; —C(S)NR°2; —
C(S)SR°, —SC(S)SR°, -(CH2)0_4OC(O)NR°2; (OR°)R°; —C(O)C(O)R°, —
C(O)CH2C(O)R°; —C(NOR°)R°; -(CH2)0_4SSR°; —(CH2)04S(O)2R°, —(CH2)(HS(O)2OR°, —
—4OS(O)2R°, —S(O)2NR°2; -(CH2)o—4S(O)R°; —N(R°)S(O)2NR°2; —N(R°)S(O)2R°, —
N(OR°)R°; —C(NH)NR°2; —P(O)2R°; -P(O)R°2; -OP(O)R°2; —OP(O)(OR°)2; SiR°3; —(C1—4
straight or branched a1ky1ene)O—N(R°)2; or —(C14 ht or branched alkylene)C(O)O—N(R°)2,
wherein each R0 may be substituted as defined below and is independently hydrogen, C1-
6 tic, , —O(CH2)0_1Ph, -CH2-(5-6 membered heteroaryl ring), or a 5—6—membered
saturated, partially unsaturated, or aryl ring having 0—4 heteroatoms independently selected from
nitrogen, , or sulfur, or, notwithstanding the definition above, two independent
occurrences of R0, taken together with their intervening atom(s), form a 3—12—membered
saturated, lly unsaturated, or aryl mono— or bicyclic ring having 0—4 atoms
independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined
below.
Suitable lent substituents on R0 (or the ring formed by taking two
independent occurrences of RO together with their intervening atoms), are independently
halogen, —(CH2)0_2R°, —(haloR°), —(CH2)0_2OH, 0_2OR', —(CH2)0_
2CH(OR')2; —O(haloR'), —CN, —N3, —(CH2)0_2C(O)R', —(CH2)0_2C(O)OH, —(CH2)o—2C(O)OR', —
(CH2)0_2SR°, —(CH2)0_2SH, 0_2NH2, —(CH2)0_2NHR°, —(CH2)0_2NR°2, —N02, , —
OSiR'3, -C(O)SR', —(C1_4 straight or branched ne)C(O)OR°, or —SSR° n each R' is
unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is
independently selected from C1_4 aliphatic, —CH2Ph, —O(CH2)0_1Ph, or a 5—6—membered
saturated, partially unsaturated, or aryl ring having 0—4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R°
include =0 and =S.
Suitable divalent substituents on a saturated carbon atom of an “optionally
substituted” group include the following: =0, =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*,
=NNHS(O)2R*, =NR*, =NOR*, —O(C(R*2))2_3O—, or —S(C(R*2))2_3S—, wherein each independent
occurrence of R* is selected from hydrogen, C1_6 aliphatic which may be tuted as defined
below, or an unsubstituted 5—6—membered saturated, partially unsaturated, or aryl ring having 0—
4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent
substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group
include: —O(CR*2)2_3O—, wherein each independent occurrence of R* is selected from hydrogen,
C1—6 aliphatic which may be substituted as defined below, or an unsubstituted 5—6—membered
saturated, lly unsaturated, or aryl ring having 0—4 heteroatoms independently selected from
nitrogen, oxygen, or sulfur.
Suitable substituents on the aliphatic group of R* e halogen, —
R', -(haloR°), -OH, —OR°, —O(haloR°), —CN, —C(O)OH, —C(O)OR°, —NH2, —NHR°, —NR°2, or
—N02, wherein each R' is unsubstituted or where preceded by “halo” is substituted only with one
or more halogens, and is independently C1_4 aliphatic, —CH2Ph, —O(CH2)0_1Ph, or a 5—6—
membered saturated, partially unsaturated, or aryl ring having 0—4 heteroatoms independently
selected from nitrogen, oxygen, or sulfur.
] le substituents on a substitutable nitrogen of an “optionally substituted” group
include —Rl, —NRl2, —C(O)RT, —C(O)ORl, —C(O)C(O)Rl, —
C(O)CH2C(O)RT, -S(O)2Rl, -S(O)2NRT2, —C(S)NRl2, —C(NH)NRT2, or —N(Rl)S(O)2Rl, wherein
each R;r is independently hydrogen, C14 tic which may be substituted as defined below,
unsubstituted —OPh, or an unsubstituted 5—6—membered saturated, partially unsaturated, or aryl
ring having 0—4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or,
notwithstanding the definition above, two independent occurrences of RT, taken together with
their intervening atom(s) form an unsubstituted 3—12—membered saturated, lly unsaturated,
or aryl mono— or bicyclic ring having 0—4 heteroatoms independently selected from nitrogen,
oxygen, or .
Suitable substituents on the aliphatic group of RT are ndently halogen, —
R', -(haloR'), —OH, —OR', oR'), —CN, —C(O)OH, —C(O)OR', —NH2, —NHR', —NR'2,
or -N02, wherein each R' is tituted or where preceded by “halo” is substituted only with
one or more halogens, and is independently C1_4 aliphatic, —CH2Ph, —O(CH2)0_1Ph, or a 5—6—
membered saturated, partially unsaturated, or aryl ring having 0—4 heteroatoms independently
selected from nitrogen, , or sulfur.
As used herein, the term “pharmaceutically able salt” refers to those salts
which are, within the scope of sound medical judgment, le for use in t with the
tissues of humans and lower animals without undue toxicity, irritation, allergic response and the
like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable
salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically
acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1—19, incorporated herein by
reference. Pharmaceutically acceptable salts of the compounds of this invention e those
derived from suitable nic and organic acids and bases. Examples of pharmaceutically
acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid
or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,
succinic acid or malonic acid or by using other methods used in the art such as ion ge.
Other pharmaceutically acceptable salts include adipate, te, ascorbate, aspartate,
benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, onate, dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,
2—hydroxy—ethanesulfonate, ionate, e, laurate, lauryl sulfate, malate, maleate,
malonate, methanesulfonate, 2—naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate,
palmitate, pamoate, pectinate, persulfate, 3—phenylpropionate, phosphate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate, p—toluenesulfonate, undecanoate, valerate salts,
and the like.
Salts derived from appropriate bases include alkali metal, alkaline earth metal,
ammonium and N+(C14alkyl)4 salts. Representative alkali or alkaline earth metal salts include
, m, ium, calcium, ium, and the like. Further pharmaceutically
acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and
amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, loweralkyl sulfonate and aryl ate.
Unless ise , structures depicted herein are also meant to include all
isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the
structure; for example, the R and S configurations for each asymmetric center, Z and E double
bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as
well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present
nds are within the scope of the invention. Unless otherwise stated, all tautomeric forms
of the nds of the invention are within the scope of the invention. Additionally, unless
otherwise stated, structures depicted herein are also meant to include compounds that differ only
in the presence of one or more isotopically enriched atoms. For example, compounds having the
present structures ing the replacement of hydrogen by deuteiium or tritium, or the
replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention.
Such compounds are useful, for example, as ical tools, as probes in biological assays, or as
therapeutic agents in accordance with the present invention. In certain embodiments, a d
moiety, R1, of a provided compound comprises one or more deuterium atoms.
As used herein, the term “inhibitor” is defined as a compound that binds to and/or
modulates or inhibits a target RNA with measurable affinity. In certain embodiments, an
inhibitor has an ICso and/or binding constant of less than about 100 nM, less than about 50 nM,
less than about 1 uM, less than about 500 nM, less than about 100 nM, less than about 10 nM, or
less than about 1 nM.
The terms “measurable y” and “measurably inhibit,” as used herein, mean a
measurable change in a downstream biological effect between a sample comprising a compound
of the present invention, or ition f, and a target RNA, and an lent sample
comprising the target RNA, in the absence of said compound, or ition thereof.
The term “RNA” (ribonucleic acid) as used herein, means lly-occurring or
synthetic oligoribonucleotides ndent of source (e.g., the RNA may be produced by a
human, animal, plant, virus, or ium, or may be synthetic in ), biological context (e.g.,
the RNA may be in the nucleus, circulating in the blood, in vitro, cell lysate, or isolated or pure
form), or physical form (e.g., the RNA may be in single-, double-, or triple-stranded form
(including RNA-DNA hybrids), may include epigenetic modifications, native post-
transcriptional modifications, artificial modifications (e.g., obtained by chemical or in vitro
modification), or other modifications, may be bound to, e.g., metal ions, small molecules,
proteins such as chaperones, or co-factors, or may be in a denatured, partially denatured, or
folded state including any native or unnatural secondary or tertiary structure such as
quadruplexes, hairpins, triplexes, three way junctions (3WJs), four way junctions (4WJs),
parallel-Y junctions, ns, bulge loops, pseudoknots, and internal loops, etc., and any
transient forms or structures adopted by the RNA). In some embodiments, the RNA is 20, 22,
50, 75, or 100 or more nucleotides in length. In some embodiments, the RNA is 250 or more
nucleotides in length. In some embodiments, the RNA is 350, 450, 500, 600, 750, or 1,000,
WO 06074
2,000, 3,000, 4,000, 5,000, 7,500, , 15,000, 25,000, 50,000, or more nucleotides in length.
In some embodiments, the RNA is between 250 and 1,000 nucleotides in length. In some
ments, the RNA is a pre-RNA, pre-miRNA, or pretranscript. In some embodiments, the
RNA is a non—coding RNA (ncRNA), messenger RNA (mRNA), micro-RNA (miRNA), a
ribozyme, riboswitch, lncRNA, lincRNA, snoRNA, snRNA, scaRNA, piRNA, ceRNA, pseudo-
gene, viral RNA, fungal RNA, parasitic RNA, or bacterial RNA. The term “target nucleic acid”
or “target RNA,” as used herein, means any type of nucleic acid or RNA, respectively, having a
secondary or ry structure capable of binding a small molecule compound described herein.
In some embodiments, the structure is a 3WJ that is bound to or stabilized by a disclosed
compound or a 3WJ that is stabilized by the binding of the compound at another site on the target
nucleic acid (e.g. RNA). All 3WJ structures are encompassed by the present invention without
nce to their conformation or attendant tertiary interactions. For e, the 3WJ may be a
cis 3WJ, a trans 3WJ, a parallel—Y junction, or r form of 3WJ. The target RNA may be
inside a cell, in a cell , or in isolated form prior to contacting the compound.
The term “effector” or “effector nucleic acid,” as used , means a nucleic acid
that binds to a target nucleic acid and regulates or modulates its activity. Exemplary effectors
include small RNAs acting in trans to induce 3WJ formation. In some ments, ors
are selected from various natural forms of small RNA such as miRNA, Piwi-interacting RNA
(piRNA), and small nucleolar RNA (snoRNA). These small RNAs can base pair with a target
such as mRNA in a manner that results in the formation of a 3WJ. The base pairing between the
effector and target is often incomplete, meaning that the two sequences have some, but not
complete, complementarity. Perfect complementarity would result in the formation of a fully
double-stranded structure that lacks a 3WJ. In order to form a 3WJ, the effector (e.g., a miRNA)
and target (e.g., an mRNA) would lly form a stretch of base pairing of at least 4
nucleotides (nt) followed by a base-pairing stem of at least 4 nt and a loop of ed nt,
ed by a second stretch of base pairing between the effector and target sequences. The
stem-loop can be formed either in the effector or the target RNA.
The term “cis 3WJ” or “cis three-way junction,” as used herein, refers to a 3WJ
formed between portions of a single nucleic acid, such as a single strand of mRNA, precursor, or
protein—bound complex thereof.
The term “trans 3WJ” or “trans three-way junction,” as used herein, refers to a 3WJ
formed between two or more nucleic acids, such as an miRNA/mRNA complex sharing partial
sequence complementarity.
4. General s ofProviding the Present nds
The compounds of this invention may be prepared or isolated in general by synthetic
and/or semi-synthetic methods known to those skilled in the art for analogous compounds and by
methods described in detail in the Examples and Figures, herein. For example, various
compounds of the present invention may be synthesized by reference to Figures 5-31 or 77-94 or
96 of US. Provisional Patent Application USSN 62/289,671, which is hereby incorporated by
reference in its entirety.
In the schemes and chemical reactions depicted in the detailed description, Examples,
and Figures, where a particular protecting group (“PG”), leaving group , or
transformation ion is depicted, one of ordinary skill in the art will appreciate that other
protecting groups, leaving groups, and transformation conditions are also suitable and are
contemplated. Such groups and transformations are described in detail in March’s Advanced
Organic Chemistry: Reactions, isms, and Structure, M. B. Smith and J . March, 5th
Edition, John Wiley & Sons, 2001, Comprehensive Organic Transformations, R. C. Larock, 2““1
Edition, John Wiley & Sons, 1999, and Protecting Groups in Organic Synthesis, T. W. Greene
and P. G. M. Wuts, 3r01 edition, John Wiley & Sons, 1999, the entirety of each of which is hereby
incorporated herein by nce.
] As used herein, the phrase “leaving group” (LG) includes, but is not limited to,
halogens (e. g. fluoride, chloride, bromide, iodide), sulfonates (eg. mesylate, tosylate,
esulfonate, brosylate, nosylate, e), ium, and the like.
] As used herein, the phrase “oxygen protecting group” includes, for example, carbonyl
protecting groups, hydroxyl protecting groups, etc. Hydroxyl protecting groups are well known
in the art and include those described in detail in Protecting Groups in Organic Synthesis, T. W.
Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, the entirety of which is
incorporated herein by reference. Examples of suitable hydroxyl protecting groups e, but
are not limited to, , allyl , ethers, silyl ethers, alkyl ethers, arylalkyl ethers, and
alkoxyalkyl ethers. es of such esters include formates, acetates, carbonates, and
sulfonates. Specific examples include forrnate, benzoyl forrnate, chloroacetate, trifluoroacetate,
methoxyacetate, triphenylmethoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate, 4-
oxopentanoate, 4,4-(ethylenedithio)pentanoate, pivaloate thylacetyl), crotonate, 4-
methoxy-crotonate, benzoate, p-benzylbenzoate, 2,4,6-t1imethylbenzoate, carbonates such as
methyl, 9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(t1imethylsilyl)ethyl, 2-
(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl. Examples of such silyl ethers e
trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, propylsilyl, and other
trialkylsilyl ethers. Alkyl ethers include methyl, benzyl, p-methoxybenzyl, 3,4-
dimethoxybenzyl, trityl, t-butyl, allyl, and allyloxycarbonyl ethers or derivatives. alkyl
ethers include acetals such as methoxymethyl, methylthiomethyl, (2-methoxyethoxy)methyl,
benzyloxymethyl, beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyranyl ethers. Examples of
kyl ethers include benzyl, p-methoxybenzyl (MPM), 3,4-dimethoxybenzyl, O-nitrobenzyl,
p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, and 2- and 4-picolyl.
Amino protecting groups are well known in the art and include those described in
detail in Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3r01 edition,
John Wiley & Sons, 1999, the entirety of which is incorporated herein by reference. Suitable
amino protecting groups include, but are not d to, aralkylamines, ates, cyclic
imides, allyl amines, amides, and the like. Examples of such groups e t-butyloxycarbonyl
(BOC), ethyloxycarbonyl, methyloxycarbonyl, trichloroethyloxycarbonyl, allyloxycarbonyl
(Alloc), benzyloxocarbonyl (CBZ), allyl, phthalimide, benzyl (Bn), fluorenylmethylcarbonyl
(Fmoc), formyl, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, acetyl,
roacetyl, benzoyl, and the like.
] One of skill in the art will appreciate that various functional groups present in
compounds of the ion such as aliphatic groups, alcohols, carboxylic acids, esters, amides,
aldehydes, halogens and nitriles can be interconverted by ques well known in the art
including, but not limited to ion, oxidation, esterification, hydrolysis, partial oxidation,
partial reduction, halogenation, dehydration, partial hydration, and hydration. “March’s
Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, MB. and March, J John Wiley &
., Sons,
New York: 2001, the entirety of which is incorporated herein by reference. Such
onversions may require one or more of the aforementioned techniques, and certain methods
for synthesizing compounds of the invention are described below in the Exempliflcation and
. Uses, Formulation inistration
Pharmaceutically acceptable compositions
] According to another embodiment, the invention provides a composition comprising
a compound of this invention or a pharmaceutically acceptable derivative thereof and a
pharrnaceutically acceptable carrier, adjuvant, or vehicle. The amount of compound in
compositions of this invention is such that is effective to measurably inhibit or te a target
RNA, or a mutant thereof, in a biological sample or in a patient. In certain embodiments, the
amount of compound in compositions of this invention is such that is effective to measurably
inhibit or modulate a target RNA, in a biological sample or in a t. In certain embodiments,
a composition of this invention is formulated for administration to a patient in need of such
ition. In some ments, a composition of this invention is formulated for oral
administration to a patient.
The term “patient,” as used herein, means an animal, ably a mammal, and most
preferably a human.
The term “pharmaceutically acceptable carrier, nt, or vehicle” refers to a non-
toxic carrier, adjuvant, or vehicle that does not destroy the pharmacological activity of the
compound with which it is formulated. Pharmaceutically acceptable rs, adjuvants or
vehicles that may be used in the compositions of this invention include, but are not limited to,
ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, um hydrogen phosphate, potassium hydrogen phosphate, sodium
chloride, zinc salts, colloidal , magnesium trisilicate, polyvinyl pyrrolidone, ose-based
substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene—block polymers, polyethylene glycol and wool fat.
A “pharmaceutically acceptable derivative” means any xic salt, ester, salt of an
ester or other derivative of a compound of this invention that, upon administration to a recipient,
is e of ing, either directly or ctly, a compound of this invention or an
inhibitorily active metabolite or residue thereof.
Compositions of the present invention may be administered orally, parenterally, by
inhalation spray, topically, rectally, nasally, buccally, lly or via an implanted reservoir.
The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-
articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial
ion or infusion techniques. Preferably, the compositions are administered orally,
intraperitoneally or intravenously. Sterile injectable forms of the compositions of this invention
may be s or oleaginous suspension. These suspensions may be formulated according to
techniques known in the art using suitable dispersing or wetting agents and suspending agents.
The sterile injectable preparation may also be a sterile injectable on or suspension in a non-
toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution
and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally
employed as a solvent or suspending medium.
For this purpose, any bland fixed oil may be employed including synthetic mono- or
di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the
ation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or
castor oil, especially in their polyoxyethylated versions. These oil ons or suspensions may
also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or
similar dispersing agents that are commonly used in the formulation of pharrnaceutically
acceptable dosage forms including emulsions and sions. Other commonly used
surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers
which are commonly used in the manufacture of pharrnaceutically acceptable solid, liquid, or
other dosage forms may also be used for the es of formulation.
Pharmaceutically acceptable itions of this invention may be orally
administered in any orally acceptable dosage form including, but not limited to, capsules, tablets,
s suspensions or solutions. In the case of tablets for oral use, carriers commonly used
include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also
typically added. For oral administration in a capsule form, useful ts include lactose and
dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient is
combined with emulsifying and suspending agents. If desired, certain sweetening, ng or
coloring agents may also be added.
Alternatively, pharmaceutically acceptable compositions of this invention may be
administered in the form of suppositories for rectal administration. These can be prepared by
mixing the agent with a le non-irritating excipient that is solid at room temperature but
liquid at rectal temperature and ore will melt in the rectum to release the drug. Such
materials include cocoa butter, x and polyethylene glycols.
Pharmaceutically acceptable compositions of this invention may also be stered
topically, especially when the target of treatment includes areas or organs readily accessible by
topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable
topical formulations are readily prepared for each of these areas or organs.
Topical application for the lower intestinal tract can be effected in a rectal
suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal
patches may also be used.
For l applications, ed pharmaceutically acceptable itions may be
formulated in a le ointment containing the active component suspended or dissolved in one
or more rs. Carriers for topical administration of compounds of this invention include, but
are not limited to, mineral oil, liquid petrolatum, white petrolatum, ene glycol,
polyoxyethylene, ypropylene compound, emulsifying wax and water. Alternatively,
provided pharmaceutically acceptable itions can be formulated in a suitable lotion or
cream containing the active components suspended or dissolved in one or more pharmaceutically
acceptable carriers. Suitable carriers include, but are not d to, mineral oil, sorbitan
monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl
alcohol and water.
For ophthalmic use, provided pharmaceutically acceptable compositions may be
formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as
solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as
benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutically acceptable
compositions may be formulated in an nt such as petrolatum.
Pharmaceutically acceptable compositions of this invention may also be administered
by nasal aerosol or inhalation. Such itions are prepared according to techniques wellknown
in the art of pharmaceutical formulation and may be prepared as solutions in ,
employing benzyl alcohol or other suitable vatives, absorption promoters to enhance
bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
Most preferably, pharmaceutically acceptable compositions of this invention are
formulated for oral stration. Such formulations may be stered with or without
food. In some ments, ceutically acceptable compositions of this invention are
administered without food. In other embodiments, pharmaceutically acceptable compositions of
this invention are administered with food.
The amount of compounds of the present invention that may be combined with the
carrier materials to produce a composition in a single dosage form will vary depending upon the
host treated, the particular mode of administration. ably, provided compositions should be
formulated so that a dosage of between 0.01 - 100 mg/kg body weight/day of the inhibitor can be
administered to a patient receiving these compositions.
It should also be understood that a specific dosage and treatment regimen for any
particular patient will depend upon a variety of factors, ing the ty of the specific
compound employed, the age, body weight, general health, seX, diet, time of administration, rate
of excretion, drug combination, and the judgment of the treating physician and the severity of the
particular disease being treated. The amount of a compound of the present invention in the
composition will also depend upon the ular compound in the composition.
Uses of Compounds and Pharmaceutically Acceptable Compositions
Compounds and compositions bed herein are generally useful for the
modulation of a target RNA to retreat an RNA-mediated e or condition.
The activity of a compound utilized in this invention to modulate a target RNA may
be assayed in vilro, in vivo or in a cell line. In vitro assays include assays that ine
modulation of the target RNA. Alternate in vitro assays quantitate the ability of the compound to
bind to the target RNA. Detailed conditions for assaying a compound utilized in this invention
to modulate a target RNA are set forth in the Examples below.
As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing,
alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or
more symptoms thereof, as described herein. In some embodiments, treatment may be
administered after one or more symptoms have developed. In other embodiments, treatment may
be administered in the absence of ms. For example, treatment may be administered to a
susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms
and/or in light of c or other susceptibility factors). Treatment may also be continued after
symptoms have resolved, for example to prevent or delay their recurrence.
] Provided compounds are tors of a target RNA and are therefore useful for
treating one or more disorders associated with or affected by (e.g., downstream of) the target
RNA. Thus, in n embodiments, the present ion provides a method for treating an
diated disorder comprising the step of administering to a patient in need thereof a
compound of the present invention, or pharmaceutically acceptable composition thereof.
As used herein, the terms “RNA-mediated” disorders, diseases, and/or conditions as
used herein means any disease or other deleterious condition in which RNA, such as an
overexpressed, underexpressed, , misfolded, pathogenic, or ongogenic RNA, is known to
play a role. ingly, another embodiment of the present invention s to treating or
lessening the severity of one or more diseases in which RNA, such as an overexpressed,
underexpressed, mutant, misfolded, pathogenic, or ongogenic RNA, is known to play a role.
In some embodiments, the t invention provides a method for treating one or
more disorders, diseases, and/or conditions wherein the er, disease, or condition includes,
but is not limited to, a cellular proliferative disorder.
Cellular Proliferatl've Disorders
] The present invention features methods and compositions for the diagnosis and
prognosis of cellular proliferative disorders (e.g., cancer) and the treatment of these disorders by
ting a target RNA. Cellular proliferative disorders described herein e, e.g., cancer,
obesity, and proliferation-dependent diseases. Such ers may be diagnosed using methods
known in the art.
2017/040514
Cancer
] Cancer includes, in one embodiment, without limitation, leukemias (e,g., acute
ia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia,
acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia,
acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, c lymphocytic
leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease),
Waldenstrom's macroglobulinemia, le myeloma, heavy chain disease, and solid tumors
such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, elioma, Ewing’s tumor,
leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian
cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat
gland carcinoma, ous gland carcinoma, papillary carcinoma, ary arcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's
tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung
carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
endroglioma, schwannoma, meningioma, melanoma, lastoma, and retinoblastoma).
In some embodiments, the cancer is melanoma or breast .
] Cancers includes, in another embodiment, without limitation, mesothelioma,
hepatobilliary (hepatic and billiary duct), bone cancer, pancreatic cancer, skin cancer, cancer of
the head or neck, cutaneous or intraocular melanoma, ovarian cancer, colon cancer, rectal cancer,
cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal, and duodenal),
uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of
the cervix, carcinoma of the , carcinoma of the vulva, Hodgkin's e, cancer of the
esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the d
gland, cancer of the yroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer
of the urethra, cancer of the penis, prostate cancer, testicular cancer, chronic or acute leukemia,
chronic myeloid leukemia, cytic lymphomas, cancer of the bladder, cancer of the kidney
or ureter, renal cell carcinoma, carcinoma of the renal pelvis, non hodgkins's ma, spinal
axis tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer,
multiple myeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a
combination of one or more of the foregoing cancers.
In some embodiments, the t invention es a method for treating a tumor in
a patient in need thereof, sing administering to the patient any of the compounds, salts or
pharmaceutical compositions described herein. In some ments, the tumor comprises any
of the s described herein. In some embodiments, the tumor comprises melanoma cancer. In
some embodiments, the tumor comprises breast cancer. In some embodiments, the tumor
comprises lung cancer. In some embodiments the the tumor comprises small cell lung cancer
(SCLC). In some embodiments the the tumor comprises non-small cell lung cancer (NSCLC).
In some embodiments, the tumor is treated by arresting further growth of the tumor.
In some embodiments, the tumor is treated by reducing the size (e.g., volume or mass) of the
tumor by at least 5%, 10%, 25%, 50 %, 75%, 90% or 99% relative to the size of the tumor prior
to treatment. In some embodiments, tumors are treated by reducing the quantity of the tumors in
the patient by at least 5%, 10%, 25%, 50 %, 75%, 90% or 99% ve to the quantity of tumors
prior to treatment.
Other Proliferative Diseases
Other proliferative diseases include, e. g., obesity, benign prostatic hyperplasia,
psoriasis, abnormal nization, lymphoproliferative disorders (e.g., a disorder in which there
is abnormal proliferation of cells of the lymphatic system), c rheumatoid arthritis,
arteriosclerosis, restenosis, and diabetic retinopathy. Proliferative diseases that are hereby
incorporated by reference include those described in US. Pat. Nos. 5,639,600 and 7,087,648.
Inflammatory Disorders andDiseases
Compounds of the invention are also useful in the treatment of inflammatory or
allergic conditions of the skin, for example psoriasis, contact dermatitis, atopic itis,
alopecia areata, erythema multiforma, itis herpetiformis, derma, vitiligo,
hypersensitivity is, urticaria, bullous pemphigoid, lupus erythematosus, ic lupus
erythematosus, pemphigus vulgaris, pemphigus eus, paraneoplastic pemphigus,
epiderrnolysis bullosa acquisita, acne vulgaris, and other inflammatory or ic conditions of
the skin.
nds of the invention may also be used for the treatment of other diseases or
conditions, such as diseases or conditions having an inflammatory component, for example,
treatment of diseases and conditions of the eye such as ocular allergy, conjunctivitis,
keratoconjunctivitis sicca, and vernal conjunctivitis, diseases affecting the nose including allergic
rhinitis, and atory disease in which mune reactions are implicated or having an
mune component or etiology, including autoimmune hematological disorders (e.g.
tic anemia, aplastic anemia, pure red cell anemia and idiopathic thrombocytopenia),
systemic lupus erythematosus, rheumatoid tis, polychondritis, scleroderma, Wegener
granulamatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, Steven-Johnson
syndrome, idiopathic sprue, autoimmune inflammatory bowel disease (e.g. tive colitis and
Crohn’s disease), irritable bowel syndrome, celiac disease, periodontitis, hyaline membrane
disease, kidney disease, glomerular disease, alcoholic liver disease, multiple sclerosis, endocrine
mopathy, Grave’s disease, sarcoidosis, alveolitis, c hypersensitivity pneumonitis,
multiple sclerosis, primary biliary cirrhosis, uveitis (anterior and posterior), n’s syndrome,
keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial lung fibrosis, psoriatic
arthritis, systemic juvenile idiopathic tis, cryopyrin-associated periodic me, nephritis,
vasculitis, diverticulitis, interstitial cystitis, glomerulonephritis (with and t nephrotic
syndrome, e. g. including idiopathic nephrotic syndrome or minal change pathy), chronic
granulomatous disease, endometriosis, leptospiriosis renal disease, glaucoma, retinal disease,
ageing, headache, pain, complex regional pain syndrome, cardiac rophy, musclewasting,
catabolic disorders, obesity, fetal growth retardation, hyperchlolesterolemia, heart disease,
chronic heart failure, elioma, anhidrotic ecodermal dysplasia, Behcet’s disease,
incontinentia pigmenti, Paget’s disease, pancreatitis, hereditary periodic fever syndrome, asthma
(allergic and non-allergic, mild, moderate, severe, bronchitic, and exercise-induced), acute lung
injury, acute respiratory distress me, eosinophilia, hypersensitivities, anaphylaxis, nasal
sinusitis, ocular allergy, silica induced diseases, COPD (reduction of damage, airways
inflammation, bronchial hyperreactivity, remodeling or disease progression), ary disease,
cystic fibrosis, acid-induced lung injury, pulmonary hypertension, polyneuropathy, cataracts,
WO 06074
muscle inflammation in conjunction with systemic sclerosis, inclusion body is,
myasthenia gravis, thyroiditis, n’s disease, lichen planus, Type 1 diabetes, or Type 2
diabetes, appendicitis, atopic dermatitis, asthma, allergy, blepharitis, bronchiolitis, bronchitis,
bursitis, cervicitis, cholangitis, cholecystitis, chronic graft rejection, colitis, conjunctivitis,
Crohn’s disease, cystitis, dacryoadenitis, dermatitis, dermatomyositis, alitis, endocarditis,
endometritis, enteritis, colitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis,
gastroenteritis, Henoch-Schonlein purpura, hepatitis, hidradenitis suppurativa, immunoglobulin
A nephropathy, interstitial lung disease, laryngitis, mastitis, meningitis, myelitis myocarditis,
myositis, nephritis, oophoritis, orchitis, is, otitis, pancreatitis, parotitis, pericarditis,
peritonitis, pharyngitis, pleuritis, phlebitis, pneumonitis, pneumonia, polymyositis, tis,
prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendonitis,
tonsillitis, ulcerative colitis, uveitis, vaginitis, vasculitis, or is.
In some embodiments the inflammatory disease which can be treated according to the
methods of this invention is an disease of the skin. In some embodiments, the inflammatory
disease of the skin is selected from contact dermatitits, atompic dermatitis, alopecia areata,
erythema multiforma, dermatitis herpetiformis, scleroderma, Vitiligo, hypersensitivity angiitis,
urticaria, bullous pemphigoid, gus vulgaris, pemphigus foliaceus, paraneoplastic
pemphigus, molysis bullosa acquisita, and other inflammatory or allergic conditions of the
skin.
In some embodiments the atory disease which can be treated according to the
methods of this invention is selected from acute and chronic gout, c gouty arthritis,
psoriasis, psoriatic tis, rheumatoid arthritis, Juvenile rheumatoid arthritis, Systemic jubenile
idiopathic arthritis , Cryopyrin Associated ic Syndrome (CAPS), and osteoarthritis.
In some embodiments the inflammatory disease which can be d according to the
methods of this invention is a TH17 ed disease. In some embodiments the THl7 mediated
disease is selected from Systemic lupus matosus, Multiple sclerosis, and inflammatory
bowel disease (including Crohn’s disease or ulcerative colitis).
In some embodiments the inflammatory disease which can be treated according to the
methods of this invention is selected from Sjogren’s syndrome, allergic disorders, osteoarthritis,
conditions of the eye such as ocular allergy, conjunctivitis, keratoconjunctivitis sicca and vernal
ctivitis, and diseases affecting the nose such as ic rhinitis.
Metabolic Disease
In some embodiments the invention provides a method of treating a metabolic
disease. In some embodiments the metabolic disease is selected from Type 1 diabetes, Type 2
diabetes, metabolic syndrome or obesity.
The compounds and compositions, according to the method of the t invention,
may be administered using any amount and any route of administration effective for treating or
lessening the ty of a cancer, an autoimmune disorder, a proliferative disorder, an
inflammatory disorder, a neurodegenerative or ogical disorder, schizophrenia, a bone-
related disorder, liver disease, or a cardiac disorder. The exact amount ed will vary from
subject to subject, depending on the species, age, and general condition of the subject, the
severity of the infection, the particular agent, its mode of administration, and the like.
Compounds of the invention are ably formulated in dosage unit form for ease of
administration and uniformity of dosage. The expression "dosage unit form" as used herein
refers to a physically discrete unit of agent appropriate for the patient to be d. It will be
understood, however, that the total daily usage of the compounds and itions of the
present invention will be decided by the ing physician within the scope of sound medical
judgment. The specific effective dose level for any particular patient or organism will depend
upon a variety of s including the disorder being treated and the severity of the disorder, the
activity of the specific compound employed; the specific composition employed; the age, body
weight, general health, sex and diet of the patient; the time of administration, route of
administration, and rate of ion of the specific compound employed; the duration of the
treatment, drugs used in combination or dental with the specific compound employed, and
like factors well known in the medical arts. The term “patient”, as used herein, means an animal,
preferably a mammal, and most preferably a human.
Pharmaceutically acceptable compositions of this invention can be administered to
humans and other s orally, rectally, parenterally, intracisternally, intravaginally,
intraperitoneally, topically (as by powders, nts, or drops), bucally, as an oral or nasal
spray, or the like, depending on the severity of the infection being treated. In certain
embodiments, the compounds of the invention may be administered orally or parenterally at
dosage levels of about 0.01 mg/kg to about 50 mg/kg and preferably from about 1 mg/kg to
about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired
therapeutic .
] Liquid dosage forms for oral administration include, but are not limited to,
pharrnaceutically acceptable emulsions, microemulsions, ons, suspensions, syrups and
elixirs. In on to the active nds, the liquid dosage forms may contain inert diluents
commonly used in the art such as, for example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl te, propylene , 1,3-butylene glycol, dimethylformamide, oils (in
ular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol,
tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures
thereof. s inert diluents, the oral compositions can also include adjuvants such as wetting
agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
able preparations, for example, sterile injectable aqueous or oleaginous
suspensions may be formulated ing to the known art using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation may also be a e injectable
solution, sion or emulsion in a nontoxic parenterally acceptable diluent or solvent, for
example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may
be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally ed as a solvent or suspending medium. For
this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid are used in the preparation of injectables.
Injectable formulations can be sterilized, for example, by filtration through a
bacterial-retaining filter, or by incorporating izing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water or other sterile injectable
medium prior to use.
In order to prolong the effect of a compound of the present invention, it is often
desirable to slow the absorption of the compound from subcutaneous or intramuscular injection.
This may be accomplished by the use of a liquid suspension of crystalline or amorphous material
with poor water solubility. The rate of absorption of the compound then depends upon its rate of
dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively,
delayed absorption of a parenterally administered compound form is accomplished by dissolving
or suspending the compound in an oil vehicle. Injectable depot forms are made by forming
microencapsule matrices of the compound in radable polymers such as polylactide-
polyglycolide. Depending upon the ratio of compound to polymer and the nature of the
particular polymer employed, the rate of compound release can be controlled. es of other
biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable
formulations are also prepared by entrapping the compound in liposomes or microemulsions that
are compatible with body tissues.
itions for rectal or vaginal administration are preferably suppositories which
can be prepared by mixing the compounds of this ion with suitable non-irritating
excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are
solid at ambient ature but liquid at body temperature and ore melt in the rectum or
vaginal cavity and release the active compound.
Solid dosage forms for oral administration include capsules, s, pills, powders,
and granules. In such solid dosage forms, the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium ate
and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic
acid, b) binders such as, for example, carboxymethylcellulose, alginates, n,
polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating
agents such as agar-agar, calcium carbonate, potato or tapioca starch, c acid, certain
silicates, and sodium ate, e) solution retarding agents such as paraffin, f) tion
accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example,
cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i)
lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium
lauryl e, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form
may also comprise buffering agents.
Solid itions of a similar type may also be employed as fillers in soft and hard-
fllled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules,
pills, and granules can be prepared with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art. They may optionally contain
ying agents and can also be of a composition that they release the active ingredient(s) only,
or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
Examples of embedding itions that can be used include polymeric substances and waxes.
Solid itions of a similar type may also be employed as fillers in soft and hard-filled
gelatin capsules using such excipients as e or milk sugar as well as high molecular weight
polethylene glycols and the like.
The active compounds can also be in micro-encapsulated form with one or more
excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and
granules can be prepared with coatings and shells such as enteric coatings, release controlling
coatings and other coatings well known in the pharmaceutical formulating art. In such solid
dosage forms the active nd may be d with at least one inert t such as
sucrose, lactose or starch. Such dosage forms may also se, as is normal practice,
additional substances other than inert diluents, e. g., tableting lubricants and other tableting aids
such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and
pills, the dosage forms may also comprise buffering agents. They may optionally contain
opacifying agents and can also be of a composition that they release the active ingredient(s) only,
or preferentially, in a certain part of the intestinal tract, optionally, in a d manner.
Examples of embedding compositions that can be used e polymeric substances and waxes.
Dosage forms for topical or transdermal administration of a compound of this
invention include nts, pastes, creams, s, gels, powders, solutions, sprays, inhalants
or patches. The active component is admixed under sterile conditions with a pharmaceutically
acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic
formulation, ear drops, and eye drops are also contemplated as being within the scope of this
invention. onally, the present invention contemplates the use of transdermal patches,
which have the added advantage of providing controlled delivery of a compound to the body.
Such dosage forms can be made by ving or dispensing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux of the compound across the
skin. The rate can be controlled by either providing a rate controlling membrane or by
dispersing the compound in a polymer matrix or gel.
According to one embodiment, the invention relates to a method of modulating the
activity of a target RNA in a biological sample comprising the step of contacting said biological
sample with a compound of this invention, or a composition comprising said compound.
According to another embodiment, the invention relates to a method of modulating
the activity of a target RNA in a biological sample comprising the step of contacting said
biological sample with a compound of this invention, or a composition comprising said
compound. In certain embodiments, the invention relates to a method of irreversibly inhibiting
the ty of a target RNA in a biological sample comprising the step of contacting said
biological sample with a compound of this invention, or a composition comprising said
compound.
The term “biological sample”, as used herein, includes, without limitation, cell
cultures or extracts thereof, biopsied material obtained from a mammal or extracts thereof, and
blood, saliva, urine, feces, semen, tears, or other body fluids or extracts f,
Another embodiment of the present invention s to a method of modulating the
activity of a target RNA in a patient comprising the step of administering to said patient a
compound of the t invention, or a composition comprising said compound.
According to another embodiment, the invention s to a method of inhibiting the
activity of a target RNA in a t sing the step of stering to said t a
compound of the present invention, or a composition comprising said compound. According to
certain embodiments, the ion relates to a method of irreversibly inhibiting the activity of a
target RNA in a patient sing the step of administering to said patient a compound of the
present invention, or a composition comprising said compound. In other embodiments, the
present invention provides a method for treating a disorder mediated by a target RNA in a patient
in need thereof, comprising the step of administering to said patient a compound according to the
present invention or ceutically able composition thereof. Such ers are
described in detail herein.
EXEMPLIFICATION
As ed in the Examples below, in certain exemplary embodiments, compounds
are prepared according to the following general procedures and used in biological assays and
other procedures described lly herein. It will be appreciated that, although the general
methods depict the synthesis of certain compounds of the present invention, the following
general methods, and other methods known to one of ordinary skill in the art, can be applied to
all compounds and subclasses and s of each of these compounds, as described herein.
Similarly, assays and other analyses can be adapted according to the knowledge of one of
ordinary skill in the art.
Example 1: nding Small Molecules
Historical efforts to identify small molecule ligands that bind to RNA have focused
on base-pairing or on canonical structural motifs in duplex RNA: intercalation between bases
and/or groove binding. But these motifs do not support ive g of small les to
specific RNAs. However, RNA folds into an enormous variety of complex tertiary structures
that present s conducive to small molecule binding — small molecules that are
complementary to the shape and electrostatics presented by those pockets. Insofar as the details
of shape and ostatics reflect the underlying sequence of the RNA, small molecules can
achieve selectivity, much as they do when binding protein pockets.
Indeed, there are now several reports of drug-like small molecules that bind to RNA,
many of them proved (see Table 4 below).
Small Molecule Ligands by Class
Though a range of small molecule chemotypes has been demonstrated to bind to
folded RNA (Guan & Disney, ACS Chem. Biol. 2012 7, 73-86), hereby incorporated by
reference, there are limited reports of high—throughput screening of large libraries (>105
compounds) to fy RNA-binding ligands. Accordingly there are also few reports of small
molecules synthetically optimized for RNA binding. The t invention paves the path to a
remedy for these deficiencies. Below is a table summarizing the broad chemotypes which have
demonstrable RNA binding and will serve as the ng point to optimize and validate our
screening , which will in turn enable the systematic screening of essentially all known
chemotypes against RNA structures of therapeutic interest.
Table 4: RNA-binding Small Molecules
-approve ac en eac e . .
Linezolid
antibiotic ribosomal RNA 2007, 26, 393-402
FDA-approved Bacterial Leach et al. Mol. Cell
Tedizolid
antibiotic ribosomal RNA 2007, 26, 393-402
Bacterial Brodersen et al. Cell
. FDA-approved
Tetracycline
antibiotic 30$ mal RNA 2000, 1 03, 1143-1154
FDA-approved Bacterial Fourmy et al. Science
Aminoglyc051des, ,
antibiotics 16S mal RNA 1996, 274, 1367-1371
proved for Jenison et al. Science
Theophylline Aptameric RNA
COPD and asthma 1994, 263, 1425-1429
These discoveries revealed a molecular mechanism of action that was not pated.
The intentional design of small molecules that bind to folded RNA has been d only rarely
e of substantial technical challenges, with one notable example being the design of
cene-based ligands able to bind selectively to RNA three—way junctions (Barros et al.,
Angew. Chem. Int. Ed. 2014, 53, 13746-13750. Triptycene-based ligands will thus provide
another chemotype with RNA binding ability to serve as another starting point in the described
ing methods.
The s provide many exemplary scaffolds and specific target compound genera,
which may be ed or altered using methods known in the art.
As shown in Figure 8 (top right structure), while tripartite scaffolds represent a
promising platform for binding 3WJs, they do not need to be symmetric, as the 3WJ will itself
not usually be ric. In the pictured example, the triangle, square, and pentagon stand for
cyclopropyl, cyclobutyl, or cyclopentyl, and may be substituted with the various nucleic acid
binding groups disclosed herein.
Example 2: Synthesis of Warhead Type IA
Scheme: Synthesis of Warhead Type IA
Triphosgene,
Dioxane, RT 6h 0
Step-1 NAG
O H
Warhead_Type_1A
MW 207
2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]oxazinecarb0xylic acid, Warhead
Type IA.
To a solution of oterephthalic acid (2.0 g, 11.05 mmol) in 1,4—Dioxane (160
mL) was added triphosgene (3.28 g, 11.05 mmol) at room temperature. The resulting reaction
mixture was stirred for 6 h at room temperature. The reaction mixture was poured in DM water
(400 mL) and extracted with ethyl acetate (3 X 150 mL). The organic layers were combined,
washed with brine and concentrated under d pressure to afford Warhead_Type_1A (2.2
g, 96.2%) as an off white solid. 1H NMR (400 MHz, DMSO-d6) 5 13.67 ppm (1H, broad), 11.89
ppm (1H, broad), 8.03-8.01 ppm (1H, d), 7.73-7.68 ppm (2H, m). MS (ESI—MS): m/z calcd for
C9H5N05 [MH]' , found 206.17.
Example 3: Synthesis of d Type 1B
Scheme: Synthesis of Warhead Type 1B
O 0
K2003' DMS
KOH, THF,
0/ Acetone Water OH
’ HO
NH2 Step--1 Step-2 NH
0 0 l
(A) (1)
MW: 223.08 ( )
MW: 195.05
Step3 Triphosgene
Dioxane RT
HO NAG
O l
Warhead_Type-1 B
MW 221 .03
1,4-dimethyl hylamino)benzene-1,4-dicarboxylate (1).
] To a solution of dimethyl 2—aminobenzene-1,4-dicarboxylate (10.0 g, 0.05 mol) in
acetone (150 mL) was sequentially added potassium carbonate (19.8 g, 0.143 mol) and
dimethylsulphate (18.1 g, 0.143mol) at room temperature. The resulting reaction mixture was
stirred at 60 °C for 24h. The reaction e was slowly cooled to room temperature and diluted
with water (200 mL). The resulted mixture was then extracted with ethyl acetate (4 x 750 mL).
The organic layers were combined, washed with brine and concentrated under reduced re
to get crude 1 as a brown solid. The crude mixture was purified by column chromatography on
silica gel (7% EtOAc/hexanes) to yield 1 (4.5g, 42%) as a pale yellow solid. MS (ESI—MS): m/z
calcd for C11H13NO4 [MH]+ 224.08, found 2242.
2-(methylamino)benzene—1,4-dicarb0xylic acid (2).
To a solution of dimethyl hylamino)benzene-1,4-dicarboxylate (1) (4.5 g, 0.02
mol) in THF (100 mL) and water (50 mL) was added potassium hydroxide (3.4g, 0.06 mol) at
room temperature. The ing reaction mixture was stirred at 70 °C for 4h. The reaction
mixture was cooled to room temperature, diluted with water (200 mL) and ed using
ium ate. The resulted mixture was then extracted with ethyl acetate (4 x 75 mL). The
organic layers were combined, washed with brine and concentrated under d pressure to get
crude 2 (3.0g, 76.33%) as a buff white solid. The crude mixture was used in next step without
further purification. 1H NMR (400 MHz, DMSO-d6) 5 13.14 ppm (1H, s), 7.87-7.85 ppm (1H, d,
J=8.0 Hz), 7.21—7.21 ppm (1H, d, J=1.6 Hz), 7.10—7.07 (1H, dd, J=8.0), 2.87 (1H, 5). MS (ESI-
MS): m/z calcd for C9H9NO4 [MH]+ 196.05, found 196.21.
1-methyl-2,4-diox0-2,4-dihydro-1H-3,1-benzoxazine—7-carboxylic acid, Warhead
Type 1B.
To a suspension of 2-(methylarnino) benzene-1,4-dicarboxylic acid (2) (3.0 g, 0.015
mol) in ydrofuran (90 mL) was added triphosgene (2.28 g, 0.076 mol) at room
temperature. The resulting reaction mixture was stirred at 30 °C for 30 min. The reaction
mixture was cooled to room temperature, diluted with water (50 mL) and extracted with ethyl
acetate (3 x 100 mL). The organic layers were combined, washed with brine and concentrated
under reduced pressure to get crude Warhead Type 1B as a yellow solid. The crude mixture
was purified by trituration using diethyl ether to yield Warhead Type 1B (31 g, 91.17%) as
yellow solid. 1H NMR (400 MHz, DMSO-dg) 6 13.78 ppm (1H, s), .09 (1H, d, J=8.4),
7.82-7.80 (2H, m), 3.51 (3H, S). MS (ESI—MS): m/z calcd for C10H7N05 [MH]' 220.03, found
onal warheads similar to this type include N—methylisatoic anhydride, 1-
methylnitroisatoic anhydride, and 1—methylnitroisatoic ide. These are commercially
ble.
Example 4: Synthesis of Warhead Type 2
Scheme: Synthesis of Warhead Type 2
o o o 0
Triphosgene CH3" K2003 BB“
OH ,
Dioxane O DMF In DCM
O O
o NH2 Step-1 \o m’go Step-2 \o ill/KO Step"?
HO Iii/KO
(1) (2) (3)
MW: 193.03 MW: 207.05 MW: 193.03
Step-4 @Orm
K2003
Acetone
do 10% dec, H2
THF: EtOAc
HO Q/ m0
o l O
Warhead_Type 2A (4)
MW: 341.08
MW: 251.04
7-meth0xy-2H-benzo[d] [1,3]0xazine—2,4(1H)—di0ne (1).
To a solution of 2-aminomethoxybenzoic acid (20 g, 119.73 mmol) in 1,4-dioxane
(400 mL) was added triphosgene (17.8 g, 59.86 mmol) at room temperature. The resulting
reaction mixture was d at room temperature for 6 h. The reaction mixture was poured in
DM water (1 L) and extracted with ethyl acetate (3 X 350 mL). The organic layers were
combined, washed with brine and concentrated under reduced pressure to afford 1 (20.5 g, 88%)
as off White solid. 1H NMR (400 MHZ, DMSO-do) 5 11.66 ppm (1H, broad), 7.85—7.83 ppm (1H,
d, J=8.8 Hz), 6.85-6.83 ppm (1H, dd, J=2.4, 6.4 Hz), 6.59-6.58 ppm (1H, d, J=2.4 Hz), 3.86 ppm
(3H, s). MS (ESI—MS): m/z calcd for C9H7NO4 [MH]' , found 192.16.
7-meth0xy-l-methyl-ZH-benzo[d][1,3]0xazine-2,4(lH)—di0ne (2).
To a solution of 7-methoxy-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (1) (20.5 g,
106.2 mmol) in N,N—dimethyl formamide (200 mL) was added K2C03 (14.65 g, 106.2 mmol) at
room temperature and the ing reaction e was stirred for 10 min. To this, methyl
iodide (18.08 g, 127.44 mmol) was added drop wise at room temperature. The reaction mixture
was poured into DM water (1 L) and ted with ethyl acetate (3 X 350 mL). The organic
layers were combined, washed with brine and concentrated under d pressure to get crude
2. The crude was purified by triturating with hexane to yield 2 (17.9 g, 93.23 %) as off white
solid. The product was used in the next step without further purification. 1H NMR (400 MHz,
DMSO-dp) 6 7.95-7.93 ppm (1H, d, J=8.4 Hz), 6.94-6.91 ppm (1H, dd, J=2.4, 6.4 Hz), 6.86-6.85
ppm (1H, d, J=2 Hz), 3.94 ppm (3H, s), 3.46 ppm (3H, s). MS (ESI—MS): m/z calcd for
C10H9NO4 [MH]+ 208.05, found 208.2.
7-hydr0xy-l-methyl-ZH-benzo[d] [1,3]0xazine-2,4(1H)—dione (3).
To a solution of 7-methoxymethyl-2H-benzo[d][1,3]oxazine-2,4(1H)—dione (2) (10
g, 48.30 mmol) in dichloromethane (500 mL) at 0 oC, BBr3 (1 M solution in dichloromethane)
(72.44 mL, 72.44 mmol) was added dropwise. The resulting reaction mixture was stirred at 0 °C
for 1 h and slowly brought to room temperature and further stirred for 24 h. The reaction
mixture was diluted with n-Hexane (500mL) and the residues ed were d. The
collected solid was washed with n-Hexane (3 X 50mL) and dried under reduced pressure. The
solid was further suspended in water (1 L) and extracted with dichloromethane (5 X 350mL).
The organic layers were combined, washed with brine and concentrated under reduced pressure
to get 3 (7.9 g, 84.74 %) as a brown solid. 1H NMR (400 MHz, MeOD) 8 7.96-7.94 ppm (1H, d,
J=8.8 Hz), 6.78-6.75 ppm (1H, dd, J=2, 64 Hz), 6.69-6.69 ppm (1H, d, J=2.4 Hz), 3.52 ppm
(3H, s). MS (ESI—MS): m/z calcd for C9H7NO4 [MH]' 192.04, found 191.96.
Benzyl 2-((1-methyl-2,4-di0xo-1,4-dihydr0-2H-benz0[d][1,3]0xazin
yl)0xy)acetate (4).
] To a on of 7-hydroxymethyl-2H-benzo[d][1,3]oxazine-2,4(1H)-dione (3) (7.9
g, 40.93 mmol) in acetone (800 mL) was added K2C03 (14.12 g, 5 mmol) and the
reaction mixture was stirred for 20 min at room temperature. To this, benzylbromoacetate
(11.251 g, 49.111 mmol) was added dropwise at room temperature and the resulting on
mixture was further stirred for 5 h. The reaction mixture was filtered and residues collected were
washed with acetone (3 X 20mL). The filtrate was concentrated under d pressure to afford
a solid mass. The solid mass was dissolved in ethyl acetate (1 L) and washed with water (3 X
300mL). The organic layers were combined, washed with brine and concentrated under reduced
pressure to get crude 4. The crude mixture was purified by column chromatography on silica gel
(20% EtOAc/n-Hexane) to yield pure 4 (0.39 g, 62.9 %) as a yellow oil. 1H NMR (400 MHz,
DMso-d6) 5 7.94-7.92 ppm (1H, d, J=8.4 Hz), 7.38-7.35 ppm (5H, m), 6.95-6.92 ppm (1H, dd,
J=2, 6.8 Hz), 6.87-6.87 ppm (1H, d, J=2 Hz), 5.23 ppm (2H, s), 5.14 ppm (2H, s), 3.40 ppm (3H,
). MS (ESI—MS): m/z calcd for NO6 [MH]+ 342.09, found 342.28.
] 2-((1-methyl-2,4-di0x0-1,4-dihydro-2H-benz0[d][1,3]0xazinyl)0xy)acetic acid,
Warhead_type_2.
To a suspension of 10% Pd/C (dry basis) (1.25g, 5 % w/V) in a 1:1 mixture of THF:
EtOAc (400 mL) was added a solution of Benzyl methyl-2,4—dioxo-1,4-dihydro-2H-
benzo[d][l,3]oxazinyl)oxy)acetate (4) (6.5 g, 19.057 mmol) at room temperature. H2 gas was
purged into the reaction mixture for 3 h at room temperature. The reaction mixture was filtered
through a celite bed and the collected filtrate was concentrated under reduced pressure to afford
crude d_type_2. The crude mixture was puiified by triturating with n—Hexane (3 X
20mL) to yield Warhead_type_2 (0.39 g, 62.9%) as an off white solid. 1H NMR (400 MHz,
DMSO—ds) 5 13.25 ppm (1H, br s), 7.95—7.92 ppm (1H, d, J=8.4 Hz), .88 ppm (2H, m),
4.94 ppm (2H, s), 3.44 ppm (3H, s). MS (ESI—MS): m/z calcd C11H9N06 [MH]+ 252.04, found
252.47.
2017/040514
Example 5: Synthesis of ARK-1 (Ark000007)
Scheme: Synthesis of ARK-1
o NHZ NHBoc
2 HO-S-OH
ll HO O
HO 0
0 H0
H0 Ho\%HO NH \% NHBOC
NHZ 2
Amberlfle IRA-400, OH
OH O NH 02, DMSO, H20 0 ,NHBoc
2 Si: § OmNHz DM water HO/m/ 70 °C,20 hrs. HO
O O
HO O —’
HO —> HO
Ho SteP_1 NH NHBOC
Step 2
0 012—1 0 OH
O OH
OH OH
(1) (2)
CI, pyridine Step-3
hrs.
HO 0 NHBoc
HO NaNs, DMF o NHBOC
OH NH; Hgo
0% NH TFA, MDC, 120 °c MW
2 NHBOC 0
HO 30 mins. 0H 3 hrs.
0H0 0 HEO
NHBOC
<— <— NHBoc
H0 OH
””2 Step-5 0H0 Step-4 OmNHBoc
O OH NHBoc HO 0
OH HO
N3 0 NHBoc
ARK-1 ”3 0
(4) OTIPBS
Kanamycin A free base, 1.
In 250 mL beaker, kanamycin A monosulfate (5.0 g, 8.582 mmol) was dissolved in
water (100 mL) and the ing aqueous solution was passed through Amberlite® 0 -OH
form ion exchange resin. The free base was eluted using DM water and the fractions collected
were lyophilized to obtain free base 1 (3.8g, 91%) as a white solid which was used without
further purification. MS S): m/z calcd for C18H36N4011 [MH]+485.23, found 485.26.
1,3,6’,3”-tetra—N-(tert-butoxycarbonyl) kanamycin A, 2.
To a stirred solution of Kanamycin A free base (1) (3.7 g, 7.641 mmol) in DMSO
(140 mL) and water (40 L) (180 mL) was added Boc anhydride (20g, 91.692 mmol) at room
temperature and the resulting reaction mixture was heated at 70 °C for 20 h. After cooling to
room temperature, an aqueous solution of NH4OH (30 mL) was added to the resulting reaction
mixture, resulting in a precipitate. The precipitate was collected through filtration, washed with
water (2 X 350 mL) and dried under reduced pressure to afford pure 2 (5.7 g, 84%) as a white
solid. 1H N1VIR (400 MHz, DMSO-d6) 5 6.92 ppm (1H, s), 6.62 ppm (1H, s), 6.53-6.51 ppm
(1H, d, J=6.8 Hz), 6.38 ppm (1H, s), 5.40 ppm (1H, broad s), 5.27 ppm (1H, broad s), 4.71 ppm
(1H, broad s), 4.22 ppm (1H, broad s), 3.80-3.25ppm (15H, broad m), 3.07 ppm (1H, broad s),
1.82-1.75 ppm (1H, broad s), 1.37 ppm (36H, broad s); MS (ESI—MS): m/z calcd for
C38H68N4019 [MH]+ 885.44, found 907.7 (M+Na adduct).
6”-(2,4,6-Triis0propylbenzenesulfonyl)-1,3,6’,3”-tetra-N-(tert-butoxycarbonyl)
kanamycin A, 3.
To a stirred solution of 1,3,6’,3”-Tetra-N-(tert-butoxycarbonyl) kanamycin A (2) (2 g,
2.261 mmol) in pyridine (35 mL) was added a on of 2,4,6-triisopropylbenzenesulfonyl
chloride (4.11 g, 13.567 mmol) in pyridine (4 mL) at room temperature. The resulting on
mixture was d at room temperature for 20 h. After this, the reaction mixture was added
ol (30 mL) and further stirred for 30 min. The reaction mixture was then poured into a
cooled 10% HCl solution (400 mL) and extracted with ethyl acetate (4 x 200mL). The c
layers were combined, washed with brine, dried using anhydrous Na2SO4 and concentrated under
reduced pressure to get crude 3 as a yellow solid. The crude mixture was purified by column
chromatography on silica gel (2% MeOH/chloroform) to get pure 3 (0.5 g, 73%) as a light
yellow solid, MS (ESI—MS): m/z calcd for C53H90N4021S [MH]+ 1151.58, found 908.6 (M-
TTPBS fragment +Na adduct).
6”-Azido-1,3,6’,3”-tetra-N-(tert—butoxycarb0nyl)kanamycin A, 4.
A 35 mL pressure vial was charged with 6”-(2,4,6-Triisopropylbenzenesulfonyl)—
,3”—tetra-N—(tert-butoxycarbonyl) kanamycin A (3) (0.5g, 0.434 mmol), NaN3 (0.565 g,
8.691 mmol), DMF (15 mL) at room temperature. The resulting reaction mixture was irradiated
under microwave at 120 °C for 3 h. After g to room temperature, the reaction mixture was
quenched with cold water (150 mL) and extracted with ethyl e (3 X 50 mL). The organic
layers were combined, washed with brine, dried using anhydrous Na2804 and concentrated under
reduced pressure to get crude 4 as brown oil. The crude mixture was purified by preparative
HPLC using the following method to get pure 4 (0.11 g, 27%) as a light yellow solid. 1H NMR
(400 MHz, CD3OD) 5 5.11—5.02 ppm (2H, t, J=9.6 Hz), 4.37—4.35 ppm (1H, d), 3.73-3.36 ppm
(15H, m), 3.23-3.18 ppm (1H, t, J=9.2 Hz), 2.07-2.04 ppm (1H, d, J=13.2 Hz), 1.47—1.45 ppm
(36H, br s). MS (EST-MS): m/z calcd for C38H67N7018 [MHTr , found 932.67 (M+Na
adduct).
Method of preparative HPLC:
W0 2018/006074
(A) 10 mM ammonium bicarbonate in H20 (HPLC grade) and (B) MeCNzlPA
(90:10) (HPLC grade), using X-BRIDGE C18, 250*19mm,5Un with a flow rate of 19.0 mL/min
and with the following gradient:
——E_
17.00
"El.
——Efi-
——m_
6”-Azid0-kanamycin A triflouroacetate salt, ARK-l-TFA SALT.
6”-Azido-1,3,6’,3”-tetra—N—(tert-butoxycarbonyl)kanamycin A, (4) , 0.121
mmol) was dissolved in 1:1 mixture of DCMzTFA (3.2 mL) and the resulting solution was stirred
at room temperature for 30 min, The reaction e was concentrated under reduced pressure
and triturated using diethyl ether to get pure ARK-l-TFA SALT (0.12 g, 102%) as a light
yellow solid. 1H NMR (400 MHz, D20) 6 5.39-5.38 ppm (1H, d, J=3.6 Hz), 4.95—4.94 ppm (1H,
d, J=3.2 Hz), 3.796-3.71 ppm (5H, m), 3.64-3.31 ppm (11H, m), .01 ppm (1H, q,
J=14.4,9.2 Hz), .37 ppm (1H, m), 1.77—1.74 ppm (1H, q, J = 12.8 Hz), 1.09—1.02 ppm (1H,
m). MS (ESI—MS): m/Z calcd for N7Olo+3TFA [MH]+ 509.24, found 510.4. HPLC
retention time: 7.103 min.
6”-Azid0-kanamycin A hloride salt, ARK-l-HCl SALT (Ark000007).
6”-Azido-kanamycin A triflouroacetate salt, TFA SALT (0.12 g, 0.124
mmol) was dissolved in water (40 mL) and the resulting aqueous solution was passed through
Amberlite® IRA-400 -OH form ion exchange resin. The free base was eluted using DM water
and the fractions collected were lyophilized to obtain ARK-1 as a free base. The free base was
ved in 0.01 N HCl (4 mL) and the resulting solution was lyophilized to obtain pure ARK—
l-HCl-SALT (0.06g, 77%) as a yellow solid. 1H NMR (400 MHZ, D20) 6 5.41-5.40 ppm (1H, d,
J=2.4 Hz), 4.96 ppm (1H, br s), 3.90-3.76 ppm (5H, m), 3.62—3.60 ppm (2H, d, J=8.8 Hz), 3.55—
3.19 ppm (10H, m, ), 3.07-3.01 ppm (1H, m), 2.41-2.38 ppm (1H, d, J: 12), 1.82-1.73 ppm (1H,
q, J = 12.8 Hz), MS (ESI—MS): m/z calcd for C18H35N7Olo.3 HCl [MH]+ 510.24, found 510.2.
HPLC retention time: 14.897 min.
Example 6: Synthesis of ARK-7 (Ark0000013)
Scheme: Synthesis of ARK-7
HCI, NaNo2
H20, KI w
—> g/6
Step-3 | Q
Zn(CN)2, DMF
o Step-4
C Q/s
NHZ CN
CH3 (4)
NaOH,
MeOH,HZO
HZNMNMNl-POC Step-5
(23) HOOC\A
// /
HATU, DIPEA
CH i
NH/Vl‘vj/VNHBDC ‘ DMF
moor/:7 \(3
<(:OOH
Step-6
HCI in e
Step-7
ARK-7_HCI salt
2,7,15-trinitro-9,10-dihydro—9,10-[1,2]benzen0anthracene,1a.
Concentrated HNO3 (400 mL) was added se to triptycene (10 g, 39.3 mmol) at
room temperature and the resulting reaction mixture was heated at 80 CC for 16 h. The resulting
brown solution was allowed to cool to room temperature, poured into ice cold water (3 000 mL)
and stirred for 30 min. The obtained precipitates were collected, washed with cold water, and
then dried in air to get the crude mixture of 1a and 1b. The crude mixture was purified by flash
column chromatography on silica gel (20% EtOAc/hexanes) to afford pure product 121 (2.23 g,
14.10%) as a white solid. la mp: >300 °C 1H NMR (400 MHz, CDC13) 6 837—836 ppm (3H, d,
J=2 Hz), 8.08-8.06 ppm (3H, dd, J=8 Hz, J=2 Hz), 7.66-7.64 ppm (3H, d, J=8.4 Hz), 5.87 ppm
(1H, S), 5.84 ppm (1H, s), 13C NMR (400 MHz, DMSO-dg) 150.24, , 145.76, 126.10,
122.60, 119.93, 52.18, 51.48, MS (ESI-MS): m/z calcd for C20H21N306 [MH]+ 390.06, No mass
response observed.
1b mp: 178-180 °C 1H NMR (400 MHz, CDC13)6 8.36-8.35 ppm (3H, m), 8.09-8.06
ppm (3H, m), 7.69-7.65 ppm (3H, m), 5.86 ppm (1H, s), 5.85 ppm (1H, s) 13C N1V1R 150.93,
,145.72,145.33,144.92,125.97,122.54, 119.93, 55.33, 51.98, 51.74.
9,10—dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-triamine, 2.
To a solution of 2,7,15-trinitro-9,10-dihydro—9,10-[1,2]benzenoanthracene (1a) (2.23
g, 5.73 mmol) in THF (100 mL) was added Raney Nickel (1.0 g) and the resulted reaction
mixture was cooled to 0 °C. Hydrazine hydrate (4 mL) was added to the ing mixture at 0
oC. The reaction mixture was stirred at 60 0C for 1 h. The resulting reaction mixture was allowed
to cool to room temperature and filtered through celite eluting with THF. The filtrate was
concentrated under reduced pressure to afford crude 2 (1.5 g, ) as a brown solid which
was used without further purification. 1H NMR (400 MHz, CDC13) 6 709-707 ppm (3H, d,
J=7.6 Hz), 6.75-6.75 ppm (3H, d, J=2 Hz), 6.29-6.27 ppm (3H, dd, J=7.6 Hz, J=2 Hz), m
(1H, S), 5.02 ppm (1H, s), 5 ppm ( 6H, broad 5). MS (ESI—MS): m/z calcd for N3
[MH]+ 300.14, found 300.4.
] 2,7,15-trii0d0-9,10-dihydr0-9,10-[1,2]benzen0anthracene, 3.
In 100 mL round bottom flask, 9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15-
triamine (2) (0.9 g, 3.01 mmol) was dissolved in concentrated hydrochloric acid (7.5 mL) and
water (15 mL) and the resulting solution was cooled to 0 0C. To this, a solution of sodium nitrite
(0.72 g, 10.5 mmol) in water (7.5 mL) was added se over 10 min and the resulting
reaction mixture was stirred for 20 min at 0 0C. After this, a solution of potassium iodide (3.74 g,
22.58 mmol) in water (10 mL) was added drop wise to the reaction mixture at 0 °C and further
stirred for 5 min. The reaction mixture was then slowly warmed to room temperature and heated
at 80 CC for 2 h. After cooling to room temperature, the reaction mixture was diluted with water
(50 mL) and extracted with dichloromethane (3 x 25 mL). The organic layers were combined,
washed with saturated sodium bisulfate (3 x 30 mL), dried using anhydrous Na2SO4 and
concentrated under reduced pressure to get crude 3 as a brown semisolid. The crude mixture was
purified by flash column chromatography on silica gel (5% EtOAc/ hexanes) to get pure product
3 (0.57 g, 30.0%) as yellow solile NMR (400 MHz, CDC13) 6 7.74-7.73 ppm (3H, d, J=l.6
Hz), 7.39-7.36ppm (3H, dd, J=7.6 Hz, J=l.6 Hz), 7.66-7.64 ppm (3H, d, J=7.6 Hz), 5.3lppm
(1H, S), 5.26 (1H, s).
9,10—dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-tricarbonitrile, 4.
To a on of2,7,15-triiodo-9,lO-dihydro-9,l0-[1,2]benzenoanthracene (3) (0.55 g,
0.87 mmol) in DMF (5 mL) was added zinc cyanide (0.33 g, 2.79 mmol) and the resulting
reaction mixture was degassed with nitrogen gas for 20 min. To this, tetrakis (0.10 g, 0.1 mmol)
was added and the resulting on mixture was stirred at 140 °C for 16 h, After g to
room temperature, the reaction mixture was d through celite, quenched with cold water (20
mL) and extracted with romethane (3 x 30 mL). The c layers were combined,
washed with brine, dried using anhydrous Na2SO4 and concentrated under reduced pressure to
get crude 4 as a brown semisolid. The crude mixture was purified by flash column
chromatography on silica gel (25% EtOAc/hexanes) to get pure product 4 (0.2 g, 70.0%) as light
yellow solid. 1H NMR (400 MHZ, CDCl3) 8 74 ppm (3H, d, J=l.2 Hz), 7.39-7.36 ppm
(3H, dd, J=7.6 Hz, J=l.6 Hz), 7.66-7.64 ppm (3H, d, J=7.6 Hz), 5.31 ppm (1H, S), 5.26 (1H, s).
9,10—dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-tricarb0xylic acid, 5.
To a solution of ihydro-9,10-[1,2]benzenoanthracene-2,7,15-tricarbonitrile (4)
(0.40 g, 1.22 mmol) in MeOH (5 mL) was added 15% aqueous NaOH solution (5 mL, 18.24
mmol) at room temperature and the resulting reaction mixture was d at 60 °C for 16 h. After
cooling to room temperature, excess of MeOH was removed under reduced pressure and the
resulting mixture was poured in ice-cold water (50 mL). The pH of this aqueous solution was
adjusted to ~2 using 1 N HCl and the residues obtained were collected through filtration to get
crude 5 (0.30 g, 65.3%) as a white solid which was used without further purification. 1H NMR
(400 MHz, MeOD) 5 8.12 ppm (3H, d, J=1.2 Hz), 7.79—7.77 ppm (3H, dd, J=7.6 Hz, J=l.6 Hz),
7.58-7.56 ppm (3H, d, J=4 Hz), 5.832ppm (2H, S), MS (EST-MS): m/z calcd for C12H2606 [MH]'
385.07, found 385.1.
Tert—butyl (3-((3-amin0propyl)(methyl)amin0)propyl)carbamate, 221.
To a solution of Nl-(3-aminopropyl)-Nl-methylpropane-1,3-diamine (5 g, 38.48
mmol) in THF (10 mL) at 0 CC was added Boc ide (1.50 g, 6.89 mmol) se over a
period of 20 min and the resulting reaction mixture was stirred at room temperature for 16 h,
THF was removed under reduced pressure and the resulting mixture was poured in water (50
mL). The aqueous mixture was extracted with ethyl acetate (3 x 30 mL). The organic layers were
ed, washed with water, dried using anhydrous Na2804 and concentrated under reduced
pressure to get pure 2a (1.3 g, 15.4 %) as a colorless oil. 1H NMR (400 MHz, O) 6 6.80-
6.79 ppm (1H, d, J=4 Hz), 3.17 (3H, broad s) 2.94-2.89ppm (2H, dd, J=12.4, 6 Hz), 2.51 ppm
(2H, broad s), 2.28-2.21ppm (4H, m), 2.08-2.07 (2H, d, J=4 Hz), 1.50—1.44 ppm (4H, m), 1.37
(9H, 5); MS (ESI—MS): m/z calcd for C12H26N202 [MH]+246.21, No mass response observed.
N2,N7,N15-tris(3-((3-tert-butylcarbonylaminopropyl)(methyl)amin0)pr0pyl)—9,10-
dihydro-9,10-[l,2]benzenoanthracene-2,7,1S-tricarboxamide, 6.
To a on of tert-butyl (3—((3-aminopropyl)(methyl)amino)propy1)carbamate (2a)
(0.71 g, 2.91 mmol) in DMF (3 mL) was added 9,10-dihydro-9,10-[1,2]benzenoanthracene-
2,7,15-tricarboxylic acid (0.35 g, 0.91 mmol), HATU (1.1 g, 2.91 mmol), DIPEA (1.0mL, 5.82
mmol) and the resulting reaction mixture was stirred at room temperature for 2 h. The reaction
mixture was poured in water (50 mL) and extracted with dichloromethane (3 x 25 mL). The
organic layers were combined, washed with brine, dried using anhydrous Na2$O4 and
concentrated under reduced pressure to get crude 6 as brown oil. The crude mixture was purified
by preparative HPLC using the ing method to afford pure t 6 (0.2 g, 20.7%) as light
yellow solid. 1H NMR (400 MHz, ds-DMSO) 5 840-837 (3H, t, J=5.2 Hz), 7.93 (3H, s) 7.55-
7.49ppm (6H, dd, J=16, 7.6 Hz ), 6.78 ppm (3H, broad s), 5.87 ppm (2H, broad s), 3.23-3.21
ppm (6H, m), 2.93-2.90 (6H, m), 2.30-2.22 (12H, m), 1.61-1.58 (6H, m), 1.50-1.46 (6H, m),
1.31 (27H, s). MS (ESI-MS): m/z calcd for (:591‘189l\l909[l\/H‘I]+ 1068.68, found 1068.9.
Method of preparative HPLC:
(A) 10mM NH4HC03 in water (B) MeCN:MeOH:IPA :10), using WATERS
X-BRIDGE C18 250mm*19 mm, 5.0 uM with the flow rate of 15.0 mL/min and with the
ing gradient:
"E!-
—m_1000_
N2,N7,N15-tris(3-((3-aminopropy1)(methy1)amin0)pr0py1)—9,10-dihydr0-9,10-
[1,2]benzenoanthracene-2,7,15-tricarboxamide, ARK-7.
To a solution of N2,N7,N15-tris(3-((3-tert-
butylcarbonylaminopropy1)(methy1)amino)propyl)-9, 10-dihydro-9, 10-[1,2]benzenoanthracene-
-tricarboxamide (6) (0.2 g) in 1,4-dioxane (5 mL) was added 4 M HCl in dioxane (1 mL)
at room temperature and the resulting reaction mixture was stirred for 2 hours. The mixture was
concentrated under d pressure to get pure hydrochloride salt of ARK-7 (0.072 g, 50.3%)
as a light yellow solid. 1H NMR (400 MHz, D20) 6 7.70 ppm (3H, s), 7.42-7.40 ppm (3H, d,
J:7.6 Hz), 7.34-7.32 ppm (3H, d, J: 8 Hz), 5.73 ppm (1H, s), 5.71 (1H, s), 3.34-3.30 ppm (6H,
t), 3.23-3.03 ppm (12H, m), 2.97-2.93 ppm (6H, t), 2.76 ppm (9H, s), 2.06-1.92 ppm (12H, m),
MS (ESI—MS): m/zcalcd for C44r165N903 [MH]+768.52, found 768.7. HPLC retention time: 4.277
Example 7: Synthesis of ARK-8 00014)
ARK-8 (Ark0000014)
ARK—8 was synthesized following the method for ARK—7 above to provide
intermediate 5. This was then coupled with ediate 2a below and converted to ARK-8 as
described below.
Tert—butyl (7-aminoheptyl)carbamate, 221.
] To a solution of heptane-1,7—diamine (5 g, 38.46mmol) in THF (10 mL) at 0 °C was
added Boc anhydride (1.68 g, 7.69 mmol) dropwise over a period of 20 min and the resulting
reaction mixture was stirred at room temperature for 16 h. THF was removed under reduced
pressure and the resulting e was poured into water (50 mL). The aqueous mixture was
extracted with ethyl acetate (3 x 25mL). The organic layers were combined, washed with water,
dried using anhydrous NazSO4 and concentrated under reduced pressure to get pure 2a (1 g, 11.3
%) as a colourless oil. 1H NMR (400 MHz, CDC13) 6 6.80-6.77 (1H, t, J=5.2 Hz), 2.91-2.85 (2H,
dd, , 6.8 Hz) .44ppm (2H, m), 1.36 ppm (11H, s), 1.31ppm (4H, s), 1.23 (6H, s),
MS (ESI—MS): m/z calcd for C12H26N202 [l\/1H]Jr 231.20, found 231.5.
N2,N7,N15-tris(7-tert—butylcarbonylaminoheptyl)—9,10-dihydr0-9,10-
[1,2]benzenoanthracene—2,7,15-tricarb0xamide, 6.
To a solution of Tert-butyl (7-aminoheptyl)carbamate (2a) (0.51 g, 2.24 mmol) in
DMF (3 mL) was added ihydro-9,10-[1,2]benzenoanthracene-2,7,15-tricarboxylic acid
(0.27 g, 0.70 mmol), HATU (0.85, 2.24 mmol), DIPEA (0.77 mL, 4.47 mmol) and the resulting
reaction mixture was stirred at room temperature for 2 h. The reaction mixture was poured into
water (50 mL) and extracted with romethane (3 x 25mL). The organic layers were
combined, washed with brine, dried using anhydrous Na2804 and concentrated under reduced
pressure to get crude 6 as a brown semisolid. The crude mixture was purified by flash column
chromatography on silica gel (0.5% MeOH/chloroform) to afford pure product 6 (0.65 g, 91.5%)
as light yellow solid. 1H NMR (400 MHz, DMSO) 5 8.34-8.32 (3H, d, J=8.8 Hz), 7.93 (3H, s)
7.53 ppm (6H, s), 6.75 ppm (3H,broad s), 5.87 ppm (1H, s), 5.76 ppm (1H, s), 3.20-3.14 (6H, d,
J= 24 Hz), 2.29 (6H, s), 1.3? (27H, 5), 1.25—1.24 (30H, m),MS (ESI—MS): m/z calcd for
C59H86N609 [MH]+1023.65, found 1045.5(M+23).
N2,N7,N15-tris(7-aminoheptyl)-9,lO-dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-
tricarboxamide, ARK-8.
To a solution of N2,N7,N15-tris(7-tert-butylcarbonylaminoheptyl)-9,10-dihydro-9,10-
[1,2]benzenoanthracene—2,7,15-tricarboxamide (6) (0.7 g) in oxane (5 mL) was added 4 M
HCl in dioxane (3 mL) at room temperature and the ing reaction mixture was stirred for 2
hours. The mixture was concentrated under reduced pressure to get crude hydrochloride salt of
ARK-8 as a yellow solid. The crude mixture was purified by ative HPLC using ing
method to afford pure ARK-8_HCl salt (0.2 g, 40.5%)as a white solid. 1H NMR (400 MHz,
D20) 6 7.62 ppm (3H, broad s), 7.13ppm (3H,broad s), 7.01 ppm (3H, broad s), 5.53ppm (1H,
S), 5.2 (1H, s), 2.92ppm (6H, broad s), m (6H, broad s), 1.22ppm (6H, broad s), m
(6H, broad s), 0.76ppm (6H, broad s), MS (ESI—MS): m/zcalcd for C44H62N603 [MH]+724.0,
found 723.6. HPLC retention time: 4.947 min.
Method of preparative HPLC:
(A) 0.05% HCl in water (B) MeCN:MeOH:IPA (65:25:10) (HPLC GR), using X
SELECT FLUORO PHENYL COLUlVlN 250*19 mm, 5.0 nM with the flow rate of 22.0
mL/rnin and with the following gradient:
——m_
——m_
Example 8: Synthesis of ARK-9 (Ark000015), ARK-10 (Ark000016), ARK-11 (Ark000017),
and ARK-12 (Ark000018)
ARK-9
m} _‘
g ‘ H:
‘~ N: 3‘ "6‘. /
_ ,w“
”\,xWW~ v
N \r I M
"3‘ ‘3
s :3
{3 5‘ g:
mg Egg/IxM .
“I figs“? 3‘ N3?“ .. .
**‘ W‘
r 1» .‘
”r N‘vwf
‘\?\at ("a “Nix“ “SJ““FNN’: 5> :3
s : Kama?“ \§
$5 ’ 2" K NR
m,' «544" "in ‘3
(3 IN
as (\v \
NH}?
ARK-10
ARK-12
ARK-9 was prepared ously to ARK-7 above h compound 2. Compound
2 was then coupled with Boc—L-Lys(Boc)-OH as described below and then deprotected to
provide ARK-9 (ARK-10 (ArkOOOOl6) was provided analogously by substituting Boc-D-
Lys(Boc)—OH). In a similar way, ARK-11 (Ark000017) and ARK-12 (Ark000018) were
provided by coupling with protected L or D-His amino acids.
Hexa-tert-butyl ((5S,5'S,5"S)—((9,10-dihydro-9,10-[1,2]benzenoanthracene—2,7,15
triyl)tris(azanediyl))tris(6-0xohexane—6,1,5-triyl))hexacarbamate, 3.
] To a solution of 9,10-dihydro-9,10—[l,2]benzenoanthracene—2,7,15-triamine (2) (0.1
g, 0.3344 mmol) in DMF (1 mL) were added Boc-L-Lys(Boc)-OH (0.37g, 1.07 mmol), HATU
(0.406, 1.07 mmol) and DIPEA (0.258g, 2.006 mmol) at room temperature. The reaction mixture
was stirred at room temperature for 60 min. The resulting on mixture was poured into ice-
cold water. The obtained solid precipitate was collected by ion and dried under reduced
pressure to afford crude 3 (0.38 g, 88.57%) as a white solid which was used without further
ation. MS (ESI—MS): m/z calcd for C68H101N9015 [MH]+ 4, found 1185.0 (M-100).
(2S,2'S,2"S)—N,N',N"-(9,10—dihydr0-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(2,6-diamin0hexanamide), ARK-9.
The crude product hexa-tert-butyl ((5S,5'S,5"S)—((9,10—dihydro—9,10-
[1,2]benzenoanthracene-2,7,15 triyl)tris(azanediyl))tris(6-oxohexane-6,1,5-triyl))hexacarbamate
(3) (0.3 g, 0.234 mmol) obtained from previous step was suspended in 4 M HCl in dioxane and
stirred at room temperature for 2 h. The resulting on mixture was concentrated under
reduced pressure to afford crude ARK-9 hydrochloride salt as a white solid. The crude product
was purified by preparative HPLC using the method shown below to afford pure salt of ARK-9
(0.19 g, 46.91%) as a White solid. The pure salt of ARK—9 was dissolved in DM water (4 mL)
and passed through Amberlite® [RA-400 -OH form ion ge resin. The free base was eluted
using DM water and the fractions collected were lyophilized to obtain free base (0.15 g) as a
white solid. The free base (0.05 g) was treated with aqueous 1 N HCl (3 mL) and lyophilized the
material to generate hydrochloride salt of ARK-9 (0.05 g, 83.33%) as a white solid. 1H NMR
(400 MHz, D20) 5 7.56-7.55 ppm (3H, d, J=1.6 Hz), .39 ppm (3H, d, J=8.0 Hz), 7.01-6.99
ppm (H, dd, J=8 Hz, J=1.6 Hz), 5.62 ppm (1H, S), 5.59 ppm (1H, s), 4.01—3.98 ppm (3H, t),
2.88-2.84 ppm (6H, t), 1.90-1.86 ppm (6H, m), 1.61-1.57 ppm (6H, 3), 1.40-1.36 ppm (6H, m),
MS (ESI—MS): m/z calcd for C22H27N502 [MH]+684.4, found 684.7. HPLC retention time: 5.092
Method of preparative HPLC:
(A) 0.1% TFA in water and (B) MeCN:MeOH:IPA (65:25:10) (HPLC grade), using
X SELECT FLUORO PHENYL COLUMN 250 X 19 mm, 5.0 um with the flow rate of 12.0
mL/min and with the following gradient:
——E_
"El.
—m_m-
—m_m-
Synthesis of ARK-10 (Ark000016):
] Hexa-tert-butyl ((5R,5'R,5"R)—((9,10-dihydro—9,10-[1,2]benzenoanthracene—
2,7,15-triyl)tris(azanediyl))tris(6-0xohexane—6,1,5-triyl))hexacarbamate, 3.
To a solution of 9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15-triamine (2) (0.3
g, 1.00 mmol) in DMF (5 mL) was added Boc—D—Lys(Boc)—OH (1.1 g, 3.210 mmol), HATU (1.2
g, 3.210 mmol) and DIPEA (0.774 g, 6.00 mmol) at room temperature. The reaction mixture was
stirred at room temperature for 60 min. The resulting reaction mixture was poured into ice-cold
water. The obtained solid precipitates were collected by filtration and dried under reduced
pressure to afford crude 3. The crude mixture was purified by preparative HPLC using following
method to afford pure 3 (0.25 g, 19.53%) as a white solid. MS (ESI—MS): m/z calcd for
C68H101N9015 [MH]+ 1283.74, found 1185.0 (M-lOO; tection of one Boc group).
] Method of preparative HPLC:
(A) 10 mM um bicarbonate in water (HPLC grade) and (B) ACN: MeOH:
IPA (65:25:10) (HPLC GR), using X BRIDGE 30mm*5um with a flow rate of 28.0
mL/min and with the following gradient:
__E_
"El.
(2R,2'R,2"R)—N,N',N"-(9,10-dihydro-9,10-[1,2]benzenoanthracene—2,7,15-
tris(2,6-diamin0hexanamide), ARK-10.
] The crude product hexa-tert-butyl ((5R,5'R,5"R)—((9,10-dihydro-9,10-
[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(6—oxohexane-6, 1,5-triyl))hexacarbamate
(3) (0.25g, 0.1947mmol) obtained from previous step was suspended in 4 M HCl in dioxane and
stirred at room temperature for 2 hours. The resulting on mixture was concentrated under
reduced re to afford crude ARK—10 hydrochloride salt as a white solid. The crude product
was purified by preparative HPLC using the method shown below to afford pure salt of ARK-10
(0.14 g, 26.41%) as a white solid. The pure salt of ARK-10 was dissolved in DM water (4 mL)
and passed through Amberlite® IRA-400 -OH form ion exchange resin. The free base was eluted
using DM water and the fractions collected were lized to obtain free base (0.07g) as a
white solid. The free base (0.07 g) was treated with aqueous 1 N HCl (3 mL) and lyophilized to
generate hydrochloride salt of ARK-10 (0.085g, 92.39%) as a light brown solid. 1H NMR (400
MHz, D20) 6 754—753 ppm (3H, d, J=2 Hz), 7.38-736 ppm (3H, d, J=8.0 Hz), 6.99-6.97 ppm
(3H, dd, J=8 Hz, J=2 Hz), 5.60 ppm (1H, S), 5.56 (1H, s), 3.99-3.96 ppm (3H, t), 2.86-2.82 ppm
(6H, t), 1.89-1.82 ppm (6H, m), 1.61-1.53 ppm (6H, m), 1.40-1.34 ppm (6H, m). MS S):
m/z calcd for C22H27N502 [MH]+ 684.4, found 684.6. HPLC retention time: 6.393 min.
Method of preparative HPLC:
(A) 0.1% TFA in water (HPLC grade) and (B) MeCN: MeOH: [PA (65:25: 10)
(HPLC GR), using X SELECT PFP C18,250*19 mm, Sum with the flow rate of 15.0mL/min and
with the following gradient:
__I!_
——m_
__E_
__E_
——m_
Synthesis of ARK-11 and ARK-12.
] Tri—tert—butyl ((2S,2'S,2"S)—((9,10—dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-
triyl) tris (azanediyl) )tris (3-(1H-imidazolyl)0xopropane—1,2-diyl))tricarbamate.
To a stirred solution of 9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15—triamine
(2) (0.3 g, 1.0 mmol) in DMF (6 mL) was added Boc—L-Histidine (0.82g, 3.2 mmol), HATU
(1.22g, 3.2 mmol), and DIPEA (0.8g, 62 mmol) at room temperature. The resulting on
mixture was d overnight at room ature. The reaction mixture was poured in ice-cold
water and residues obtained were collected through filtration, dried under reduced pressure to get
crude 3 (0.65g, 65%) as light brown solid which was directly used in the next step without
ation. MS (ESI—MS): m/z calcd for (:531‘1621\I1209[lVH‘I]+ 1011.15, found 1011.9.
(2S,2'S,2"S)—N,N',N"-(9,10-dihydr0-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(2-amin0(1H-imidaz01yl)pr0panamide) hloride, _HCl Salt.
To a stirred solution of tri—tert-butyl ((2S,2'S,2"S)—((9,lO—dihydro-9,10-
[1,2]benzenoanthracene-2,7,15-triyl) tris (azanediyl))tris (3-(1H-imidazolyl)—l-oxopropane-
1,2-diyl))tricarbamate (3) (0.65g, 0.643 mmol) in dichloromethane (8 mL) was added 4N HCl in
Dioxane (5 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 3h.
The reaction mixture was concentrated under reduced pressure to get crude ARK—11. The crude
mixture was purified by preparative HPLC using following method to afford pure product ARK-
11_TFA salt , 64.42%) as colorless viscous oil. The ARK—11_TFA salt was dissolved in
methanol (10 mL). To this, r bound tetraalkylammonium carbonate and the resulting
mixture was stirred at room temperature for 30 min. The mixture was filtered through celite and
the resulting filtrate was concentrated under reduced pressure to get ARK-11_Free base. The free
base was dissolved in 0.01 N HCl (10 mL) and resulting solution was lyophilized to obtain pure
ARK-11_HCl salt (0.16g, 61.06%) as white solid. 1H NMR (400 MHZ, D20) 5 8.56 ppm (3H,
s), 7.51 ppm (3H, s), .31 ppm (6H, m), 6.93-6.91 ppm (3H, s), 561-558 ppm (2H, s), 4.26
ppm (3H, s), 3.36-3.34 ppm (6H, m), 3.21 ppm (2H, s), MS S): m/z calcd for
C38H33N1203 [MI-I]+ 7108, found 7122. HPLC retention time: 5770 min.
Method for preparative HPLC:
] (A) 0.1% TFA in water (HPLC grade) and (B) 10% IPA in acetonitrile (HPLC grade),
using WATERS X-BRIDGE C18, 250mm*30mm*5um with the flow rate of 35.0mL/min and
with the following gradient:
_Efl__10.0
] Tri-tert—butyl ((2R,2'R,2"R)—((9,10-dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-
triyl) tris (azanediyl)) tris (3-(1H-imidazolyl)—1-0xopropane—1,2-diyl))tricarbamate.
To a stirred solution of 9,lO-dihydro-9,10—[1,2]benzenoanthracene—2,7,15-triamine
(2) (0.25 g, 0.84 mmol) in DMF (6 mL) was added Boc-D-Histidine (0.68g, 2.67 mmol), HATU
(1.01 g, 2.67 mmol), DTPEA (0.69g, 5.35 mmol) at room ature. The resulting reaction
mixture was stirred over night at room temperature. The reaction mixture was poured in ice-cold
water and residues obtained were collected through filtration, dried under reduced pressure to get
crude 3 (0.75g, 88.9%) as white solid which was ly used in the next step without
ation. MS (ESI-MS): m/z calcd for C53H62N1209[1V1H]+ 1011.48, found 1011.6.
(2R,2'R,2"R)-N,N',N"-(9,10-dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-
triyl)tris(2-amino(1H-imidazoly1)propanamide) hydrochloride, ARK-12_HC1 Salt.
To a stirred solution of tri-tert-butyl ((28,2‘S,2"S)-((9,10-dihydro-9,10-
[1,2]benzenoanthracene—2,7,15-triyl) tris (azanediy1))tris (3-(1H—imidazol-4—y1)—1-oxopropane-
1,2-diyl))tricarbamate (3) (0.75 g, 0.742 mmol) in dichloromethane (8 mL) was added 4N HCl in
Dioxane (5 mL) at 0 °C. The resulting reaction mixture was stirred at room temperature for 3 h.
The reaction e was concentrated under d pressure to get crude . The crude
mixture was purified by preparative HPLC using following method to afford pure product ARK-
12_TFA salt (0.70 g, 72.53%) as white solid. The pure salt of ARK-12 was dissolved in DM
water (4 mL) and passed h Amberlite® IRA-400 -OH form ion exchange resin. The free
base was eluted using DM water and the fractions collected were lyophilized to get free base
(0.06 g) as a white solid. The free base (0.06 g) was dissolved in aqueous 1 N HCl solution (3
mL) and lyophilized the material to generate hydrochloride salt of ARK-12 (0.07 g, 10.16%) as
a white solid. 1H NMR (400 MHZ, D20) 5 8.54 ppm (3H, s), 7.50 ppm (3H, s), 7.37-7.35 ppm
(3H, d, J=8 Hz), 7.28 ppm (3H, S), 6.90-6.88 ppm (3H, dd, J=7.6 Hz), 5.59 ppm (1H, s), 5.56
ppm (1H, s), 4.25—4.22 ppm (3H, t, J=7.2 Hz ), 3.33—3.31 ppm (6H, d, J:7.2 Hz). MS (ESI—MS):
m/zcalcd for C38H38N1203 [MH]+711.32, found 684.6. HPLC retention time: 6.347 min.
Method for preparative HPLC:
0.1% TFA in water (HPLC grade) and (B) 10% IPA in acetonitrile (HPLC ,
using WATERS X-BRIDGE C18, 250mm*30mm*5ttm with the flow rate of 35.0mL/min and
with the following gradient:
Example 9: Synthesis of ARK-77 and ARK-77A (Ark000033 and Ark000034)
Scheme: Synthesis of Int-13
@1102
$00 BOC NosyhN NosykN
N NH HATU DMF
\/\ /\/ 1 l l
/ ‘
° \ _. ”Wowfl ”_. ”AWN “3
ARK-20 0
Step _10 Step .11 TFA salt 0
MW:232.17 <10) (11)
MW: 555.21
MW: 455.16
BocHNMg 0
BocHNM
WNf" (12)
N (\3 MW: 1531.81
ARK-18 .
MW: 1094.66 ”°5Y'
enol,
Step -13 K2003,ACN
60°C
meo\ (13)
Tert—butyl(2-(2-((ZS,4S)azid0-N-methyl((2-
henyl)sulfonyl)pyrrolidinecarboxamido) ethoxy)ethyl)(methyl)carbamate, 10.
To a solution of ARK-20 (2.0 g, 8.614 mmol) in N,N—dimethylformamide (40 mL)
were sequentially added (28,4S)azido((2-nitrophenyl)sulfonyl)pyrrolidinecarboxylic
acid (2.34 g, 6.89 mmol), HATU (2.62 g, 6.89 mmol) and N,N—diisopropylethylamine (3.33 g,
.84 mmol) at room temperature. The ing reaction mixture was stirred for l h at room
temperature. The reaction mixture was poured in ice-cold water and extracted with ethyl acetate
(3 >< 100 mL). The organic layers were combined, washed with brine and trated under
reduced pressure to get crude 10 (3.5 g, 91.6 %) as brown semisolid. The crude mixture was used
in next step without further purification. MS (ESI—MS): m/z calcd for C22H33N7OgS [MH]+
, found 573.43(M+18, water adduct).
(2S,4S)—4-azido-N-methyl-N-(Z-(Z-(methylamino)ethoxy)ethyl)—1-((2-
nitrophenyl)sulf0nyl) pyrrolidine—2-carb0xamide_TFA Salt, 11.
To a solution of tert—butyl (2-(2-((2S,4S)—4-azido—N—methyl((2—
nitrophenyl)sulfony1)pyrrolidine—2-carboxamido) ethoxy)ethy1)(methy1)carbamate (10) (3.5 g,
6.30 mmol) in dichloro methane (30 mL) was added trifluoro acetic acid (3.15 mL, 31.52 mmol)
at room temperature. The resulted reaction mixture was stirred at room temperature for 2 h. The
reaction mixture was filtered through celite bed and filtrate thus collected was concentrated
under d re to get crude 11 (4.3 g, quantitative yield) as a brown oil which was used
in next step without further purification. MS S): m/z calcd for C17H25N706STFA [MH]+
456.16, found 45632,
Tri—tert—butyl (((9-(3-((2-(2-((2S,4S)—4-azido—N-methyl((2-
nitrophenyl)sulf0nyl)pyrrolidine—2-carboxamid0)eth0xy)ethyl)(methyl)amin0)—3-
0x0propyl)—9,10-dihydr0-9,10[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(8-
0x00ctane—8,1-diyl))tricarbamate, 12.
To a solution of (2S,4S)—4-azido-N-methyl-N-(2-(2-(methylamino)ethoxy)ethy1)—1-
((2-nitrophenyl)sulfonyl) pyrrolidinecarboxamide_TFA Salt (11) (1.25 g, 2.19 mmol) in N,N—
dimethylformamide (30 mL) were sequentially added 3-(2,7,15-tris(8-((tertbutoxycarbonyl
)amino)octanamido)—9,10—[1,2]benzenoanthracen-9(1OH)—y1)propanoic acid
(ARK-18) (2.0 g, 1.83 mmol), HATU (0.833 g, 2.192 mmol) and N,N—diisopropylethylamine
(0.942 g, 7.31 mmol) at room temperature. The resulting reaction mixture was stirred for 1 h at
room temperature. The reaction mixture was poured in ice-cold water and extracted with ethyl
acetate (3 X 100 mL). The c layers were ed, washed with brine and trated
under reduced pressure to get crude 12. The crude mixture was purified by column
chromatography on silica gel (3.2% methanol/chloroform) to yield 12 (2.3 g, 82.17 %) as a dark
yellow solid. MS S): m/z calcd for C79H113N13016S [MH]+ 1532.81, found 1433.19 (M-
100, one Boc group fell off).
rt—butyl (((9-(3-((2-(2-((2S,4S)—4—azid0-N-methylpyrrolidine—Z-
carboxamido)eth0xy)ethyl) (methyl)amino)—3-ox0pr0pyl)—9,10-dihydro-
9,10[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl)) tris(8-0x00ctane—8,1-
diyl))tricarbamate, 13.
To a solution of tri-tert-butyl (((9-(3-((2-(2—((2S,4S)—4-azido—N—methyl-l-((2-
nitrophenyl)sulfonyl) pyrrolidine—2-carboxamido)ethoxy)ethyl)(methyl)amino)oxopropyl)-
9,10-dihydro-9,10[l,2] benzenoanthracene-2,7, 1 5-triyl)tris(azanediyl))tris(8-oxooctane-8, 1 -
diyl))tricarbamate (12) (2.2 g, 1.44 mmol) in acetonitrile (30 mL) were sequentially added
potassium carbonate (0.99 g, 7.18 mmol) and thiophenol (0.44 mL, 4.31 mmol) at room
temperature. The resulted reaction e was stirred at 80 0C for 2 h. The reaction mixture was
d through celite bed and the collected filtrate was concentrated under reduced pressure to
get crude 13 as yellow oil. The crude mixture was subjected to reverse phase chromatography to
yield 13 (1.1 g, 56.88%) as a light yellow solid. The yellow solid was further subjected to
preparative HPLC d mentioned below) purification ed by lyophilization to yield
pure 13 (0.41g, 52.17%) as a white amorphous powder. MS (ESI—MS): m/z calcd for
C73H110N12012 [MH]+ 1347.84, found 8.
Method for preparative HPLC:
(A) 10 mM NH4HC03 IN WATER (HPLC GRADE) and (B) 100% Acetonitrile
(HPLC GRADE) in water (HPLC GRADE), using X-BRIDGE C18, 250mm*30mm*5um with
the ing flow rate and gradient:
rate
—__m_
———m_
Scheme: Synthesis of ARK-77
0 BOCHNM w
HO i I 6
o T BOCHNW/jerHg QNEx/MNHBOC
Warhead-1B N\
HATU DIPEA DMF \NfOH
—» TH)
Step -14 i1 0
(13) 0‘,
MW: 1346.86 0
(14)
0 MW: 1549.86
HCI in dioxane
Step -15
ARK-77_HCI salt
MW: 1249.70
Tri—tert—butyl (((9-(3-((2-(2-((2S,4S)—4-azido-N-methyl(1-methyl-2,4-dioxo-1,4-
dihydro-ZH-benzo [d] [1,3]0xazine—7-carbonyl)pyrrolidine—2-
amid0)eth0xy)ethyl)(methyl)amino)—3-oxopropyl)—9,10-dihydro-9,10-
[1,2]benzenoanthracene—2,7,l5-triyl)tris(azanediyl))tris(8-0x00ctane—8,1-diyl))tricarbamate,
To a solution of tri-tert-butyl (((9-(3—((2-(2-((ZS,4S)—4—azido—N—methylpyrrolidine-Z-
amido)ethoxy)ethyl) (methyl)amino)-3 -oxopropyl)—9,10-dihydro-
9,10[1,2]benzenoanthracene-2,7,15-t1iyl)tris(azanediyl)) tris(8-oxooctane—8,1-diyl))tricarbamate
(13) (0.2 g, 0.148 mmol) in N,N—dimethylformamide (8 mL) were sequentially added l-methyl-
2,4-dioxo—2,4-dihydro-1H—3,1-benzoxazine—7-carboxylic acid ad_type_1B) (0.039 g,
0.178 mmol) and HATU (0.068 g, 0.178 mmol) at room temperature. The reaction mixture was
stirred for 5 minutes. To this, N,N—diisopropylethylamine (0.038 g, 0.297 mmol) was added
dropwise and the resulted reaction mixture was further stirred for 30 minutes at room
temperature. The reaction mixture was diluted by ethyl acetate (100 mL) and washed with ice-
cold water (3 X 30mL). The organic layers were combined, washed with brine and concentrated
under reduced pressure at 25 °C to get crude 14. The crude mixture was purified by preparative
HPLC d mentioned below) followed by lization to yield 14 (0.12g, 52.17%) as a
white amorphous powder. MS (ESI—MS): m/z calcd C83H115N13016 [MH]+ 1550.86, found
2 (M-100, one Boc group fell off).
Method for ative HPLC:
(A) 100% Acetonitrile (HPLC GRADE) and (B) 100% Tetrahydrofuran (HPLC
GRADE), using SUNFIRE SILICA, 150mm*19mm*5um with the flow rate of 19.0mL/min and
with the following gradient:
N,N',N"-(9-(3-((2-(2—((2S,4S)azido-N-methyl(1-methyl-2,4-di0xo-1,4-
dihydro-2H-benz0 [d] [1,3]oxazinecarb0nyl)pyrrolidinc
carboxamido)eth0xy)ethyl)(methyl)amino)—3-0xopropyl)—9,10-dihydr0-9,10-
[1,2]benzenoanthracene-2,7,15—triyl)tris(8-amin00ctanamide), _HCl salt.
To a solution of tri—tert-butyl (((9-(3—((2-(2-((2S,4S)—4-azido—N—methyl-l-(1-methyl-
2,4-dioxo- l ,4-dihydro-2H-benzo[d] [ l ,3]oxazine—7-carbonyl)pyrrolidine
carboxamido)ethoxy)ethyl)(methyl)amino)—3 -oxopropyl)—9, 10-dihydro-9, 10-
[1,2]benzenoanthracene-2,7,15-triyl)tris(azanediyl))tris(8-oxooctane-8,1-diyl))tricarbamate (14)
(0.079 g, 0.051 mmol) in 1,4-dioxane (3.0 mL) was added 4 M HCl in dioxane solution (1.5 mL)
at room ature and the ing reaction mixture was stirred for 30 minutes under nitrogen
atmosphere. During this, solid residue started to precipitate out. The suspension was further
stirred for 30 minutes and finally allowed to stand at room temperature. The solid residues
d to t on bottom of the flask. The solvent was decanted and the es left were
triturated with acetonitrile (3 X 3 mL). Finally the solid was dried under reduced pressure at 25
°C to get pure ARK-77_HCl_Salt (0.054g, 69.28%) as a white amorphous powder. 1H NMR
(400 MHz, DMSO-d6) 8 9.91 ppm (3H, broad), 8.09-8.03 ppm (1H, m), 7.90 ppm (8H, broad),
7.67 ppm (3H, broad), 7.37-7.33 ppm (2H, m), 7.29-7.27 ppm (3H, m), 7.23 ppm (3H, m), 5.38
ppm (1H, s), 5.01 ppm (1H, m), 4.86-4.79 ppm (1H, m), 4.31-4.23 ppm (1H, m), 4.09 ppm (1H,
m), 3.79-3.64 ppm (4H, m), 3.48 ppm (14H, m), 3.44-3.40 ppm (4H, m), 3.18 ppm (1H, s), 3.08-
3.01 ppm (6H, m), 2.77-2.66 ppm (7H, m), 2.25 ppm (6H, broad s), 1.53 ppm (12H, broad s),
1.27 ppm (18H, broad 3). MS (ESI—MS): m/z calcd for C68H91N13010 [MH]+ 0, found
125 1 .48.
Scheme: Synthesis of A
BocHNM/W O
O NHO
HO N’ko WNHg/SQ0 NH
BocHNW BOCHN
NHBoc H B NHLK/MNHBOC
Warhead_1A °——
HATU, DIPEA, DMF \NfoH
\(H Step -14 o
“95H N:
” °
(13) o
MW: 1346.86 0
(14)
MW: 1535.84
HCI in dioxane
Step -15
HzNWNHv
HzNWNHuNfliA/WNHz
MN”H
H N\
o? \\/
ARK-77A_HCI salt
MW: 1235.69
Tri-tert—butyl (((9-(3-((2-(2-((2S,4S)—4-azido(2,4-dioxo-1,4-dihydro-2H-
benzo [d] [1,3]0xazine—7—carb0nyl)—N-methylpyrrolidine—Z-
carboxamido)ethoxy)ethyl)(methyl)amino)—3-oxopropyl)—9,1 0-dihydro-9,10-
[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(8-0x00ctane—8,1-diyl))tricarbamate,
To a solution of tri—tert-butyl (((9-(3-((2-(2—((2S,4S)azido—N—methylpyrrolidine-2—
carboxamido)ethoxy)ethyl) (methyl)amino)-3 -oxopropyl)—9,10-dihydro-
9,10[1,2]benzenoanthracene-2,7,15-triy1)tris(azanediyl)) -oxooctane-8,1-diyl))tricarbamate
(13) (0.156 g, 0.116 mmol) in N,N—dimethylformamide (6 mL) were sequentially added 2,4-
dioxo—l,4-dihydro-2H—benzo[d][1,3]oxazinecarboxylic acid (Warhead_type_1A) (0.029 g,
0.139 mmol) and HATU (0.053 g, 0.139 mmol) at room temperature. The reaction mixture was
stirred for 5 minutes. To this, N,N—diisopropylethylamine (0.03 g, 0.232 mmol) was added drop
wise and the resulted reaction mixture was further stirred for 30 minutes at room temperature.
The reaction mixture was diluted by ethyl acetate (100 mL) and washed with ld water (3 X
30mL). The organic layers were combined, washed with brine and concentrated under reduced
re at 25 0C to get crude 14. The crude mixture was d by preparative HPLC using
following method to yield pure 14 (0.093 g, 52.17 %) as a white ous powder. The prep-
fraction was concentrated by reduced pressure at 25 °C under nitrogen atmosphere. MS (ESI—
MS): m/z calcd C82H113N13016[1V1H]+ 4, found 1 (M—100, one Boc group fell off).
Method for preparative HPLC:
(A) 100% Acetonitrile (HPLC GRADE) and (B) 100% Tetrahydrofuran (HPLC
GRADE), using SUNFIRE SILICA, 150mm*19mm*5um with the following flow rate and
following gradient:
rate
———m-
———m_
———m_
———m_
___m_
N,N',N"-(9-(3-((2-(2—((2S,4S)—4-azido(2,4—di0x0-1,4-dihydro-2H-
benzo [d] [1,3]0xazine—7—carbonyl)—N-methylpyrrolidine—Z-
carboxamid0)eth0xy)ethyl)(methyl)amino)—3-0xopropyl)—9,10-dihydro-9,10-
[1,2]benzenoanthracene—2,7,15-triyl)tris(8-amin00ctanamide), ARK-77A_HCI salt.
To a solution of tri-tert—butyl (((9-(3-((2-(2-((2$,4S)—4—azido-1—(2,4-dioxo-1,4-
dihydro—2H-benzo[d][1,3]oxazinecarbonyl)—N—methylpyrrolidine
carboxamido)ethoxy)ethyl)(methyl)amino)—3 -oxopropyl)—9,10-dihydro-9,10-
[l,2]benzenoanthracene—2,7, l 5-triyl)tris(azanediyl))tris(8-oxooctane-8, l -diyl))tricarbamate (14)
(0.06 g, 0.039 mmol) in 1,4-Dioxane (Dry) (3 ml) was added 4 M HCl in dioxane (1.2 mL) at
room temperature and the resulting reaction e was stirred for 30 minutes under en
atmosphere. The solid al was stable at the bottom of the flask and the solvent was decanted
under inert atmosphere, then the solid material was triturating with acetonitrile (HPLC Grade) (3
X 3 mL). The remaining solid was concentrated by reduced pressure at 25 °C under en
atmosphere to afford pure ARK-77A_HCl_Salt (0.054g, 69.28%) as a white amorphous
powder. 1H NMR (400 MHz, DMso—dé) 5 12.04—11.95 ppm (1H, d), 9.91 ppm (3H, broad),
7.98-7.96 ppm (1H, m), 7.89 ppm (7H, broad), 7.71-7.67 ppm (3H, broad), 7.29—7.27 ppm (4H,
d), 7.23 ppm (3H, broad), 5.38 ppm (1H, s), 503-501 ppm (1H, m), 4.86-4.79 ppm (1H, m),
4.30-4.23 ppm (1H, m), 4.07 ppm (1H, m), 3.76 ppm (1H, m), 3.35-3.44 ppm (2H, m), 3.17 ppm
(1H, s), 3.08-3.04 ppm (5H, m), 2.99-2.84 ppm (1H, m), 2.79-2.68 ppm (7H, m), 2.25-2.23 ppm
(6H, t), 1.53 ppm (12H, broad), 1.27 ppm (18H, broad). MS (ESI-MS): m/z calcd for
C67H89N13010 [MH]+ 9, found 1238.46.
Example 10: sis of ARK-78 and ARK-78A (Ark000035 and Ark000037)
: Synthesis of Int-13
o:é:o 0
$06 Nosyh
N 0 ~ N
/ \/\O/\/ \/\NH \ Nosy|\
MF N N
o N TFA MDC ‘
DlPEA W,” \/\O/\/ 3
—>‘ \NH\/o\/\o/\/N N3
ARK-21 309 0
519.340 Step -11
MW:276.37 (10) (11)
MW: 599.18 MW: 499,18
Int-11
HATU, DMF
/ 6 DIPEA
O BocHN/VVW
BocHNWVYNHa Q WNHBOC —> O
0 NH step -12
ARK-18 WM
MW: 109456 Ng’CCosyl0 (12)
MW: 1575.84
J Thiophenol,
K2003” ACN
Step 13 60°C
NfCfi: (13)
MW: 1390.85
Tert—butyl (2-(2—(2-((ZS,4S)azido-N—methyl—1-((2-
nitrophenyl)sulfonyl)pyrrolidine-Z-carboxamid0)eth0xy)eth0xy)ethyl)(methyl)carbamate,
To a solution of ARK-21 (2.4 g, 8.68 mmol) in methylformamide (30 mL)
were sequentially added (ZS,4S)azido((2-nitrophenyl)sulfonyl)pyrrolidinecarboxylic
acid (2.96 g, 8.68 mmol), HATU (3.96 g, 10.42 mmol) and N,N—diisopropylethylamine (3.36 g,
26.05 mmol) at room temperature. The resulted reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was poured in ice-cold water and extracted with ethyl acetate
(3 X 100 mL). The organic layers were combined, washed with brine and concentrated under
d pressure to get crude 10 (4.0 g, 76.9 %) as yellow Viscous liquid. The crude e was
used in next step without further purification. MS S): m/z calcd for C24H37N7O9 S [MH]+
600.18, found 617.5 (M+18).
(2S,4S)—4-azido-N-methyl-N-(2-(2-(2-(methylamino)eth0xy)eth0xy)ethyl)—1-((2
nitrophenyl)sulfonyl)pyrrolidine-Z-carboxamide_TFA Salt, 11.
To a solution of tri—tert-butyl ((2R,2'R,2"R)—((9,lO-dihydro-9,10-
[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(3—(1H-imidazol-4—yl)—1-oxopropane-1,2-
diyl))tricarbamate (10) (4.0 g, 6.67 mmol) in dichloro methane (20 mL) was added trifluoro
acetic acid (2.58 mL, 33.38 mmol) at room temperature. The resulted reaction mixture was
stirred at room temperature for 2 h. The reaction mixture was filtered through celite bed and
filtrate thus collected was concentrated under reduced pressure to get crude 11 (7.5 g,
Quantitative yield) as a brown oil which was used in next step without further purification. MS
(ESI—MS): m/z calcd for C19H29N7O7 S [MH]+ 500.18, found 50031.
Tri—tert—butyl (((9-(1-((2S,4S)azid0((2-nitrophenyl)sulf0nyl)pyrr0lidin
yl)—2,1 l-dimethyl-l,12-di0x0-5,8-dioxa-2,1 l-diazatetradecan-l4-yl)—9,10-dihydr0-9,10—
[1,2]benzenoanthracene-2,7,15-triyl)tris(azanediyl))tris(8-0xooctane-8,1-diyl))tricarbamate,
To a on of (2S,4S)azido-N-methyl-N—(2-(2-(2-
(methylamino)ethoxy)ethoxy)ethyl)((2—nitrophenyl)sulfonyl)pyrrolidine—2-carboxamide_TFA
Salt (11) (2.69 g, 4.38 mmol) in methylformamide (40 mL) were sequentially added 3-
(2,7,15-tris(8-((tert-butoxycarbonyl)amino)octanamido)—9,10-[1,2]benzenoanthracen-9(10H)-
yl)propanoic acid 8) (4.0 g, 3.65 mmol), HATU (1.67 g, 4.38 mmol) and N,N—
diisopropylethylamine (1.41 g, 10.96 mmol) at room temperature. The ed reaction mixture
was stirred for 1 h at room temperature. The reaction mixture was poured in ice—cold water and
extracted with ethyl acetate (3 X 100 mL). The organic layers were combined, washed with brine
and concentrated under d pressure to get crude 12. The crude mixture was purified by
column tography on silica gel (4.3% methanol/chloroform) to yield 12 (4.7 g, 81.6 %) as
a dark yellow solid. MS (ESI—MS): m/z calcd for C81H117N13017S [MH]+ 1576.84, found 1578.4.
rt—butyl (((9-(1-((2S,4S)—4-azidopyrrolidin-Z-yl)—2,11-dimethyl-1,12-di0xo-
,8-dioxa-2,1l-diazatetradecanyl)-9,10-dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-
triyl)tris(azanediyl))tris(8-0xooctane-8,1-diyl))tricarbamate, 13.
To a solution of tri-tert-butyl (((9-(1—((ZS,4S)azido-l-((2-
nitrophenyl)su1fonyl)pyrrolidinyl)-2, 1 1-dimethyl-1,12-dioxo-5,8-dioxa—2,1 1-diazatetradecan-
-9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15—triyl)tris(azanediyl))tris(8-oxooctane-
8,1-diyl))tricarbamate (12) (4.7 g, 2.98 mmol) in acetonitrile (50 mL) were sequentially added
potassium carbonate (2.06 g, 14.91 mmol) and thiophenol (0.92 mL, 8.95 mmol) at room
temperature. The resulted reaction mixture was stirred at 80 0C for 2 h. The reaction e was
filtered h celite bed and the collected filtrate was concentrated under d pressure to
get crude 13 as yellow oil. The crude mixture was subjected to reverse phase chromatography to
yield 13 (1.9 g, 45.8%) as a light yellow solid. The yellow solid was r subjected to
preparative HPLC (method mentioned below) purification followed by lyophilization to yield
pure 13 (0.34 g, 8.2 %) as a white amorphous powder. MS (ESI-MS): m/z calcd for C53H62N1209
[MH]+ 1391.86, found 1392.3.
Method for preparative HPLC:
(A) 10mM NH4HC03 in water (HPLC grade) and (B) 100% acetonitrile (HPLC
grade) in water (HPLC grade), using X-BRIDGE C18, 250mm*30mm*5um with the flow rate of
.0 mL/min and with the following gradient:
——m_
Scheme: Synthesis of ARK-78
O BOCHN 0‘ G
HomeO NH/6
BOCHNW Qo NHBOC
0 NH
NHBoc
O l O:
Warhead_1 B ofo
HATU, DIPEA, DMF H
—> 0
Step -14 N 40
(13) 1 2 °
MW: 1390.86 0
(14)
MW: 1593.88
Step _15 J HCI in e
\N 1
O//k0 <0
ARK-78_HCI_SaIt
MW: 129373
Tri—tert—butyl (((9-(1-((ZS,4S)azido(1-methyl-2,4-di0xo-1,4-dihydro-2H-
benzo [d] [1,3]oxazine—7-carbonyl)pyrrolidin-2—yl)—2,1 l-dimethyl-l,12-di0x0-5,8-di0xa-2,1 1-
diazatetradecanyl)-9,10-dihydro-9,10-[1,2]benzenoanthracene—2,7,15-
triyl)tris(azanediyl))tris(8-0xooctane-8,1-diyl))tricarbamate, 14.
To a solution of tri—tert-butyl tri-tert-butyl 1-((2S,4S)azidopyrrolidin-Z-yl)—
2,1 l-dimethyl-l,12-dioxo-5,8-dioxa-2,l l-diazatetradecan—l4-yl)—9, l 0-dihydro-9, 10-
[l,2]benzenoanthracene—2,7,lS-triyl)tris(azanediyl)) tris(8-oxooctane-8,l-diyl))tricarbamate (13)
(0.14 g, 0.1 mmol) in N,N—dimethylformamide (5 mL) were sequentially added l-methyl-2,4-
2,4-dihydro-lH-3,l-benzoxazinecarboxylic acid (Warhead_type_1B) (0.027 g, 0.12
mmol) and HATU (0.046 g, 0.12 mmol) at room temperature. The reaction mixture was stirred
for 5 minutes. To this, N,N—diisopropylethylamine (0.026 g, 0.201 mmol) was added dropwise
and the resulted reaction mixture was further stirred for 30 minutes at room ature. The
reaction mixture was diluted by ethyl acetate (100 mL) and washed with ice-cold water (3 X 30
mL). The organic layers were combined, washed with brine and concentrated under reduced
pressure at 25 °C to get crude 14 (0.1 g, 62.5%) as a light yellow solid which was used in the
next step without further purification. MS (ESI-MS): m/z calcd 9N13017 [MH]+ 8,
found 1496.61 (M-100).
N,N',N"-(9-(1-((ZS,4S)azid0(1-methyl-2,4-diox0-1,4-dihydro-ZH-
benzo [d] [1,3]oxazine—7—carbonyl)pyrr01idin-2—yl)—2,1 l-dimethyl-l,12-di0x0-5,8-di0xa-2,1 1-
etradecanyl)-9,10-dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-triyl)tris(8-
aminooctanamide), ARK-78_HC1 salt.
To a solution of tri-tert-butyl (((9-(1-((2S,4S)azido(1-methyl-2,4-dioxo-1,4-
dihydro—2H-benzo[d][1,3]oxazinecarbonyl)pyrrolidin—2-yl)—2, l 1-dimethyl-1,12—dioxo-5, 8-
dioxa-2,1 1-diazatetradecanyl)-9,10—dihydro-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))t1is(8—oxooctane-8,1—diyl))tricarbamate (14) (0.067 g, 0.042 mmol) in 1,4-
dioxane (3.0 mL) was added 4 M HCl in dioxane solution (1.5 mL) at room temperature and the
resulted on mixture was stirred for 30 minutes under nitrogen atmosphere. During this,
solid residue started to precipitate out. The suspension was r d for 30 minutes and
finally d to stand at room temperature. The solid residues started to deposit on bottom of
the flask. The solvent was decanted and the residues left were ated with acetonitrile (3 X 3
mL). Finally the solid was dried under reduced pressure at 25 °C to get pure ARK-78_HCl_Salt
(0.045g, 76.3 %) as a white amorphous powder. 1H NMR (400 MHz, DMSO-d6) 5 9.91 ppm
(3H, broad s), 8.11-7.97 ppm (1H, m), 7.89 ppm (8H, broad s), 7.66 ppm (3H, broad s), 7.37-
7.34 ppm (2H, broad s), 7.29-7.22 ppm (6H, m), 5.39 ppm (1H, s), 4.97 ppm (1H, m), 4.82 ppm
(1H, m), 4.28 ppm (2H, m), 4.03 ppm (1H, m), 3.74 ppm (1H, m), 3.64 ppm (3H, broad s), 3.57
ppm (12H, broad s), 3.50-3.47 ppm (5H, m), 3.15-3.03 ppm (7H, m), 2.90-2.85 ppm (2H, (1),
275-272 ppm (7H, m), 2.25—2.23 ppm (6H, broad s), 1.54 ppm (12H, broad s), 1.27 ppm (17H,
broad s). MS (ESI—MS): m/z calcd for C70H95N13011 [MH]+1294.73, found 1295.41.
Scheme: Synthesis of ARK-78A
Warhead_1A
HATU, DIPEA, DMF
Step -14
(13)
MW: 1390.86
(14)
MW: 1579.88
Step -15 \ HCI in dioxane
HZNMA/ENHv
HZNWNHC/SQNEKNVVNHZ
o‘/‘O
Nfi/Oo
o//kO/§o
ARK-78A_HCI salt
MW: 1279.71
“Hm—bu I 9' 1' 25:45 '4-azid0 2,4-di0x0-1,4-dih dr0-2H-y
benzo [d] [1,3]oxazine—7-carbonyl)pyrr01idin-2—yl)—2,1 l-dimethyl-l,12-di0x0-5,8-di0xa-2,1 1-
diazatetradecanyl)-9,10-dihydro-9,10-[1,2]benzenoanthracene—2,7,15-
tris(azanediyl))tris(8-0x00ctane-8,1-diyl))tricarbamate, 14.
To a solution of tri—tert-butyl (((9-(1—((2S,4S)—4-azidopyrrolidin-Z-yl)—2,1l-dimethyl—
1,12-dioxo-5,8-dioxa-2,l l-diazatetradecan-l4-yl)—9,lO-dihydro-9,10-[1,2]benzenoanthracene-
-triyl)tris(azanediyl)) tris(8-oxooctane-8,l-diyl))tricarbamate (13) (0.075g, 0.05 mmol) in
N,N—dimethylformamide (4 mL) were sequentially added oxo—1,4-dihydro-2H-
benzo[d][1,3]oxazine-7—carboxylic acid (Warhead_type_1A) (0.013 g, 0.065 mmol) and
2017/040514
HATU (0.024 g, 0.065 mmol) at room temperature. The on mixture was stirred for 5
s. To this, N,N—diisopropylethylamine (0.014 g, 0.108 mmol) was added drop wise and
the resulted reaction mixture was further stirred for 30 minutes at room temperature. The
reaction mixture was diluted by ethyl acetate (100 mL) and washed with ice-cold water (3 X
30mL). The organic layers were combined, washed with brine and concentrated under reduced
pressure at 25 °C to get crude 14. The crude mixture was purified by preparative HPLC using
following method to yield pure 14 (0.04g, 52 %) as a white amorphous powder. The prep-
fraction was concentrated by reduced pressure at 25 °C under nitrogen atmosphere. MS (ESI-
MS): m/z calcd for C84H117N13017 [MH]+ 1580.88, found 1481.75(M-100).
] Method for preparative HPLC:
(A) 100% Acetonitrile (HPLC GRADE) and (B) 100% Tetrahydrofuran (HPLC
, using SUNFIRE , 150mm*19mm*5um with the flow rate of 16.0mL/min and
with the following gradient:
N,N',N"-(9-(1-((2S,4S)—4-azido(2,4-dioxo-1,4-dihydro-2H-
benzo [d] [1,3]oxazine-7—carbonyl)pyrrolidinyl)-2,1 l-dimethyl-l,12-dioxo-5,8-di0xa-2,1 1-
diazatetradecanyl)-9,10-dihydro-9,10-[1,2]benzenoanthracene—2,7,15-triyl)tris(8-
aminooctanamide), A_HC1 salt.
To a solution of tri—tert-butyl (((9-(1-((2S,4S)azido(2,4-dioxo-1,4-dihydro-2H-
benzo[d][1,3]oxazine-7—carbonyl)pyrrolidinyl)—2, 1 1-dimethyl—1,12-dioxo-5,8-dioxa-2, 1 1-
etradecanyl)—9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))t1is(8—oxooctane-8,1—diyl))tricarbamate (14) (0.04 g, 0.025mmol) in 1,4-
Dioxane (AR Grade) (2 mL) was added 4 M HCl in dioxane (1 mL) at room temperature and the
resulting reaction mixture was stirred for 30 minutes under nitrogen atmosphere. The solid
material stable at the bottom of the flask, the solvent was decanted under inert atmosphere, the
solid material was triturating with acetonitrile (HPLC Grade) (3 X 3 mL). The remaining solid
was concentrated by reduced pressure at 25 °C under nitrogen atmosphere to afford pure ARK-
78A_HCl_Salt (0.032g, 91.43 %) as a white amorphous . 1H NMR (400 MHz, DMSO-
d6) 5 11.99-11.95 ppm (1H, t), 9.91-9.90 ppm (3H, d), 8.01-7.94 ppm (1H, m), 7.87 ppm (8H,
broad s), 7.66 ppm (3H, broad s), 7.32—7.22 ppm (7H, m), 7.16-7.11 ppm (1H, m), 5.39 ppm (1H,
s), 4.99—4.95 ppm (1H, t), 4.83-4.82 ppm (1H, m), .22 ppm (1H, m), 4.15—3.98 ppm (1H,
m), 3.76-3.71 ppm (1H, m), 3.64-3.61 ppm (4H, m), 3.52 ppm (2H, broad s), 3.34-3.32 ppm (2H,
m), 3.15 ppm (2H, m), 3.10-3.03 ppm (7H, m), 2.89-2.86 ppm (1H, d), 2.76-2.72 ppm (7H, m),
2.26-2.23 ppm (6H, t), 1.53 ppm (12H, broad s), 1.27 ppm (17H, broad 3). MS (ESI—MS): m/z
calcd for C69H93N13011 [MH]+.1280.71, found 1281.50.
Example 11: Synthesis of ARK-79 and ARK-79A 0036 and Ark000038)
Synthesis of Int-13
11wN
o=é:o ‘7
6N01 Nosyl\
130C EEOC N
1 Nosyl\ N
/N\flo/\/o\/\O/\/NH\ HATU, DMF N o N
/ WON \/\o/V N
DIPEA 3TFA, MDC |
—> —> /NH/\O/\/OV\O/\/N 3
ARK—22 Step .1 o (10) Step -11
MW:320.23 MW: 643.26
(11)
MW: 543.21
Int-11
HATU, DMF
DIPEA
B HNWWNH $1 c NHWWNHBM —>Step -12
ARK-18 0:4
MW: 1094.66
Nosyl/N\/ ’Na
(12)
MW: 1619.87
Thiophenol,
Step -13 KZCOSHACN
60°C
/\/\/\/\H/
H110 ‘Na (13)
MW: 1434.89
utyl S,4S)azido((2-nitrophenyl)sulfonyl)pyrrolidin-2—yl)—2-
methyloxo-5,8,11-trioxaazatridecanyl)(methyl)carbamate, 10.
2017/040514
To a solution of ARK-22 (3.1 g, 9.68 mmol) in N,N—dimethylformamide (40 mL)
were sequentially added (2S,4S)azido((2-nitrophenyl)sulfonyl)pyrrolidinecarboxylic
acid (3.96 g, 11.62 mmol), HATU (4.414 g, 11.62 mmol) and isopropylethylamine (2.5 g,
19.36 mmol) at room temperature. The resulted reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was poured in ice-cold water and extracted with ethyl acetate
(3 X 100 mL). The organic layers were combined, washed with brine and concentrated under
reduced pressure to get crude 10 (4 g, 64.2%) as yellow solid. The crude mixture was used in
next step without further purification. MS (ESI-MS): m/z calcd for C26H411\I70108[l\/H‘I]+ 644.26,
found 544.36 .
(2S,4S)—4-azido-N-methyl((2-nitr0phenyl)sulfonyl)—N-(5,8,11-tri0xa
azatridecanyl) pyrrolidinecarboxamide_TFA Salt, 11.
To a solution of tert—butyl S,4S)—4-azido((2-nitrophenyl)sulfonyl)pyrrolidin-
2-methyl—1-oxo-5,8,11-trioxaazatridecanyl)(methyl) carbamate (10) (3 g, 4.66
mmol) in dichloro methane (20 mL) was added trifluoro acetic acid (1.8 mL, 23.32 mmol) at
room temperature. The resulted reaction mixture was stirred at room temperature for 2 h. The
reaction e was filtered through celite bed and filtrate thus collected was concentrated
under reduced pressure to get crude 11 (3.1 g, quantitative yield) as a dark yellow oil which was
used without further purification. MS (ESI—MS): m/z calcd for C21H33N7OsSTFA [MH]+ 544.21,
found 544.47.
Tri-tert—butyl (((9-(1-((2S,4S)azido((2-nitr0phenyl)sulfonyl)pyrr0lidin
yl)—2,14—dimethyl-1,15-di0x0-5,8,11-tri0xa-2,14-diazaheptadecanyl)—9,10-dihydro-9,10-
enzenoanthracene-2,7,l5-triyl)tris(azanediyl))tris(8-ox00ctane-8,1-diyl))tricarbamate,
To a on of (2S,4S)—4-azido-N-methyl((2-nitrophenyl)sulfonyl)-N-(5,8,11-
trioxaazatridecany1) pyrrolidinecarboxamide_TFA Salt (11) (2.88 g, 4.38 mmol) in
N,N-dimethylformamide (40 mL) were sequentially added 3-(2,7,15-tris(8-((tert-
butoxycarbonyl)amino)octanamido)—9,10-[1,2]benzenoanthracen—9(1OH)—yl)propanoic acid
(ARK-18) (4.0 g, 3.65 mmol), HATU (1.67 g, 4.38 mmol) and N,N—diisopropylethylamine
(2.36 g, 18.27 mmol) at room ature. The resulted reaction mixture was stirred for 1 h at
room temperature. The reaction mixture was poured in ice-cold water and extracted with ethyl
2017/040514
acetate (3 X 100 mL). The organic layers were combined, washed with brine and concentrated
under reduced pressure to get crude 12. The crude mixture was purified by column
chromatography on silica gel (5.4% methanol/chloroform) to yield 12 (5.9 g, 99.7 %) as a dark
yellow solid. MS (ESI-MS): m/z calcd for C83H121N13OISS [MH]+ 1620.87, found 1522.31(M-
100, one Boc group fell off).
Tri—tert—butyl (((9—(1-((2S,4S)—4-azidopyrrolidin-Z-yl)—2,14-dimethyl-1,15—di0xo-
,8,11-tri0xa-2,14-diazaheptadecanyl)-9,10-dihydro-9,10-[1,2]benzenoanthracene-
2,7,15-triyl)tris(azanediyl))tris (8-0xooctane—8,1-diyl))tricarbamate, 13.
To a solution of tri-tert-butyl (((9-(1—((2S,4S)—4-azido-l-((2-
nitrophenyl)sulfonyl)pyrrolidinyl)-2,l4-dimethyl-1,15—dioxo-5,8, 1 1-trioxa-2,14—
diazaheptadecan-l7-yl)—9,10-dihydro-9,10-[1,2] benzenoanthracene—2,7, 15-
triyl)tris(azanediyl))t1is(8-oxooctane-8,1—diyl))tricarbamate (12) (5.9 g, 3.64 mmol) in
acetonitrile (60 mL) were sequentially added potassium carbonate (2.51 g, 18.21 mmol) and
thiophenol (1.11 mL, 10.93 mmol) at room temperature. The resulted reaction mixture was
stirred at 80 °C for 2 h. The reaction mixture was d through celite bed and the collected
filtrate was concentrated under reduced pressure to get crude 13 as yellow oil. The crude mixture
was subjected to reverse phase chromatography to yield 13 (1.9 g, 36.3%) as a light yellow solid.
The yellow solid was r subjected to preparative HPLC (method ned below)
purification followed by lyophilization to yield pure 13 , 9.8%) as a white amorphous
powder. MS (ESI—MS): m/z calcd for (:771‘11181\IIZOI4[IVE—I]+ 1435.89, found 1437.41.
Method for ative HPLC:
(A) 100% Acetonitrile (HPLC GRADE) IN WATER (HPLC GRADE) and (B)
lOmM 3 IN WATER (HPLC GRADE), using GRACE DENIL C18,
250mm*25mm*5um with the flow rate of 22.0 mL/min and with the following gradient:
__IE_
WO 06074
Scheme: Synthesis of ARK-79
/\/\/\/\[/ 0 BocHNW
BocHN ‘ |
H000 N/V) NH C 6/ Q 0
NH6/6 o | BocHNM WNHBOC
BocHNWl/ WNHBOC O NH
0 NH Warhead_1B
HATU, DIPEA‘ DMF
N\ —> /\/
H Step -14 of0
HMO "N3 0
(13)
MW: 1434.89
O/ D/EO
(14)
MW: 1637.91
Step -15 HCI in dioxane
0/0 D
ARK-79_HCI_SaIt
MW: 1337.75
Tri—tert—butyl (((9-(1-((2S,4S)-4—azido(1-methyl-2,4-dioxo-1,4-dihydro-2H-
benzo [d] [1,3]oxazine—7—carb0nyl)pyrrolidin-2—yl)—2,14-dimethyl-1,15-di0x0-5,8,1 1-trioxa-
iazaheptadecanyl)-9,10-dihydr0-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))tris(8-0xooctane-8,1-diyl))tricarbamate, 14.
To a solution of tfi-tert-butyl (((9-(1—((2S,4S)—4-azidopyrrolidin-Z-yl)—2,l4-dimethyl-
1,15-di0X0-5,8,ll-trioxa-Z,l4-diazaheptadecanyl)-9,lO-dihydro-9,10—
[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris (8—oxooctane-8,l—diyl))tficarbamate (13)
(0.1 g, 0.07 mmol) in N,N—dimethylformamide (4 mL) were sequentially added l-methyl-2,4-
dioxo—2,4-dihydro-1H—3,1-benzoxazinecarboxylic acid (Warhead_type_1B) (0.039 g, 0.18
mmol) and HATU (0.018 g, 0.084 mmol) at room temperature. The reaction mixture was d
for 5 minutes. To this, isopropylethylamine (0.018 g, 0.14 mmol) was added drop wise
and the resulted reaction mixture was further stirred for 30 minutes at room temperature. The
reaction mixture was diluted by ethyl acetate (100 mL) and washed with ice-cold water (3 x 30
mL). The organic layers were ed, washed with brine and concentrated under reduced
pressure at 25 °C to get crude 14. The crude mixture was purified by ative HPLC (method
mentioned below) followed by lyophilization to yield 14 (0.053 g, 46.5%) as a white amorphous
powder. MS (ESI—MS): m/z calcd C87H123N13018 [MH]+ 1638.91, found 1540.40 (M-100).
Method for preparative HPLC:
(A) 100% Acetonitrile (HPLC GRADE) and (B) 100% Tetrahydrofuran (HPLC
GRADE), using SUNFIRE SILICA, 250mm*19mm*5um with the flow rate of 15.0 mL/min and
with the following gradient:
] N,N',N"-(9-(1-((2S,4S)azido(1-methyl-2,4-dioxo-1,4-dihydro-2H-
benzo [d] [1,3]oxazine-7—carbonyl)pyrrolidinyl)-2,14—dimethyl-1,15-di0x0-5,8,1 1-tri0xa-
iazaheptadecanyl)-9,10-dihydr0-9,10-[1,2]benzenoanthracene-2,7,15-triyl)tris(8-
aminooctanamide), ARK-79_HCl salt.
To a solution of tri-tert-butyl (((9-(1-((2S,4S)azido(1-methyl-2,4-dioxo-1,4-
dihydro—2H—benzo[d][1,3]oxazinecarbonyl)pyrrolidin—2—yl)—2,l4-dimethyl-1,15—dioxo-5, 8,1 1-
trioxa-2,14-diazaheptadecanyl)-9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))tris(8—oxooctane-8,1—diyl))tricarbamate (14) (0.035 g, 0.021 mmol) in 1,4-
dioxane (3.0 mL) was added 4 M HCl in dioxane solution (1 mL) at room temperature and the
resulted reaction mixture was stirred for 30 minutes under nitrogen atmosphere. During this,
solid residue d to precipitate out. The suspension was r stirred for 30 minutes and
finally allowed to stand at room ature. The solid residues started to deposit on bottom of
the flask. The solvent was decanted and the residues left were triturated with acetonitrile (3 x 3
mL). Finally the solid was dried under reduced pressure at 25 °C to get pure ARK-79_HCI_Salt
(0.025 g, 80.6%) as a white ous powder. 1H NMR (400 MHz, DMSO-d6) 6 9.89 ppm
(3H, broad s), 8.10-8.08 ppm (1H, m), 7.89 ppm (9H, broad s), 7.66 ppm (3H, broad s), 7.38-
7.37 ppm (1H, d), 7.33—7.22 ppm (6H, m), 5.38 ppm (1H, s), 4.95-4.90 ppm (1H, m), 4.25 ppm
(1H, m), 4.06 ppm (1H, m), 3.75 ppm (1H, m), 3.63-3.57 ppm (10H, d), 3.38-3.33 ppm (5H, m),
3.10-3.04 ppm (7H, m), 2.88-2.84 ppm (1H, d), 2.74-2.72 ppm (7H, broad s), 2.25-2.23 ppm
(6H, t), 1.60-1.53 ppm (12H, (1), 1.27 ppm (18H, broad 3). MS (ESI—MS): m/Z calcd for
C72H99N13012 [MH]+ 5, found 1339.55.
: Synthesis of ARK-79A
Warhead_1A
HATU, DIPEA, DMF
Step -14
QNOMNS (13)
MW: 1434.89
(14)
MW: 1623.89
Step -15 HCI in dioxane
0:O1 030
ARK-79A_HCI_SaIt
MW: 1323.74
Tri—tert—butyl (((9-(1-((ZS,4S)azid0(2,4-di0xo-1,4-dihydr0-2H-
benzo [d] [1,3]oxazine—7—carbonyl)pyrrolidinyl)-2,14-dimethyl-1,15-di0x0-5,8,1 1-tri0xa-
2,14-diazaheptadecanyl)-9,10-dihydr0-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))tris(8-0xooctane-8,1-diyl))tricarbamate, 14.
] To a solution of tri—tert-butyl (((9-(1—((2S,4S)—4-azidopyrrolidinyl)—2,l4-dimethy1—
1,15-dioxo-5,8,l 1-trioxa—2, 14-diazaheptadecanyl)-9,lO-dihydro-9,10—
[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris (8-oxooctane-8,1-diyl))tricarbamate (13)
(0.2g, 0.139 mmol) in N,N—dimethylformamide (8 mL) were tially added 2,4-dioxo-1,4-
dihydro—2H-benzo[d][1,3]oxazinecarboxylic acid (Warhead_type_1A) (0.035 g, 0.167
mmol) and HATU (0.064 g, 0.167 mmol) at room temperature. The reaction mixture was stirred
for 5 minutes. To this, N,N—diisopropylethylamine (0.036 g, 0.279 mmol) was added dropwise
and the resulted reaction mixture was further stirred for 30 minutes at room ature. The
reaction e was diluted by ethyl acetate (100 mL) and washed with ice-cold water (3 X 30
mL). The organic layers were combined, washed with brine and concentrated under reduced
pressure at 25 0C to get crude 14. The crude mixture was purified by preparative HPLC using
following method to yield pure 14 (0.04g, 17.7 %) as a off white amorphous powder. The prep-
fraction was concentrated by reduced pressure at 25°C under en atmosphere. MS (ESI—
MS): m/z calcd C36H121N13013 [MH]+ 1624.89, found 1525.76 (M—100; one Boc group fell off).
Method for preparative HPLC:
(A) 100% Acetonitrile (HPLC GRADE) and (B) 100% Tetrahydrofuran (HPLC
GRADE), using SUNFIRE , 150mm*19mm*5um with the flow rate of 18.0mL/min and
with the following gradient: 98% A and 2% for 20 min.
N,N',N"-(9-(1-((2S,4S)azido(2,4-dioxo—1,4-dihydro-2H-
benzo [d] [1,3]oxazinecarb0nyl)pyrrolidinyl)-2,1 thyl-1,15-di0x0-5,8,1 l-trioxa-
2,14-diazaheptadecanyl)-9,10-dihydr0-9,10-[1,2]benzenoanthracene-2,7,15-triyl)tris(8-
aminooctanamide), ARK-79A_HC1 salt.
To a solution of tri-tert-butyl (((9-(1-((2S,4S)azido(2,4-dioxo-1,4-dihydro-2H-
benzo[d][1,3]oxazinecarbonyl)pyrrolidiny1)-2, ethy1-1,15-dioxo-5,8,1 l-trioxa-2,14-
diazaheptadecan-l7-yl)-9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))tris(8-oxooctane-8,1-diyl))tricarbamate (14) (0.04 g, 0.024 mmol) in 1,4-
Dioxane (Dry) (3 ml) was added 4 M HCl in dioxane (1.2 mL) at room temperature and the
resulting reaction mixture was stirred for 30 minutes under nitrogen atmosphere. The solid
al stable at the bottom of RBF, the solvent was decant under inert atmosphere, the solid
material was tn'turating with acetonitrile (HPLC Grade) (3 X 3 mL). The remaining solid was
trated by d pressure at 25 °C under nitrogen atmosphere to afford pure ARK-
79A_HCl_Salt (0.033 g, 94.28 %) as an off white amorphous powder. 1H NMR (400 MHz,
6) 5 11.97-11.95 ppm (1H, d), 9.90 ppm (3H, broad s), 802—798 ppm (1H, m), 7.88
ppm (8H, broad s), 7.66 ppm (3H, broad s), 7.32—7.22 ppm (7H, m), 7.16—7.08 ppm (1H, m), 5.39
ppm (1H, s), 496-491 ppm (1H, m), 4.80 ppm (1H, m), 4.28-4.20 ppm (1H, m), 4.05 ppm (1H,
m), 3.75—3.73 ppm (1H, m), 3.64 ppm (3H, broad s), 3.57 ppm (11H, broad s), 3.54 ppm (2H, m),
3.39-3.38 ppm (2H, m), 3.34-3.32 ppm (3H, d), 3.16 ppm (2H, broad s), 3.08-3.02 ppm (8H, m),
2.87-2.83 ppm (1H, d), 2.76-2.68 ppm (7H, m), 2.27—2.23 ppm (6H, t), 1.60-1.53 ppm (12H,
broad s), 1.27 ppm (18H, broad s). MS (ESI—MS): m/z calcd for C71H97N13012 [MH]+1324.74,
found 1325.50.
e 12: sis of ARK-80, ARK-89, ARK-125 (Ark000024, 027, and
Ark000030)
Scheme: Synthesis of Int-13
WONN
o:é=o ‘3
Boo Nosyl\ Nosyk
‘ Boo
N N
N NH HATU.DMF l
/ WON ‘
DIPEA /NWONNQ‘N3 TFA,MDC
—, l —>/NH/\0/\/NYQ‘N3l
ARK-20 0
Step 40 Step .11 TFA salt 0
MW:232.17 (10) (11)
MW: 555-21
MW: 455.16
NH BocHNM
BucHNM 0
Int-11
s HATU,DMF, DIPEA BocHN/WWNH
NH / o WNHBOC
BocHNW a QNHWNHBOC —> 0 NH
Step _12
N3 HN‘
0“ NJ”
(3 (12)
ARK“ N \<o MW: 1531.81
MW: 1094.66 ””5“
Thiophenol,
Step -13 KZCOZACN
60°C
MNJ‘\ o
<13)
m o MW:1346.84
Tert—butyl (2-(2-((2S,4S)—4—azido-N-methyl((2-
nitrophenyl)sulfonyl)pyrrolidinecarb0xamid0) ethoxy)ethyl)(methyl)carbamate, 10.
To a solution of ARK-20 (1.0 g, 4.307 mmol) in N,N—dimethylformamide (20 mL)
were sequentially added (28,4S)azido((2-nitrophenyl)sulfonyl)pyrrolidinecarboxylic
acid (1.17 g, 3.44 mmol), HATU (1.96 g, 5.17 mmol) and N,N—diisopropylethylamine (1.67 g,
12.92 mmol) at room temperature. The resulted reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was poured in ice-cold water and ted with ethyl e
(3 X 100 mL). The organic layers were combined, washed with brine and concentrated under
reduced pressure to get crude 10 (2.52 g, quantitative yield) as brown semisolid. The crude
mixture was used in next step without further purification. MS (ESI—MS): m/z calcd for
C22H33N708S [MH]+ 556.21, found 573.43 (M+18).
(2S,4S)—4-azido-N-methyl-N-(Z-(Z-(methylamin0)eth0xy)ethyl)((2-
nitrophenyl)sulf0nyl) pyrrolidine—2-carb0xamide_TFA Salt, 11.
To a solution of tri utyl (2-(2-((2S,4S)-4—azido-N—methyl((2-
henyl)sulfonyl)pyrrolidinecarboxamido) ethoxy)ethyl)(methyl)carbamate (10) (2.5 g,
4.50 mmol) in dichloro methane (15 mL) was added trifluoro acetic acid (1.72 mL, 22.51 mmol)
at room temperature. The resulted reaction mixture was stirred at room temperature for 2 h. The
reaction e was filtered through celite bed and filtrate thus collected was concentrated
under reduced pressure to get crude 11 (4.12 g, quantitative yield) as a brown oil which was used
without further purification. MS S): m/z calcd for C17H25N706STFA [MH]+456.16,
found 456.32.
Tri-tert—butyl (((9-(3-((2-(2-((2S,4S)-4—azido-N-methyl((2-
henyl)sulf0nyl)pyrrolidine—2-carb0xamid0)ethoxy)ethyl)(methyl)amino)—3-
0x0pr0pyl)—9,10-dihydr0-9,10[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(8-
oxooctane-S,1-diyl))tricarbamate, 12.
To a solution of (2S,4S)—4—azido-N—methyl—N—(2-(2—(methylamino)ethoxy)ethyl)—1-
trophenyl)sulfonyl) pyrrolidinecarboxamide_TFA Salt (11) (1.75 g, 3.07 mmol) in
N,N-dimethylformamide (30 mL) were sequentially added 3-(2,7,15-t1is(8—((tert-
butoxycarbonyl)amino)octanamido)—9,10-[1,2]benzenoanthracen-9(10H)—yl)propanoic acid
(ARK-18) (2.8 g, 2.56 mmol), HATU (1.17 g, 3.07 mmol) and N,N-diisopropylethylamine
(0.66 g, 5.12 mmol) at room temperature. The resulted reaction mixture was stirred for 1 h at
room temperature. The reaction mixture was poured in ice-cold water and ted with ethyl
e (3 X 100 mL). The c layers were combined, washed with brine and concentrated
under reduced pressure to get crude 12. The crude mixture was purified by column
chromatography on silica gel (15% methanol/chloroform) to yield 12 (1.48 g, 37.8 %) as a dark
yellow solid. MS (ESI-MS): m/z calcd for C79H113N13016S [lVlH]+ 1532.81, found 1433.19 (M—
100; one Boc group fell off).
2017/040514
Tri—tert—butyl (((9-(3-((2-(2-((2S,4S)azid0-N-methylpyrrolidine—Z-
carboxamid0)eth0xy)ethyl) (methyl)amino)—3-oxopropyl)—9,10-dihydro-
9,10[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl)) tris(8-0x00ctane—8,1-
diyl))tricarbamate, 13.
To a solution of tri-tert-butyl (((9-(3-((2-(2-((2S,4S)—4-azido—N—methyl-l-((2-
nitrophenyl)sulfonyl) idine—2-carboxamido)ethoxy)ethyl)(methyl)amino)-3 opyl)-
9, 10-dihydro-9, 10[1,2] benzenoanthracene-2,7,15-triyl)tris(azanediyl))tris(8-oxooctane-8,1—
diyl))tricarbamate (12) (1.48 g, 0.97 mmol) in acetonitrile (15 mL) were sequentially added
potassium carbonate (0.67 g, 4.83 mmol) and thiophenol (0.3 mL, 2.89 mmol) at room
temperature. The resulted reaction mixture was stirred at 80 0C for 2 h. The reaction mixture was
filtered h celite bed and the collected filtrate was concentrated under reduced pressure to
get crude 13 as yellow oil. The crude mixture was subjected to reverse phase chromatography to
yield 13 (0.76 g, 58.4 %) as a light yellow solid. MS (EST-MS): m/z calcd for C73H110N12012
[MH]+ 1347.84, found 1349.28.
: Synthesis of ARK-80
F F
o BOCHNM
F F
o i\
r0 N o BOCHNWVYNH WNHBOC
NHBoc o NH
Wartype-Z \
Int-A
’ g
N3 \ o
Step-14 Nf
° °3w. 3 (13) och \°
MW:1345.86 0
(14)
9.87
Step -15 jHCI in dioxane
HzNMNHOO
HzN/WWNH 3/6Q WNH;
o NH
N3\(\‘Y<\N_/‘OgN
\ ofo o
ARK-80_HC| salt
MW: 1
O F F
F 0
F F
Hon” dfi; + EDCHCLTHF F F dig
O N O —> O
o ' F F 0
OH Step-A T50 ll“
Warhead-2 Warhead-2_lnt-A
MW 417.03
Perfluorophenyl 2-((1-methyl-2,4-di0x0-l,4-dihydr0-2H-benz0[d][1,3]0xazin
yl)oxy)acetate, Int-A.
To a solution of Warhead-2 (0.04 g, 0.17 mmol) in ydrofuran (1 mL) was
added N—(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hloride (0.035 g, 0.17 mmol) at
0 °C under nitrogen atmosphere. The reaction mixture was stirred at 0 0C for 10 min. To this, a
solution of pentafluorophenol (0.03 g, 0.17 mmol) in tetrahydrofuran (0.5 mL) was added
dropwise at 0 0C under nitrogen atmosphere. The resulted reaction mixture was further stirred at
0 °C for 1 h. The reaction mixture was directly used in the next step without work up and
isolation. MS (ESI—MS): m/z calcd C17H8F5NO6 [MH]+ 418.03, the compound did not show
2017/040514
mass response. Note: Intermediate-A was not isolated — the reaction mass was transferred as
such to the next step reaction mass.
Tri—tert—butyl (((9-(3-((2-(2-((2S,4S)—4-azido-N-methyl(2-((1-methyl-2,4-dioxo-
1,4-dihydr0-2H-benzo[d] [1,3]oxazinyl)0xy)acetyl)pyrrolidine
carboxamido)eth0xy)ethyl)(methyl)amino)—3-ox0propyl)—9,10-dihydro-9,10-
[1,2]benzenoanthracene—2,7,15—triyl)tris(azanediyl))tris(8-0xooctane—8,1-diyl))tricarbamate,
To a solution of tri-tert-butyl (((9-(3—((2-(2-((2S,4S)—4-azido—N—methylpyrrolidine
carboxamido)ethoxy)ethyl) (methyl)amino)-3 opyl)—9, l O-dihydro-
9, l0[l,2]benzenoanthracene-2,7,15-triyl)tris(azanediyl)) tris(8-oxooctane-8, ))tricarbamate
(13) (0.27 g, 0.17 mmol) in tetrahydrofuran (4 mL) was added pentafluorophenyl [(l—methyl-
2,4-dioxo-1,4-dihydro-2H-3,l-benzoxazinyl)oxy]acetate (Warhead_type_2) (0.071 g, 0.17
mmol) and the resulted reaction mixture was further stirred for 1h at room temperature. The
reaction mixture was trated under reduced pressure to get crude 14 (0.38 g, Quantitative
yield) as a yellow solid which was used in the next step without further ation. MS (ESI—
MS): m/z calcd C34H117N13017 [lVIH]+ 1580.87, found 1482.29 (M—100; one Boc group fell off).
N,N',N"-(9-(3-((2-(2-((2S,4S)—4-azido-N-methyl(2-((1-methyl-2,4—dioxo-1,4-
dihydro-ZH-benzo [d] [1,3]oxazinyl)oxy)acetyl)pyrrolidine—2—
carboxamid0)eth0xy)ethyl)(methyl)amino)—3-oxopropyl)—9,10-dihydro-9,10-
[1,2]benzenoanthracene—2,7,l5—triyl)tris(8-aminooctanamide), ARK-80_HC] salt.
To a solution of tri-tert-butyl (((9-(3-((2-(2-((2S,4S)azido-N—methyl-l-(2-((l-
methyl-2,4-dioxo-l,4-dihydro-2H-benzo[d][l,3]oxazinyl)oxy)acetyl)pyrrolidine
amido)ethoxy)ethyl)(methyl)amino)-3 -oxopropyl)—9, lO-dihydro-9, 10-
[1,2]benzenoanthracene-2,7,15-triyl)tris(azanediyl))tris(8-oxooctane-8,l-diyl))tricarbamate, (14)
(0.38 g, 0.025 mmol) in tetrahydrofuran (5.0 mL) was added 4 M HCl in dioxane solution (2
mL) at room temperature and the resulted reaction e was stirred for 4 h under en
atmosphere. The reaction mixture was concentrated under reduced pressure to get crude ARK-
80_HCl_Salt as a yellow solid. The crude mixture was purified by preparative HPLC using
ing method to get pure ARK-80_HCl salt (0.012 g, 3.6%) as a white amorphous powder.
1H NMR (400 MHz, DMSO-d6) 6 9.93-9.91 ppm (3H, broad s), 7.92-7.85 ppm (10H, broad s),
7.65 ppm (4H, broad s), 7.40 ppm (2H, broad 5), 727-7. 15 ppm (8H, m), .71 ppm (3H, m),
6.54 ppm (1H, s), 5.36 ppm (1H, s), 5.10-5.02 ppm (3H, m), 4.83 ppm (2H, m), 4.66-4.56 ppm
(2H, m), 4.39—4.28 ppm (2H, m), 4.06-4.01 ppm (2H, m), 3.58-3.55 ppm (4H, m), 3.47-3.41 ppm
(7H, m), 3.13-2.94 ppm (9H, m), 2.71-2.66 ppm (8H, m), 2.22 ppm (7H, broad s), .50
ppm (12H, d), 1.26 ppm (18H, 3). MS (ESI—MS): m/z calcd for C69H93N13011 [MH]+ 1280.71,
found 1281.43.
Method for preparative HPLC:
(A) 0.05% HCl IN WATER (HPLC GRADE) and (B) 100% Acetonitrile (HPLC
GRADE), using X-BRIDGE, 250mm*19mm*5pm with the flow rate of 19.0mL/min and with
the following gradient:
__IE_
Scheme: Synthesis of ARK-89
0 BOCHNM
0:§/F 0
COOH \N
HATU, DMF
DIPEA S
N3 \ f0
—> N
meo (13> 5‘” '14
g 0 MW: 1346.86 W0
(14)
MW: 1560.85
Step .15 l HCI in dioxane
_HCI salt
MW: 1260.70
Tri—tert—butyl (((9-(3-((2-(2-((ZS,4S)azido-l-(3-(4-
(fluorosulfonyl)phenyl)pr0panoyl)—N-methylpyrrolidine—Z-
amido)eth0xy)ethyl)(methyl)amino)—3-ox0propyl)—9,10-dihydro-9,10-
[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(8-0x00ctane—8,1-diyl))tricarbamate,
To a solution of tri-tert-butyl (((9-(3—((2-(2-((2$,4S)—4-azido—N—methylpyrrolidine
carboxamido)ethoxy)ethyl) (methyl)amino)-3 -oxopropyl)—9,10-dihydro-
9,10[1,2]benzenoanthracene-2,7,15-triy1)tris(azanediyl)) tris(8-oxooctane—8, 1-diyl))tricarbamate
(13) (0.31 g, 0.23 mmol) in N,N—dimethylformamide (6 mL) were sequentially added 3-(4-
(fluorosulfonyl)phenyl)propanoic acid (0.043 g, 0.18 mmol) and HATU (0.070 g, 0.18 mmol)
at room temperature. The reaction mixture was stirred for 5 s. To this, N,N—
diisopropylethylamine (0.036 g, 0.276 mmol) was added drop wise and the resulted reaction
mixture was further stirred for 1h at room temperature. The reaction mixture was diluted by ethyl
acetate (100 mL) and washed with ice-cold water (3 x 30mL). The c layers were
combined, washed with brine and concentrated under reduced pressure at 25 0C to get crude 14
2017/040514
(0.45 g, Quantitative yield) as a dark yellow solid which was used in the next step without
further purification. MS (ESI—MS): m/z calcd C82H117FN120158[MH]+ 1561.85, found 1463.45
(M-100, one Boc group fell off).
N,N’N”-(9-(3-((2-(2-((2S,4S)—4-azid0(3-(4-(11u0r0sulfonyl)phenyl)pr0panoyl)—
N-methylpyrrolidine—Z-carboxamido)eth0xy)ethyl)(methyl)amino)—3-ox0propyl)—9,10-
dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(8-aminooctanamide),
ARK-89_HC1 salt.
To a solution of tri-tert-butyl (((9-(3-((2-(2-((2S,4S)-4—azido-1—(3-(4-
(fluorosulfonyl)phenyl)propanoyl)-N-methylpyrrolidine
carboxamido)ethoxy)ethyl)(methyl)amino)—3 -oxopropyl)—9, 10-dihydro-9, 10-
[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(8-oxooctane-8,1-diyl))tricarbamate (14)
(0.45 g, 0.028 mmol) in 1,4-dioxane (5.0 mL) was added 4 M HCl in dioxane (2 mL) at room
temperature. The resulting reaction mixture was stirred for 4 hours. The mixture was
concentrated under reduced pressure to get crude ARK-89_HC1_Salt as a yellow solid. The
crude mixture was purified by preparative HPLC using following method to get pure ARK-
89_HCl salt (0.053g, 12.8 %) as a yellow solid. 1H NMR (400 MHz, DMSO-ds) 5 9.95 ppm
(3H, broad s), .95 ppm (10H, m), 7.67-7.62 ppm (5H, m), 7.28-7.21 ppm (6H, m), 5.38
ppm (1H, s), 4.77 ppm (0.5H, m), .49 ppm (1H, m), 4.31—4.21 ppm (1H, m), .96
ppm (2H, m), 3.62-3.44 ppm (6H, m), 3.22-3.03 ppm (8H, m), 2.98-2.88 ppm (4H, m), 2.74-2.60
ppm (10H, m), 2.24-2.23 ppm (7H, t), .52 ppm (12H, d), 1.26 ppm (18H, 5). MS (ESI-
MS): m/Z calcd for C67H93FN1209S [MH]+ 0, found 1262.31.
Method for preparative HPLC:
(A) 0.05% HCl IN WATER (HPLC GRADE) and (B) 100% Acetonitrile (HPLC
GRADE), using X-SELECT FP, 250mm* l9mm*5um with the following gradient:
——m_
_—m_
Scheme: sis of ARK-125
F /\/\/\/\H/ 0
1 BDCHN
028:0 0
NH 3 o
O BOCHNWY 0 OW
N HBoc
WNHBDC O NH
H HO 0
HATU,DMF H
DIPEA ”Smufo
Step -14 N ‘0
F028
(13) (14)
MW: 1346.86 MW: 1532.81
Step -15 l HCI in dioxane
HZNM
HZNWNHU ‘0 fl) NH
O NH’\/\/\/\/ 2
ARK-125_HCI salt
MW: 5
Tri—tert—butyl (((9-(3-((2-(2-((2S,4S)—4-azid0(4-(fluorosulfonyl)benzoyl)—N-
methylpyrrolidine—2-carb0xamid0)ethoxy)ethyl)(methyl)amino)—3-0xopropyl)—9,10-
dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(8-0xooctane—8,1—
diyl))tricarbamate, 14.
To a solution of tri-tert-butyl 3—((2-(2-((ZS,4S)—4-azido—N—methylpyrrolidine
carboxamido)ethoxy)ethyl) (methyl)amino)-3 -oxopropyl)—9,10-dihydro-
9,10[1,2]benzenoanthracene-2,7,15-t1iyl)tris(azanediyl)) tris(8-oxooctane—8, 1-diy1))tricarbamate
(13) (0.30 g, 0.22 mmol) in N,N—dimethylformamide (10 mL) were sequentially added 4-
fluorosulfonylbenzoic acid (0.054 g, 0.27 mmol) and HATU (0.101 g, 0.27 mmol) at room
temperature. The reaction mixture was d for 5 minutes. To this, N,N—diisopropylethylamine
(0.079 g, 0.45 mmol) was added drop wise and the resulted reaction mixture was further stirred
for 1h at room temperature. The on mixture was diluted by ethyl acetate (100 mL) and
washed with ice—cold water (3 x 30 mL). The organic layers were combined, washed with brine
and concentrated under reduced pressure at 250C to get crude 14 (0.388 g, Quantitative yield) as
a yellow semi—solid which was used in the next step without further purification. MS S):
m/z calcd C80H113FNIZOISS [MH]+ 1532.81, found 1434.35 (M-100, one Boc group fell off).
N,N’,N”-(9-(3-((2-(2-((2S,4S)—4-azid0(4-(flu0rosulfonyl)benz0yl)—N-
methylpyrrolidine—2-carb0xamid0)ethoxy)ethyl)(methyl)amino)—3-0xopropyl)—9,10-
dihydro-9,10-[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(8-aminooctanamide),
ARK-125_HCI salt.
To a solution of tri-tert-butyl (((9—(3-((2-(2-((2S,4S)azido-l-(4-
(fluorosulfonyl)benzoyl)—N—methylpyrrolidine-2—carboxamido)ethoxy)ethyl)(methyl)amino)—3-
pyl)-9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15-triyl)tris(azanediyl))tris(8-
oxooctane-8,1-diyl))t1icarbamate (14) (0.38 g, 0.0025 mmol) in 1,4-dioxane (5.0 mL) was added
4 M HCl in dioxane (2 mL) at room temperature and the resulting reaction mixture was stirred
for 4 hours. The mixture was concentrated under reduced pressure to get crude ARK-
l_Salt as a yellow solid. The crude mixture was purified by preparative HPLC using
following method to get pure ARK-125_HCl_Salt (0.110g, 33.0%) as a yellow solid. 1H NMR
(400 MHz, DMSO-d6) 6 9.96-9.93 ppm (3H, broad s), 8.24-8.21 ppm (2H, m), 7.97 ppm (8H,
broad s), .82 ppm (2H, m), 7.71-7.68 ppm (3H, m), 7.32-7.19 ppm (6H, m), 5.38 ppm (1H,
s), 5.05-5.01 ppm (1H, m), 4.87-4.80 ppm (1H, m), 4.30-4.20 ppm (1H, m), 3.89 ppm (18H,
broad s), 370-355 ppm (5H, m), 3.48-3.38 ppm (3H, m), 3.18 ppm (1H, s), 308—304 ppm (6H,
m), 2.79-2.68 ppm (7H, m), 2.25 ppm (6H, broad s), 1.54-1.52 ppm (12H, d), 1.26 ppm (18H,
). MS S): m/z calcd for C65H89FN1209$ [MH]+ 1233.65, found .
Method for preparative HPLC:
(A) 0.05% HCl IN WATER (HPLC GRADE) and (B) 100% Acetonitrile (HPLC
GRADE), using X-SELECT FP, 250mm*19mm*5um with the flow rate of 19.0 mL/min and
with the following gradient:
Example 13: sis of ARK-81, ARK-90, and ARK-126 0025, Ark000028, and
Ark000031)
Scheme: Synthesis of 13
mm.2
0:5:0
\/O\ANH; N
HATU, DMF N NOSYI\N
\N/wowo/VN 3 TFA, MDC ‘
DIPEA N
—> i —> \NH'\/O\/\o/\/N 3
ARK-21 stgp.1o 50C 0 Step -11
MW:276.37 (10) (11)
MW: 599.18 MW: 499.18
wNHv Int-11
HATU, DMF
DIPEA
NH / s o
BocHNW\/\[r a Q WNHBOC ’
0 NH Step -12
ARK-18
MW: 1094.66
N3 CNN“ MW:(1I§l5.84
Thiophenol,
KZCOEHACN
Step 13 j 60°C
Nz’CC (13)
MW: 139086
Tert—butyl (2-(2-(2-((ZS,4S)—4—azid0-N-methyl((2-
nitrophenyl)sulfonyl)pyrrolidinecarboxamid0)ethoxy)eth0xy)ethyl)(methyl)carbamate,
To a solution of ARK-21 (0.9 g, 3.04 mmol) in N,N-dimethylforrnamide (6 mL)
were sequentially added (28,4S)azido((2-nitrophenyl)sulfonyl)pyrrolidinecarboxylic
acid (1.33 g, 3.91 mmol), HATU (1.4 g, 3.91 mmol) and N,N-diisopropylethylamine (0.85 g,
6.52 mmol) at room temperature. The resulted reaction mixture was stirred for 1 h at room
ature. The reaction mixture was poured in ld water and extracted with ethyl acetate
(3 X 100 mL). The organic layers were combined, washed with brine and concentrated under
reduced pressure to get crude 10 (1.5 g, 78.9%) as brown semisolid which was used in next step
without further purification. MS (ESI—MS): m/z calcd for C24H37N709 S [MH]+ 600.18, found
617.5(M+18).
(2S,4S)—4-azido-N-methyl-N-(Z-(Z-(2-(methylamino)eth0xy)eth0xy)ethyl)—1-((2
nitrophenyl)sulf0nyl)pyrrolidine—Z-carb0xamide_TFA Salt, 11.
To a solution of tri-tert-butyl((2R,2'R,2"R)—((9,10-dihydro-9,10-
enzenoanthracene—2,7, l 5-triyl)tris(azanediyl))tris(3-(lH-imidazolyl)— 1 opane- 1 ,2-
diyl))tricarbamate (10) (1.5 g, 2.5 mmol) in dichloro methane (10 mL) was added trifiuoro
acetic acid (0.96 mL, 12.52 mmol) at room temperature. The ed reaction mixture was
stirred at room temperature for 2 h. The reaction mixture was filtered through celite bed and
filtrate thus collected was concentrated under reduced pressure to get crude 11 (1.4 g, 91.50 %)
as a brown oil which was used without further purification. MS (ESI—MS): m/z calcd for
C19H29N7O7 S [MH]+500.18, found 500.31.
Tri-tert—butyl (((9-(1-((2S,4S)—4-azido((2-nitrophenyl)sulfonyl)pyrr0lidin-Z-
yl)—2,1l-dimethyl-l,12—di0x0-5,8-di0xa-2,11-diazatetradecan-l4-yl)—9,10-dihydr0-9,10—
[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(8-0x00ctane—8,1-diyl))tricarbamate,
To a solution of (2S,4S)—4-azido—N—methyl-N-(2—(2-(2-
lamino)ethoxy)ethoxy)ethyl)- l itrophenyl)sulfonyl)pyrrolidinecarboxamide_TFA
Salt (11) (0.56 g, 0.91 mmol) in N,N-dimethylformamide (4 mL) were sequentially added 3-
(2,7, l 5-tris(8-((tert-butoxycarbonyl)amino)octanamido)-9, l0-[1,2]benzenoanthracen-9( 1 0H)-
yl)propanoic acid (ARK-18) (0.5 g, 0.46 mmol), HATU (1.44 g, 0.55 mmol) and N,N-
diisopropylethylamine (0.12 g, 0.91 mmol) at room temperature. The resulted reaction e
was stirred for l h at room temperature. The reaction mixture was poured in ice-cold water and
extracted with ethyl acetate (3 X 100 mL). The organic layers were combined, washed with brine
and concentrated under reduced pressure to get crude 12. The crude mixture was purified by
column chromatography on silica gel (15% methanol/chloroform) to get 12 (0.6 g, 84.5%) as a
brown solid. MS (ESI—MS): m/z calcd for C81H117N13017S [MH]+ 1576.84, found 1578.4.
Tri—tert—butyl (((9-(1-((2S,4S)—4-azidopyrrolidin-Z-yl)—2,11-dimethyl-1,12-di0xo-
,8-dioxa-2,1l-diazatetradecanyl)-9,10-dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-
triyl)tris(azanediyl))tris(8-0xooctane-8,1-diyl))tricarbamate, 13.
To a solution of rt-butyl (((9-(1—((ZS,4S)azido-l-((2-
nitrophenyl)su1fonyl)pyrrolidinyl)-2, 1 1-dimethyl-1,12-dioxo-5,8-dioxa—2,1 1-diazatetradecan-
14-yl)-9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15—triyl)tris(azanediyl))tris(8-oxooctane-
8,1-diyl))tricarbamate (12) (0.6 g, 0.38 mmol) in acetonitrile (10 mL) were sequentially added
potassium carbonate (0.26 g, 1.90 mmol) and enol (0.12 mL, 1.14 mmol) at room
temperature. The resulted reaction mixture was stirred at 80 0C for 2 h. The reaction e was
filtered h celite bed and the collected filtrate was concentrated under reduced pressure to
get crude 13 as yellow oil. The crude mixture was subjected to reverse phase chromatography to
yield 13 (0.4 g, 84.9%) as a light yellow solid. MS (EST—MS): m/z calcd for C53HszN1209 [MH]+
1391.86, found 1392.3.
2017/040514
Scheme: Synthesis of ARK-81
F: : :F 0
MN” F F do
BDCHN y) 0W0 N’éo BOCHNW\/\gNH
Wartype-Z BOCHNWNHC HBOC6
NH 6/60 0
ElocHN/VVW WNHBUC | 00
0 NH nt-A \N
Step -14 o8
/\/ 8
o\/‘O o
0 fill d) \
N3 ”$0
a (13)
MW: 1390.86 (14)
mw- 1523 89
Step -15 HCI in dioxane
MNH0 w
Mme/$9 WNW
HZN o 0 NH
0\ S o
NZ/CéfoN H
\ o
ARK-81_HCI_Sa|t
MW: 1323.74
o FjgF
Ho‘n/\ dk + EDCHCLTHF
O N O —> O
F :IEE:)[:6Lrod
o I F
OH Step-A
Warhead-2 d2-_lnt-A
MW 417.03
Perfluorophenyl 2-((1-methyl-2,4-dioxo-1,4-dihydro-2H-benzo[d][1,3]0xazin
yl)0xy)acetate, Int-A.
To a solution of Warhead-2 (0.048 g, 0.19 mmol) in tetrahydrofuran (1 mL) was
added N—(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (0.037 g, 0.19 mmol) at
0 °C under nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 10 min. To this, a
solution of pentafluorophenol (0.036 g, 0.19 mmol) in tetrahydrofuran (0.5 mL) was added drop
wise at O 0C under nitrogen here. The resulted reaction mixture was further stirred at 0 °C
for l h. The reaction mixture was directly used in the next step without work up and isolation.
MS (ESI—MS): m/z calcd C17H8F5N06 [MH]+ 418.03, the compound did not show mass
WO 06074
se. Note: Intermediate-A was not isolated — the reaction mass was transferred as such to
the next step reaction mass.
] Tri—tert—butyl (((9-(1-((ZS,4S)azido(2-((1-methyl-2,4-di0xo-1,4-dihydro-2H-
benzo [d] [1,3]oxazinyl)0xy)acetyl)pyrrolidinyl)—2,1 l-dimethyl-1,12-di0x0—5,8-di0xa-
2,11-diazatetradecanyl)—9,10-dihydr0-9,10—[1,2]benzenoanthracene—2,7,15-
triyl)tris(azanediyl))tris(8-0x00ctane-8,1-diyl))tricarbamate, 14.
To a solution of (((9-(3-((2-(2-((ZS,4S)azido—N—methylpyrrolidine-2—
carboxamido)ethoxy)ethyl) (methyl)amino)-3 -oxopropyl)—9, l 0-dihydro-
9, l0[l,2]benzenoanthracene-2,7, l l)tris(azanediyl)) tris(8-oxooctane-8, l-diyl))tricarbamate
(13) (0.27 g, 0.19 mmol) in tetrahydrofuran (4 mL) was added solution of pentafluorophenyl
[(l -methyl-2,4-dioxo- l ,4-dihydro-2H—3, l -benzoxazinyl)oxy]acetate
(Warhead_type_2_lnt_A) (0.081 g, 0.19 mmol) and the resulted reaction mixture was stirred
for l h at room temperature. The reaction mixture concentrated under reduced pressure to get
crude 14 (0.3 g, 80.21 %) as brown solid which was used in the next step without further
purification. MS (ESI-MS): m/z calcd C86H121N13018 [MH]+ 1623.89, found 1525.46 (M-100,
one Boc group fell off).
"-(9-(1-((ZS,4S)azido(2-((1-methyl-2,4-dioxo-1,4-dihydro-2H-
benzo [d] [1,3]0xazinyl)oxy)acetyl)pyrrolidinyl)—2, 1 l-dimethyl-l ,12-dioxo—5,8-dioxa-
2,11-diazatetradecanyl)—9,10-dihydr0-9,10-[1,2]benzenoanthracene—2,7,15-triyl)tris(8-
aminooctanamide), ARK-81_HCl salt.
To a solution of tri-tert-butyl (((9-(1-((ZS,4S)azido-l-(2-((l-methyl-2,4-dioxo-l,4-
dihydro-2H-benzo[d] [ l ,3]oxaziny1)oxy)acetyl)pyrrolidin-Z-yl)-2, l l-dimethyl-1,12-dioxo-5,8-
2,l l-diazatetradecan-l4-yl)-9, lO-dihydro-9, 10-[l,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))t1is(8-oxooctane-8,l-diyl))tricarbamate (14) (0.3 g, 0.0018 mmol) in
ydrofuran (5.0 mL) was added 4 M HCl in dioxane solution (2 mL) at room ature
and the ed reaction mixture was stirred for 4 h under nitrogen atmosphere. The reaction
mixture was concentrated under reduced pressure to get crude ARK-81_HCl_Salt as a yellow
solid. The crude mixture was purified by preparative HPLC using following method to get pure
ARK-81_HCl salt (0.034g, 12.8%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) 6 9.94
ppm (3H, br S), 7.79—7.86 ppm (8H, m), 7.66 ppm (2H, S), 7.43 ppm (1H, S), 7.31-7.18 ppm
(7H, m), 6.88—6.82 ppm (1H, m), 6.78—6.76 ppm (1H, m), 5.38 ppm (1H, S), 5.11-5.02 ppm (1H,
m), 4.80 ppm (1H, br S), 4.36-4.31 ppm (1H, m), 4.03-4.01 ppm (1H, m), 3.62-3.42 ppm (15H,
m), 3.37-3.26 ppm (4H, m), 3.16 ppm (1H, s), 3.05-2.99 ppm (5H, m), 2.89 ppm (1H, s), 2.81—
2.71 ppm (7H, m), 2.24 ppm (6H, S), 1.53-1.52 ppm (12H, (1), 1.26 ppm (18H, S). MS (ESI—
MS): m/z calcd for C71H97N13012 [MH]+1323.74, found 1325.4.
Method for ative HPLC:
(A) 0.05% HCl in water (HPLC GRADE) and (B) 100% acetonitrile (HPLC
GRADE), using X-SELECT C18, 250mm*19mm*5nm with the flow rate of /min and
with the following gradient:
__E_
——m_
2017/040514
Scheme: Synthesis of ARK-90
o:s:o
BocHNM E:
0 NH
BOCHNM 0
K O
6 COOH
/ BOCHNMVVYNHa 69I BocHNWNHC A/NHBOC HAd—ILJfiEiMF o NHWNHBOC
—> \
Step -14 (N
N\ o
H X
ofo o
,\/ o S SOZF
\N N\
‘0 hrs/Cf
NH (13) o (14)
N3 MW: 1390.86 MW: 1604.87
SIBP -15 HCI in dioxane
H NMNHQ
2 o
HzN/WNHCISQNswwNHz
0‘ SOZF
ARK-90_HC| salt
MW: 1304.72
Tri—tert—butyl (((9-(1-((2S,4S)—4—azido(3-(4-
(fluorosulfonyl)phenyl)propanoyl)pyrrolidinyl)-2,1 l-dimethyl-1,12-diox0-5,8-di0xa-2,1 1-
diazatetradecanyl)-9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))tris(8-ox00ctane-8,1-diyl))tricarbamate, 14.
To a on of tri-tert-butyl 1-((2S,4S)—4-azidopyrrolidin-Z-yl)—2,11-dimethyl-
1, 12-dioxo-5,8-dioxa-2,1 1-diazatetradecanyl)-9,10-dihydro-9,10-[1,2]benzenoanthracene-
2,7,15-triyl)tris(azanediyl))tris(8-oxooctane-8,1-diyl))tricarbamate (13) (0.4 g, 0.29 mmol) in
N,N-dimethylformarnide (4 mL) were sequentially added 3-(4—(fluorosulfonyl)phenyl)propanoic
acid (0.07 g, 0.29 mmol) and HATU (0.13 g, 0.35 mmol) at room temperature. The reaction
mixture was stirred for 5 minutes. To this, N,N-diisopropylethylamine (0.08 g, 0.56 mmol) was
added drop wise and the resulted reaction mixture was further stirred for 1h at room temperature.
The reaction mixture was diluted by ethyl acetate (100 mL) and washed with ice—cold water (3 X
30mL). The organic layers were combined, washed with brine and concentrated under reduced
pressure at 250C to get crude 14 (0.4 g, 87%) as a brown solid which was used in the next step
without further purification. MS (ESI—MS): m/z calcd for C84H121FN12016S [MH]+ 1605.88,
found 1506.5 (M-100, one Boc group fell off).
”-(9-(1-((ZS,4S)azido(3-(4-
(fluorosulfonyl)phenyl)pr0pan0yl)pyrrolidin-2—yl)—2,1l-dimethyl-1,12—di0x0-5,8-di0xa-2,11-
diazatetradecanyl)-9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))tris(8-amin00ctanamide), ARK-90_HCl salt.
To a solution tri—tert-butyl (((9-(1-((2S,4S)—4-azido—1-(3-(4-
(fluorosulfonyl)phenyl)propanoyl)pyrrolidinyl)-2, 1 1-dimethy1— 1 , 12-dioxo-5,8-dioxa-2,l 1-
diazatetradecanyl)—9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))t1is(8-oxooctane-8,1—diyl))tricarbamate (14) (0.4 g, 0.0025 mmol) in 1,4-
dioxane (5.0 mL) was added 4 M HCl in e (2 mL) at room temperature. The resulting
reaction mixture was stirred for 4 hours. The mixture was concentrated under reduced pressure to
get crude of ARK-90_HCl_Salt as yellow solid. The crude mixture was purified by preparative
HPLC using following method to get pure ARK-90_HCI salt (0.035 g, 7’. 11 %) as yellow solid.
1H NMR (400 MHz, DMSO) 5 9.89 ppm (3H, broad s), 8.03-8.00 ppm (2H, t), .56 ppm
(5H, m), 7.29-7.20 ppm (6H, m), 5.33 (1H, s), 3.62-3.52ppm (6H, m) 3.49-3.44 ppm (3H, m),
3.44-3.02 ppm (6H, m), 3.05-2.99 ppm (8H, m), 2.93 ppm (3H, broad s),2.76-2.70 ppm (10H,
m), 2.23(6H, s), 1519 (14H, s), 1.52 (21H, s). MS (ESI—MS): m/z calcd for C68H95FNIZOIOS
[MH]+. 2, found 1306.3. HPLC retention time: 10.894 min.
Method for preparative HPLC:
0.05% HCl in water (HPLC grade) and (B) 100% itrile (HPLC grade), using
WATERS X-BRIDGE C18, 250mm*30mm*5um with the flow rate of 35.0 mL/min and with
the following gradient:
"turn
Scheme: Synthesis of ARK-126
F W
o:é:o BocHN b 0
© NH 3 0
BocHNW/\g a QNHWNHBOC
HO *0 o:
HATU, DMF
DIPEA of0
Step -14
N3fit)0N:
(13) SOZF
MW: 1390.86
(14)
MW: 1576.84
Step -15 l HCI in dioxane
HZN “ 0
WNH , g o
HZN a QNHWNHz
SOZF
ARK-126_HCI_SaIt
MW: 1276.68
Tri-tert—butyl (((9-(1-((ZS,4S)azido-l-(3-(4-(fluor0sulfonyl)benzoyl)pyrrolidin-
2,1l-dimethyl-1,12-di0x0-5,8-di0xa-2,1l-diazatetradecan-l4-yl)-9,10-dihydr0-9,10-
[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris(8-0xooctane—8,l-diyl))tricarbamate,
To a solution of rt-butyl (((9-(1-((2S,4S)—4-azidopyrrolidin-Z-yl)—2,ll-dirnethyl-
1,12-dioxo-5,8-dioxa-2,1 l-diazatetradecan-l4-yl)-9, lO-dihydro-9, lO-[1,2]benzenoanthracene-
2,7,l5—triyl)tris(azanediyl))tris(8-oxooctane-8,l—diyl))tricarbamate (13) (011 g, 0.072 mmol) in
N,N—dimethylformamide (4 mL) were sequentially added 4-fluorosulfonylbenzoic acid (0.018 g,
0.09 mmol) and HATU (0.033 g, 0.09 mmol) at room temperature. The reaction mixture was
stirred for 5 minutes. To this, N,N—diisopropylethylamine (0.018 g, 0.14 mmol) was added drop
wise and the resulted reaction mixture was further stirred for 1h at room temperature. The
reaction mixture was diluted by ethyl acetate (100 mL) and washed with ice-cold water (3 X 30
mL). The organic layers were combined, washed with brine and concentrated under reduced
pressure at 25 0C to get crude 14 (0.12 g, quantitative yield) as a yellow semi-solid which was
used in the next step without further purification. MS (ESI—MS): m/z calcd for C82H117FN12016S
[MH]+ 1577.84, found 1478.46(M-100, one Boc group fell off).
N,N’,N”-(9—(1-((2S,4S)—4-azid0(3—(4-(fluorosulfonyl)benzoyl)pyrrolidinyl)-
2,11-dimethyl-1,12-diox0-5,8-di0xa-2,1l-diazatetradecan-l4-yl)—9,10-dihydro-9,10-
[1,2]benzenoanthracene-2,7,15—triyl)tris(azanediyl))tris(8-amin00ctanamide), ARK-
126_HCl salt.
To a solution of tri—tert-butyl (((9-(1-((2S,4S)-4—azido-l—(3-(4-
(fluorosulfonyl)benzoyl)pyrrolidinyl)—2, 1 1-dimethyl-1,12-dioxo-5, 8-dioxa-2,1 1-
diazatetradecan—14-yl)—9,10-dihydro-9,10—[1,2]benzenoanthracene—2,7,15-
triyl)tris(azanediyl))tris(8-oxooctane-8,1-diyl))tricarbamate (14) (0.12 g, 0.0007 mmol) in 1,4-
dioxane (5.0 mL) was added 4 M HCl in dioxane (2 mL) at room temperature and the resulting
reaction mixture was stirred for 4 hours. The mixture was trated under reduced pressure to
get crude ARK-126_HCI_Salt as a yellow solid. The crude mixture was purified by preparative
HPLC using following method to get pure 6_HCl_Salt (0.03 g, 28.57 %) as a yellow
solid. 1H NMR (400 MHz, DMSO) 6 .91 ppm (3H, broad s), 826—813 ppm (2H, m), 7.87
ppm (9H, broad s), 7.78-7.76 ppm (1H, d), 7.67 ppm (3H, broad s), 7.29—7.22 ppm (6H, m), 5.39
ppm (1H, s), 5010-4969 ppm (0.5H, t), 4.86-4.82 ppm (0.5H, m), 4.72-4.60 ppm (1H, m), 4.44-
4.36 ppm (1H, m), 4.30-4.21 ppm (1H, m), 4.14-4.00 ppm (1H, m), 3.64-3.61 ppm (20H, m)
3.48-3.37 ppm (6H, m), .11 ppm (3H, m), 3.07-3.03 ppm (5H, m), .84 ppm (2H,
broad s), 2.76 -2.68 ppm (7H, m), 2.26-2.23 ppm (6H, t), 1.53 ppm (12H, s), 1.27 ppm (18H, s).
MS (ESI—MS): m/z calcd for C67H93FN12OIOS [MH]+.1277.68, found 127835.
Method for preparative HPLC:
(A) 0.05% HCl in water (HPLC grade) and (B) 100% Acetonitrile (HPLC ,
using SUFTRE C18, 150mm*19mm*5um with the flow rate of 19.0mL/min and with the
ing gradient:
__IE_
Example 14: Synthesis of ARK-82, ARK-91, and ARK-127 (Ark000026, Ark000029, and
Ark000032)
Scheme: Synthesis of 13
o:é:o ‘3
Nosy|\
BOO 53°C N
‘ Nosyl \
/Nv\O/\/O\/\O/\/NH\ HATU, DMF N o N N
/ wow \/\o/\/
DIPEA ‘ 3TFA, MDC \
I 3
o —> /NH/\o/\/OV\o/\/N
ARK-22 Step .10 (10) Step -11
MW:320.23 MW: 643.26
(11)
MW: 543.21
Bod-(NM
WWNH
NH Bod-1N u NHBoc
BocHN E a O
Int-11
6 HATU, DMF
BocHNWVYNHa Q LVWNHBW —>DIPEA
0 NH Step -12 o‘fo
o OH
OH \Nf
ARK-18 o:/
MW: 6 "
NosyI/NO 'N;
(12)
MW: 7
Thiophenol,
Step -13 K2003” ACN
60°C
H110 N; (13)
MW: 1434.89
Tert—butyl (1-((2S,4S)azido((2-nitr0phenyl)sulfonyl)pyrrolidin-Z-yl)—2-
methyloxo-5,8,11-trioxaazatridecanyl)(methyl)carbamate, 10.
WO 06074
To a solution of ARK-22 (0.41 g, 1.281 mmol) in N,N—dimethylformamide (10 mL)
were sequentially added (2S,4S)azido((2-nitrophenyl)sulfonyl)pyrrolidinecarboxylic
acid (0.52 g, 1.54 mmol), HATU (0.584 g, 1.54 mmol) and N,N—diisopropylethylamine (0.33 g,
2.56 mmol) at room temperature. The resulted reaction mixture was stirred for 1 h at room
temperature. The reaction mixture was poured in ice-cold water and extracted with ethyl e
(3 X 100 mL). The organic layers were combined, washed with brine and concentrated under
reduced pressure to get crude 10 (0.8 g, 97.2 %).as brown semisolid which was used in next step
without further purification. MS S): m/z calcd for C26H41N7OIOS [MH]+ 644.26, found
544.36 (M-100).
(2S,4S)—4-azido-N-methyl((2-nitr0phenyl)sulfonyl)—N-(5,8,11-tri0xa
decanyl) pyrrolidinecarboxamide_TFA Salt, 11.
To a solution of tri— tert-butyl (1—((2S,4S)—4-azido—1-((2-
nitrophenyl)sulfonyl)pyrrolidinyl)methyloxo-5 ,8, 1 1-trioxaazatridecan-13 -yl)(methyl)
carbamate (10) (0.8 g, 1.24 mmol) in dichloro methane (10 mL) was added trifluoro acetic acid
(0.48 mL, 6.21 mmol) at room temperature. The resulted reaction mixture was stirred at room
temperature for 2 h. The reaction e was filtered through celite bed and filtrate thus
collected was trated under reduced pressure to get crude 11 (1.05 g, Quantitative yield) as
a brown oil which was used without r purification. MS (ESI—MS): m/z calcd for
C21H33N7OsSTFA [MH]+ 544.21, found 544.47.
Tri-tert—butyl (((9-(1-((2S,4S)azido((2-nitr0phenyl)sulfonyl)pyrr0lidin
yl)—2,14—dimethyl-1,15-di0x0-5,8,11-tri0xa-2,14-diazaheptadecanyl)—9,10-dihydro-9,10-
[1,2]benzenoanthracene-2,7,l5-triyl)tris(azanediyl))tris(8-ox00ctane-8,1-diyl))tricarbamate,
To a solution of (2S,4S)—4-azido-N-methyl((2-nitrophenyl)sulfonyl)-N-(5,8,11-
trioxaazatridecanyl) pyrrolidinecarboxamide_TFA Salt (11) (0.65 g, 0.98 mmol) in
N,N-dimethylformamide (4 mL) were tially added 3-(2,7,15-tris(8-((tert-
butoxycarbonyl)amino)octanamido)—9,10-[1,2]benzenoanthracen—9(10H)—yl)propanoic acid
(ARK-18) (0.9 g, 0.822 mmol), HATU (0.375 g, 0.98 mmol) and N,N—diisopropylethylamine
(0.21 g, 1.64 mmol) at room temperature. The resulted reaction mixture was stirred for 1 h at
room temperature. The reaction e was poured in ice-cold water and extracted with ethyl
WO 06074
acetate (3 X 100 mL). The organic layers were ed, washed with brine and concentrated
under reduced pressure to get crude 12. The crude mixture was purified by column
chromatography on silica gel (1.5% methanol/chloroform) to get 12 (1.72 g, quantitative yield)
as a brown solid which was used in the next step without further ation. MS (ESI—MS): m/z
calcd for C83H121N13OISS [MH]+ 1620.87, found 1522.31 (M-100).
Tri—tert—butyl (((9-(1-((2S,4S)—4-azidopyrrolidin-Z-yl)—2,14-dimethyl-1,15—di0xo-
-tri0xa-2,14-diazaheptadecany1)-9,10-dihydro-9,10-[1,2]benzenoanthracene-
2,7,15-triyl)tris(azanediyl))tris (8-0xooctane—8,1-diyl))tricarbamate, 13.
To a solution of tri-tert-butyl (((9-(1—((2S,4S)azido((2-
nitrophenyl)sulfonyl)pyrrolidinyl)-2,l4-dimethyl-l,15—dioxo-5,8, l l-trioxa-2,14—
diazaheptadecan-l7-yl)-9,10-dihydro-9,10-[1,2] benzenoanthracene—2,7, 15-
triyl)tris(azanediyl))t1is(8—oxooctane-8,l—diyl))tricarbamate (12) (0.7 g, 0.43 mmol) in
itrile (60 mL) were sequentially added potassium carbonate (0.29 g, 2.16 mmol) and
thiophenol (0.13 mL, 1.296 mmol) at room temperature. The resulted on mixture was
stirred at 80 °C for 2 h. The reaction mixture was filtered through celite bed and the collected
filtrate was concentrated under reduced pressure to get crude 13 as yellow oil. The crude
mixture was subjected to reverse phase chromatography to yield 13 (0.39 g, 62.9%) as a light
yellow solid. The crude was purified by trituration with n—Pentane (to remove unreacted
thiophenol) to get 13 (0.39 g, 62.9%) as a yellow solid. MS (ESI—MS): m/z calcd for
C77H113N12014[1V1H]+ 1435.89, found 1437.41.
Scheme: Synthesis of ARK-82
BocHNW 0 F
s F F
F:::F O
BocHNWfNH6/ Q WNHBOC owe/CELLOo
0 NH ‘6 ‘
o:( War type-2
Int-A
0f°H SteP -14
09/ ,, N
HNO “3
mo ”Q
MW: 143489 0%,}! o
(14)
MW: 1567,92
Step 45l HCI in e
ARK-82_HCI_SaIt
MW: 1367.76
Horomko F F: : :F o
o F F
+ EDCHCLTHF F Grom’koF o
F F
o '
OH Step-A
o I
Warhead-2 Warhead-2_lnt-A
MW 417.03
Perfluorophenyl 2-((1-methyl-2,4—di0x0-l,4-dihydr0-2H-benz0[d][1,3]0xazin
)acetate, Int-A.
To a solution of Warhead-2 (0.055 g, 0.21 mmol) in ydrofuran (1 mL) was
added N—(3-Dimethylaminopropyl)-N’—ethylcarbodiimide hydrochloride (0.047 g, 0.21 mmol) at
0 °C under nitrogen atmosphere. The reaction mixture was stirred at O 0C for 10 min. To this, a
solution of pentafluorophenol (0.04 g, 0.21 mmol) in tetrahydrofuran (0.5 mL) was added drop
wise at 0 °C under nitrogen atmosphere. The resulted on mixture was further stirred at 0°C
for 1 h. The reaction mixture was directly used in the next step without work up and isolation.
MS (ESI-MS): m/z calcd C17HsF5N06 [MH]+ 418.03, the nd did not show mass
response. Note: Intermediate-A was not isolated — the reaction mass was transferred as such to
the next step reaction mass.
] Tri—tert—butyl (((9-(1-((2S,4S)azido(2—((1-methyl-2,4-dioxo-1,4-dihydro-2H-
benzo [d] [1,3]oxazin yl)0xy)acetyl)pyrrolidin-Z-yl)—2,l4-dimethyl-1,15-di0xo-5,8,1 1-
trioxa-2,14-diazaheptadecanyl)-9,10-dihydr0-9,10—[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))tris(8-0xooctane-8,1-diyl))tricarbamate, 14.
To a solution of tri-tert-butyl (((9-(1—((2S,4S)—4-azidopyrrolidinyl)—2,14-dimethyl-
1,15-dioxo-5,8,1 1-trioxa-2,14-diazaheptadecanyl)-9,10-dihydro-9,10-
enzenoanthracene—2,7,15-triyl)tris(azanediyl))tris (8-oxooctane-8,1—diyl))t1icarbamate (13)
(0.3 g, 0.21 mmol) in tetrahydrofuran (4 mL) was added solution of pentafluorophenyl [(1-
methyl-2,4-dioxo-1,4-dihydro-2H—3,1-benzoxazin—7-yl)oxy]acetate (Warhead_type_2) (0.087
g, 0.21 mmol) and the resulted reaction mixture was stirred for 1 h at room temperature. The
reaction mixture concentrated under reduced pressure to get crude 14 (0.54 g, Quantitative yield)
as brown solid which was used in the next step without further ation. MS (ESI—MS): m/z
calcd CssH125N13019 [MH]+ 1668.92, found 1570.41 (M-100, one Boc group fell off).
N,N',N"-(9-(1-((2S,4S)azido(2-((1-methyl-2,4-dioxo-1,4-dihydro-2H-
benzo [d] [1,3]0xazinyl)oxy)acetyl)pyrrolidinyl)—2, 14-dimethyl-1 ,15-dioxo—5,8,1 1-
trioxa-2,14-diazaheptadecanyl)-9,10-dihydr0-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(8-aminooctanamide), _HCl salt.
To a solution of rt-butyl (((9-(1-((2S,4S)—4-azido—1-(2-((1-methyl-2,4-dioxo-1,4-
dihydro-2H-benzo[d][1,3]oxazin yl)oxy)acetyl)pyrrolidinyl)-2,14-dimethyl-1,15-dioxo-
, 8,1 1-trioxa-2,14-diazaheptadecan-l7-yl)-9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15 -
t1iyl)tris(azanediyl))t1is(8-oxooctane-8,1-diyl))tricarbamate (14) (0.54 g, 0.0032 mmol) in
tetrahydrofuran (5.0 mL) was added 4 M HCl in dioxane solution (2 mL) at room temperature
and the resulted reaction mixture was stirred for 4 h under nitrogen atmosphere. The reaction
mixture was concentrated under reduced pressure to get crude ARK-82_HCl_Salt as a yellow
solid. The crude e was purified by preparative HPLC using following method to get pure
ARK-81_HC1 salt g, 10.2%) as a yellow solid. 1H NMR (400 MHz, 6) 6 9.95
ppm (3H, br S), 7.99 ppm (8H, broad s), 7.90—7.88 ppm (2H, d), 7.66 ppm (3H, broad s), 7.46
ppm (2H, broad s), 7.33 ppm (2H, broad s), 7.28-7.25 ppm (5H, m), 7.23-7.21 ppm (2H, d),
6.89-6.85 ppm (1H, m), 6.78-6.76 ppm (1H, m), 6.55 ppm (2H, broad s), 5.38 ppm (1H, s), 5.12-
.00 ppm (2H, m), 4.77 ppm (1H, m), 4.37-4.34 ppm (3H, m), 4.06-4.05 ppm (1H, m), 3.82 ppm
(1H, m), 3.63-3.43 ppm (15H, m), 3.09-3.01 ppm (7H, m), 296-294 ppm (1H, d), 2.82-2.80
ppm (1H, d), .64 ppm (7H, m), 2.24 ppm (7H, broad s), 1.54-1.52 ppm (12H, (1), 1.26 ppm
(18H, 5). MS (ESI—MS): m/Z calcd for C73H101N13013 [MH]+1368.76, found 137025.
Method for ative HPLC:
(A) 0.05% HCl in water (HPLC GRADE) and (B) 100% acetonitrile (HPLC
GRADE), using KINETEX BIPHENYL, 250mm*21.2mm*5pm with the flow rate of 20.0
mL/min and with the following gradient:
__IE_
2017/040514
Scheme: Synthesis of ARK-91
COOH
HATU, DMF
DIPEA
Step -14
\NfOH 2.,”
HD “3 (13)
MW: 1434.89 \%
$02F (14)
MW: 1648.89
Step -15 j HCI in dioxane
ARK-91_HCI_SaIt
MW: 1348.74
Tri—tert—butyl (((9-(1-((2S,4S)—4—azid0(3-(4-
(fluorosulfonyl)phenyl)propanoyl)pyrrolidin-2—yl)—2,14-dimethyl-1,15-diox0-5,8,1l-trioxa-
2,14-diazaheptadecanyl)-9,10-dihydr0-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))tris(8-ox00ctane-8,1-diyl))tricarbamate, 14.
To a solution of tri-tert-butyl (((9-(1-((2S,4S)—4-azidopyrrolidin-Z-yl)—2,l4-dimethyl-
l,15-dioxo-5,8,l l-trioxa-Z,14-diazaheptadecan—17-yl)-9,10-dihydro-9,10-
[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris (8-oxooctane-8,l-diyl))tricarbamate (13)
(0.30 g, 0.21 mmol) in N,N-dimethylformamide (6 mL) were sequentially added 3-(4-
(fluorosulfonyl)phenyl)propanoic acid (00.058 g, 0.25 mmol) and HATU (0.095 g, 0.25 mmol)
2017/040514
at room temperature. The reaction mixture was stirred for 5 minutes. To this, N,N-
diisopropylethylamine (0.054 g, 0.42 mmol) was added dropwise and the ed reaction
mixture was further stirred for 1h at room temperature. The on mixture was diluted by ethyl
acetate (100 mL) and washed with ice-cold water (3 x 30mL). The organic layers were
combined, washed with brine and concentrated under reduced pressure at 25 °C to get crude 14
(0.55 g, quantitative yield) as a brown solid which was used in the next step without further
purification. MS (ESI-MS): m/z calcd C86H125Fl\112()178[lVH‘I]+ 1649.89, found 1551.29 (M-100,
one Boc group fell off).
N,N',N"-(9-(1-((2S,4S)azido(3-(4-
(fluorosulfonyl)phenyl)pr0panoyl)pyrrolidinyl)-2,14-dimethyl-1,15—di0xo-5,8,11-tri0xa-
2,14-diazaheptadecanyl)-9,10-dihydr0-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))tris(8-aminooctanamide), _HCl salt.
To a solution rt-butyl (((9-(1-((2S,4S)azido—l-(3-(4-(fluorosulfonyl)phenyl)
propanoyl)pyrrolidinyl)-2,l4—dimethyl—l,15-dioxo-5, 8,1 l-trioxa-2, l4-diazaheptadecan—l 7-yl)-
9,10-dihydro-9,l0-[1,2]benzenoanthracene-2,7,15-triyl)tris(azanediyl))tris(8-oxooctane-8,ldiyl
))tricarbamate (14) (0.55 g, 0.0033 mmol) in l,4-dioxane (9.0 mL) was added 4 M HCl in
dioxane (4 mL) at room temperature. The resulting reaction mixture was stirred for 4 hours. The
mixture was concentrated under reduced pressure to get crude of ARK-91_HCl_Salt as yellow
solid. The crude mixture was purified by preparative HPLC using following method to get pure
ARK-91_HC] salt (0.09 g, 18.5 %) as yellow solid. 1H NMR (400 MHz, DMSO—d6) 8 9.94 ppm
(3H, broad s), 8.04-8.00 ppm (2H, m), 7.96 ppm (6H, broad s), 7.66 ppm (4H, broad s), 7.62-
7.52 ppm (1H, m), 7.31-7.18 ppm (6H, broad s), 5.38 ppm (1H, s), 4.71-4.66 ppm (1H, m), 4.25
ppm (9H, m), 3.40-3.99 ppm (1H, m), .49 ppm (9H, m), 3.44-3.35 ppm (5H, m), 3.31-3.24
ppm (2H, m), 3.16-3.15 ppm (2H, m), .00 ppm (6H, m), 2.95-2.91 ppm (3H, m), 2.77-2.69
ppm (7H, m), .23 ppm (6H, t), 1.54-1.52 ppm (12H, d), 1.26 ppm (18H, broad s). MS
(ESI—MS): m/z calcd for C71H101FN120118 [MH]+ 1349.74, found 8.
Method for preparative HPLC:
(A) 0.05% HCl in water (HPLC GRADE) and (B) 100% acetonitrile (HPLC
GRADE), using X-SELECT C18, 250mm*30mm,5um with the flow rate of 23.0mL/min and
with the following gradient:
2017/040514
Scheme: Synthesis of ARK-127
a HNWNH‘ Exocr-Ir\1/\/\/\/\(r 09
C?» ,F
/\/\/\/\/NH 0 H000 EUCHN/VWY Q
EccHN U\/\/\/\/NHE°°
g HWNHBDc O NH
HATU, DMF
DIPEA
Step -14 NAB
\ 0H \Nk/‘O
ANf 0d,
HN "N3 (13) \
MW: 9
0:350
F (14)
MW: 1620,87
Step -15 l HCI in dinxane
ARK-127_HCI_SaIt
MW: 1320.71
tri-tert—butyl (((9-(1-((2S,4S)—4-azid0(4—(flu0r0sulfonyl)benzoyl)pyrrolidin
yl)—2,14—dimethyl-1,15-di0X0-5,8,1 1-trioxa-2,14-diazaheptadecanyl)-9,10-dihydro-9,10-
[1,2]benzenoanthracene-2,7,15-triyl)tris(azanediyl))tris(8-0x00ctane-8,1-diyl))tricarbamate,
To a solution of tri-tert-butyl (((9-(1—((2S,4S)—4-azidopyrrolidin-Z-yl)—2, 14-dimethy1-
1,15-dioxo-5,8,1 l-trioxa-Z,14-diazaheptadecany1)-9,10-dihydro-9,10-
[1,2]benzenoanthracene—2,7,15-triyl)tris(azanediyl))tris (8-oxooctane-8,1-diyl))tricarbamate (13)
(0.05 g, 0.03 mmol) in N,N-dimethylformamide (2 mL) were sequentially added 4-
fluorosulfonylbenzoic acid (0.09 g, 0.04 mmol) and HATU (0.016 g, 0.04 mmol) at room
temperature. The on mixture was stirred for 5 minutes. To this, N,N—diisopropylethylamine
(0.09 g, 0.14 mmol) was added drop wise and the ed reaction mixture was further stirred
for 1h at room temperature. The on mixture was diluted by ethyl acetate (100 mL) and
washed with ice-cold water (3 x 30mL). The organic layers were ed, washed with brine
and concentrated under reduced pressure at 25 °C to get crude 14 (0.075 g, quantitative yield) as
a yellow semi—solid which was used in the next step without further purification. MS (ESI—MS):
m/z calcd C84H121FN12017S [MH]+ 1621.87, found 1523.47 (M-100, one Boc group fell off).
4-((2S,4S)—4—azid0-2—(methyl(12-methyl0x0(2,7,15-tris(8-
aminooctanamido)—9,l0-[1,2]benzenoanthracen-9(10H)—yl)—3,6,9-trioxa
azapentadecyl)carbamoyl)pyrrolidinecarbonyl)benzenesulfonyl fluoride, ARK-127_HCl
salt.
To a on of tri-tert-butyl (((9-(1-((2S,4S)azido-l-(4-
(fluorosulfonyl)benzoyl)pyrrolidinyl)—2,14-dimethyl-1,15-dioxo—5, 8,1 l—trioxa-2, 14-
diazaheptadecan-l7-yl)—9,10-dihydro-9,10-[1,2]benzenoanthracene-2,7,15-
triyl)tris(azanediyl))tris(8—oxooctane-8,1—diyl))tricarbamate (14) (0.075 g, 0.0005 mmol) in 1,4-
dioxane (3.0 mL) was added 4 M HCl in dioxane (1 mL) at room temperature and the resulting
on mixture was stirred for 4 hours. The mixture was concentrated under reduced re to
get crude ARK-127_HC1_Salt as a yellow solid. The crude mixture was purified by preparative
HPLC using following method to get pure ARK-127_HC1_Salt (0.014g, 21.2%) as a yellow
solid. 1H NMR (400 MHz, DMSO-d6) 5 9.89 ppm (3H, broad s), 8.26-8.22 ppm (1H, m), 8.16
ppm (1H, m), 7.89-7.85 ppm (9H, m), 7.75 ppm (1H, m), 7.69-7.66 ppm (3H, m), 7.29-7.22 ppm
(5H, m), 5.38 ppm (1H, s), 4.99-4.87 ppm (2H, m), 4.39-4.38 ppm (1H, m), 4.28-4.16 ppm (1H,
m), 4.05-4.02 ppm (1H, m), 3.81-3.74 ppm (1H, m), 3.64-3.52 ppm (9H, m), 3.38-3.28 ppm (7H,
m), .99 ppm (8H, m), 2.76-2.65 ppm (8H, m), .23 ppm (5H, t), 1.54 ppm (11H,
broad s), 1.27 ppm (18H, broad 5). MS (ESI—MS): m/z calcd for C69H97FN12OIIS [MH]+ 1321.71,
found 1322.42.
Method for preparative HPLC:
WO 06074
(A) 005% HCl in water (HPLC grade) and (B) 100% Acetonitrile (HPLC grade),
using SUNFIRE C18, 250mm*l9mm*5um with the flow rate of 20.0mL/min and with the
following gradient:
__II_
——m_
Example 15: ation of CPNQ Analogues and Other Quinoline—Based Ligands
Exemplary small molecule ligands based on CPNQ and other quinoline scaffolds
were prepared based on the tic schemes shown in s 63-71. Analytical data for the
prepared compounds are shown below in Table 5.
Table 5: Analytical Data for CPNQ Analogues and Quinoline—Based Ligands
Target Mol. HPLC HPLC LCMS LCMS
ID Weight RT Purity RT Purity
DMSO-d6: 6 9.04-9.03 ppm (1H,
dd, J: 4, 1.6 Hz), 8.66-8.63 ppm
(1H, J: 8.4, 1.2 Hz), 8.26-8.24
ppm (1H,d, J=8.4 Hz), 7.73-7.70
ppm (1H, dd, J: 8.4, 4 Hz), 7.53-
7.47 ppm (4H, m), 7.26-7.24
6.902
97.56/oo
ppm (1H, d, J=8.4 Hz),3.96 ppm
ARK-131 620.3 621.69 95.7860
min (2H, br s), 3.64 ppm (4H, brs),
3.51 ppm (3H, m), 3.43-3.39
ppm (2H, m), 3.30 ppm (2H, m),
3.23-3.18 ppm (4H, m), 2.99-
2.95 (3H, m), 2.89-2.79 ppm
(5H, m), 1.38-1.37 ppm (9H, d,
J=5.6 Hz).
6: 6 9.03 ppm (1H, br
s), 8.76-8.74 ppm (1H, d, J=8.4
Hz), 8.21-8.19 ppm (1H, d, J=8
Hz), 7.74-7.72 ppm (1H,
m),7.58-7.53 ppm (4H, m), 7.31-
7.30 ppm (1H, d, J=6 Hz), 4.67
7 792
ARK-137 654.26 655.64 min 97.73% 96.05% ppm (1H, br s), 4.28 ppm (1H, br
s), 3.97-3.91 (1H, m), 3.71 ppm
(3H, br s), 3.59 ppm (1H, br s),
3.46 ppm (1H, brs), 3.28-3.10
ppm (6H, m), 2.96-2.94 ppm
(2H, br s), 2.79-2.68 ppm (5H,
m), 1.39-1.35 ppm (9H, d,
Mol. HPLC HPLC LCMS LCMS
ID Weight RT Purity RT Purity
J=15.2 Hz).
D20: 6 7.66-7.64 ppm (2H, d,
J=7.6 Hz), .49 ppm (1H, t,
J: 14.8, 7.6 Hz), 7.43-7.39 ppm
(2H, t, J: 15.2, 7.6 Hz), 3.40-
ARK-138 249.18 3.36 ppm (2H, m), 3.25-3.18
ppm (2H, m), 3.16-3.09 ppm
(2H, m), 2.99-2.96 ppm (2H, t,
J: 15.2, 7.6 Hz), 2.80 ppm (3H,
s), 2.06-1.94 ppm (4H, m).
DMSO-d6: 6 9.05-9.04 ppm
(1H, dd, J=4, 1.6 Hz), .65
ppm (1H, dd, J=8.4, 1.2 Hz),
8.28-8.25 ppm (1H, d, J=8.4 Hz),
ARK-179 396.1 95.09% .71 ppm (1H, dd, J=8.8,
4.4 Hz), 7.57-7.51 ppm (4H, m),
7.26-7.24 ppm (1H, d, J=8.4 Hz),
3.94 ppm (2H, br s), 3.65 ppm
(2H, br s), 3.21 ppm (4H, br s).
DMSO-d6: 6 9.04-9.03 ppm
(1H, dd, J=4.4, 1.6 Hz), 8.65-
8.63 ppm (1H, dd, J=8.8, 1.6
Hz), 8.25-8.23 ppm (1H, d, J=8.4
Hz),7.73-7.70 ppm (1H, dd,
ARK-180 380.13 381.39 67.91 96.24% 4.185 98.46%
mln J=8.8, 4 Hz), 7.58-7.55 ppm (2H,
m), .29 ppm (2H, m), 7.26-
7.24 ppm (1H, d, J=8.4 Hz), 3.93
ppm (2H, br s), 3.67 ppm (2H, br
s), 3.19 ppm (4H, br s).
DMSO-d6: 6 9.04-9.03 ppm
(1H, dd, J=4, 1.6 Hz), 8.65-8.63
ppm (1H, dd, J=8.8, 1.6 Hz),
8.25-8.23 ppm (1H, d, J=8.4
7.404 Hz),7.73-7.69 ppm (3H, m),
ARK-181 440.04 441.4 97.43% 96.33%
in 7.46-7.44 ppm (2H, dd, J=6.8,
1.6 Hz ), 7.26-7.23 ppm (1H, d,
J=8.4 Hz), 3.94 ppm (2H, brs),
3.64 ppm (2H, brs), 3.21-3.17
, .
DMSO-d6: 6 9.04-9.03 ppm
(1H, d, J=2.8 Hz), 8.67-8.65 ppm
(1H, d, J=8.8 Hz), 8.26-8.24 ppm
(1H, d, J=8.4 Hz), 7.74-7.71 ppm
ARK-182 392.15 96.87% 99.00% (1H, dd, J=8.8, 4.4 Hz), 7.47-
7.44 ppm (2H, d, J=8.8 Hz),
7.26-7.24 ppm (1H, d, J=8.4 Hz),
7.03-7.01 ppm (2H, d, J: 8.4
Hz), 3.83-3.81 ppm (2H, d, J=6.4
Mol. HPLC HPLC LCMS LCMS
ID Weight RT Purity RT Purity
Hz), 3.19 ppm (4H, brs), 2.55
ppm (3H, 3)-
DMSO-d6: 6 9.04-9.03 ppm
(1H, dd, J=4, 1.2 Hz), 8.66-8.63
ppm (1H, dd, J=8.8, 1.6 Hz),
8.25-8.23 ppm (1H, d, J=8 Hz),
6.650m 7.73-7.70 ppm (1H, dd, J=8.4, 4
ARK-183
in Hz), 7.51-7.47 ppm(5H, br s),
7.26-7.24 ppm (1 H, dd, J=8.4
Hz), 3.95 ppm (2H, brs), 3.65
ppm (2H, br s), 3.20-3.19 ppm
4H, br s .
6: 6 .03 ppm
(1H, dd, J=4, 2.8 Hz), 8.65-8.63
ppm (1H, d, J=8.4 Hz), 8.26-8.24
ppm (1H, d, J=8.4 Hz), 7.80-7.71
7 5:3
ARK-184 430.06 431.35 96.37% 98.88% ppm (3H, m), 7.51-7.48 ppm(1H,
dd, J=8, 1.6 Hz), 7.26-7.24 ppm
(1H, d, J=8.4 Hz), 3.94 ppm (2H,
br s), 3.64 ppm (2H, br s), 3.22
(2H, 8), 3.16 ppm (2H, S).
DMSO-d6: 6 9.04-9.03 ppm
(1H, dd, J=4, 1.6 Hz), 8.65-8.63
ppm (1H, dd, J=8.4, 1.6 Hz),
8.25-8.23 ppm (1H, d, J=8 Hz),
7.73-7.70 ppm (1H, dd, J=8.8,
7 010
ARK-185 369.09 397.45 96.30% 4.4 Hz), .54 ppm (2H, m),
7.52-7.50 ppm (1H, m), 7.46-
7.44 ppm (1H, m), 7.26-7.24
ppm (1H, d J=8.4 Hz), 3.95 ppm
(2H, br s), 3.63 ppm (2H, br s),
3.23-3.17 ppm (4H, br d).
DMSO-d6: 6 9.00-8.99 ppm
(1H, dd, J=4, 1.2 Hz), 8.52-8.50
ppm (1H, dd, J=8.4, 1.6 Hz),
8 050 8.23-8.21 ppm (1H, d, J=8.4 Hz),
ARK-186 432.06 433.39 96.44%
m n .79 ppm (4H, m), 7.64-
7.61 ppm (1H, dd, J=8.4, 4 Hz),
7.26-7.24 ppm (1H, d, J: 8 Hz),
3.24 ppm (8H, s).
DMSO-d6: 6 9.03-9.02 ppm
(1H, dd, J=4.4, 1.6 Hz), 8.70
ppm (1H, br s), 8.26-8.24 ppm
(1H, d, J=8 Hz), 7.71-7.68 ppm
(1H, dd, J=8.8, 4.4 Hz), 7.56-
ARK-187 426.11 97.61% 99.40%
7.51 ppm (4H, m), 7.39 ppm
(1H, br s), 4.62-4.54 ppm (1H, br
s), 4.13-4.10 ppm (1H, m), 3.87
ppm (1H, br s), 3.60-3.51 ppm
(3H, m), 3.45-3.39 ppm (3H, br
Weight HPLC HPLC LCMS
RT Purity LgMS Purity
s), 3.11 ppm (1H, br s).
DMSO-d6: 6 9.02-8.99 ppm
(1H, dd, J=10.4, 3.2 Hz), 8.63-
8.61 ppm (1H, dd, J: 43.6, 8.4
Hz), 8.23-8.20 ppm (1H, m),
7.72-7.62 ppm (1H, m), 7.55-
7.47 ppm (3H, m), 7.41-7.39
ARK-189 98.36% ppm (1H, d, J=8 Hz), 7.28-7.23
ppm (1H, m), 3.93 ppm (1H, br
s), 3.85-3.82 ppm (1H, t, J:
11.2, 5.6 Hz), 3.56-3.53 ppm
(3H, m), 3.46-3.42 ppm (3H, m),
2.17 ppm (1H, br s), 2.01 ppm
(1 H, br s).
DMSO-d6: 6 8.97-8.96 ppm
(1H, d, J=2.8 Hz), 8.62-8.60 ppm
(1H, d, J: 7.6 Hz), .09
ppm (1H,dd, J: 36, 8.8 Hz),
ARK-190 408.1 409.16 6-5.75 97.92% 7.58-7.47 ppm (5H, m), 6.98-
6.81 ppm (1H, m), 4.92-4.78
ppm (1 H, m), 4.41-4.22 ppm
(2H, m), 3.81 -3. 59 ppm (3H, m),
DMSO-d6. 6 8. 96 ppm (1H, br
s), 8.61 ppm (1H, brs), 8.20-
8.09 ppm (1H, m), 7.52 ppm
ARK-191 408.1 409.21 65.14 (5H,br s), 6.98-6.82 ppm (1H,
98.38% 98.54%
mln m), 4.92-4.78 ppm (1H, m), 4.41-
4.22 ppm (2H, m), 3.79-3.61
ppm , 2.14-2.09 ppm (2H,
DMSO-d6: 6 8.66 ppm (1H, s),
8.06-8.04 ppm (1H, d, J=8.4 Hz),
7.87-7.82 ppm (2H, d, J=12.4,
ARK-194 352.11 353.44 6-492 99.65% 99.38%
mln 8.8 Hz), 7.59-7.51 ppm (5H, m),
3.85 ppm (4H br s), 3.76 ppm
DMSO-d6: 6 7. 7 ppm
(4H, m), 7.04-7. 00 ppm (1H t
, 7.6 Hz), 6.45-6.42 ppm
ARK-196 342.11 343.48 7'8.73 (2H, dd, J=8, 3.6 Hz), ,4.50-4.46
96.62% 95.57%
mln ppm (2H, t, J=16.8, 8.4 Hz), 3.75
ppm (2H, br s), 3.46 ppm (2H, br
s), 3.13-3.09 ppm (2H, t, J: 8.4
Hz), 3.03-2.97 ppm (4H, m).
Example 16: Exemplary Compound Data
Additional data for compounds whose preparation is described above as well as
ures of further exemplary compounds are provided in Table 6 below.
2017/040514
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Example 17: RNA Sequences Prepared
The following RNA sequences were designed and prepared for use in testing
compound binding (including verifying the ed binding mode or identifying the binding
mode, when not known) and validating the methods of the t invention.
Table 7: RNA Sequences ed
RNA Length
Modifications Sequence (5' to 3')
Designation (nt)
GCUGCCGGGACGGGUCCAAGAUGGA
CGGCCGCUCAGGUUCUGCUUUUACC
UGCGGCCCAGAGCCCCAUUCAUUGC
CCCGGUGCUGAGCGGCGCCGCGAGU
CGGCCCGAGGCCUCCGGGGACUGCC
GUGCCGGGCGGGAGACCGCCAUGGC
GACCCUGGAAAAGCUGAUGAAGGCC
Exon 1 of the UUCGAGUCCCUCAAGUCCUUCCAGC
HTTmRNA AGCAGCAGCAGCAGCAGCAGCAGCA
HTT.EXOn1.41
ww41CAG GCAGCAGCAGCAGCAGCAGCAGCAG
repeats (HD CAGCAGCAGCAGCAGCAGCAGCAGC
disease) AGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAACAG
CCGCCACCGCCGCCGCCGCCGCCGC
CGCCUCCUCAGCUUCCUCAGCCGCC
GCCGCAGGCACAGCCGCUGCUGCCU
CAGCCGCAGCCGCCCCCGCCGCCGC
CCCCGCCGCCACCCGGCCCGGCUGU
GGCUGAGGAGCCGCUGCACCGACC
GCUGCCGGGACGGGUCCAAGAUGGA
CGGCCGCUCAGGUUCUGCUUUUACC
UGCGGCCCAGAGCCCCAUUCAUUGC
CCCGGUGCUGAGCGGCGCCGCGAGU
CGGCCCGAGGCCUCCGGGGACUGCC
GUGCCGGGCGGGAGACCGCCAUGGC
GACCCUGGAAAAGCUGAUGAAGGCC
Exon 1 of the UUCGAGUCCCUCAAGUCCUUCCAGC
HTTmRNA AGCAGCAGCAGCAGCAGCAGCAGCA
HTT.Ex0n1.41
ww41CAG GCAGCAGCAGCAGCAGCAGCAGCAG
CAG_5Bio
s (HD CAGCAGCAGCAGCAGCAGCAGCAGC
dbease) AGCAGCAGCAGCAGCAGCAGCAGCA
GCAGCAGCAGCAGCAGCAGCAACAG
CCGCCGCCGCCGCCGCCGC
CGCCUCCUCAGCUUCCUCAGCCGCC
GGCACAGCCGCUGCUGCCU
CAGCCGCAGCCGCCCCCGCCGCCGC
CCCCGCCGCCACCCGGCCCGGCUGU
GGCUGAGGAGCCGCUGCACCGACC
RNA L72?" Modifications Sequence (5‘t0 3')
Designation
GCUGCCGGGACGGGUCCAAGAUGGA
CGGCCGCUCAGGUUCUGCUUUUACC
UGCGGCCCAGAGCCCCAUUCAUUGC
GCUGAGCGGCGCCGCGAGU
CGGCCCGAGGCCUCCGGGGACUGCC
GUGCCGGGCGGGAGACCGCCAUGGC
Exon 1 ofthe GACCCUGGAAAAGCUGAUGAAGGCC
HTT mRNA UUCGAGUCCCUCAAGUCCUUCCAGC
HTT.Ex0n1.17
with 17 CAG AGCAGCAGCAGCAGCAGCAGCAGCA
repeats GCAGCAGCAGCAGCAGCAGCAGCAA
(healthy) CAGCCGCCACCGCCGCCGCCGCCGC
CGCCGCCUCCUCAGCUUCCUCAGCC
GCCGCCGCAGGCACAGCCGCUGCUG
CCUCAGCCGCAGCCGCCCCCGCCGC
CGCCCCCGCCGCCACCCGGCCCGGC
UGUGGCUGAGGAGCCGCUGCACCGA
GCUGCCGGGACGGGUCCAAGAUGGA
CGGCCGCUCAGGUUCUGCUUUUACC
UGCGGCCCAGAGCCCCAUUCAUUGC
CCCGGUGCUGAGCGGCGCCGCGAGU
CGGCCCGAGGCCUCCGGGGACUGCC
GUGCCGGGCGGGAGACCGCCAUGGC
Exon 1 ofthe GACCCUGGAAAAGCUGAUGAAGGCC
HTT mRNA UUCGAGUCCCUCAAGUCCUUCCAGC
on1.17
with 41 CAG AGCAGCAGCAGCAGCAGCAGCAGCA
CAG_5Bio
repeats GCAGCAGCAGCAGCAGCAGCAGCAA
(healthy) CAGCCGCCACCGCCGCCGCCGCCGC
CUCCUCAGCUUCCUCAGCC
GCCGCCGCAGGCACAGCCGCUGCUG
CCUCAGCCGCAGCCGCCCCCGCCGC
CGCCGCCACCCGGCCCGGC
UGUGGCUGAGGAGCCGCUGCACCGA
Portion of the GCAGCAGCAGCAGCAGCAGCAGCAG
41CAG HTT CAGCAGCAGCAGCAGCAGCAGCAACA
AG_3
RNA having GCCGCCACCGCCGCCGC
WJ_SBio
the 3-way
junction
Portion of the GCAGCAGCAGCAGCAGCAGCAGCAG
HTT17CAG_in 17CAG HTT CAGCAACAGCCGCCACCGCCGCCGC
ternalbulge_5B RNA having CGCCGCCGCCGCCU
io the internal
bulge
220AG_hairpi A hairpin CAGCAGCAGCAGCAGCAGCAGCAGC
n_SBio consisting of a AGCAGCAGCAGCAGCAGCA
pure stretch of
RNA Length
Modifications Sequence (5' to 3')
Designation (nt)
Portion of the GCAGCAGCAGCAGCAGCAGCAGCAG
41CAG HTT CAGCAGCAGCAGCAGCAGCAGCAACA
HTT41CAG_3
RNA having ACCGCCGCCGC
the 3-way
junction
Portion of the GCAGCAGCAGCAGCAGCAGCAGCAG
17CAG HTT CAGCAACAGCCGCCACCGCCGCCGC
HTT17CAG_in
RNA having CGCCGCCGCCGCCU
ternalbulge
the internal
bulge
A hairpin CAGCAGCAGCAGCAGCAGC
22CAG_hairpi consisting of a AGCAGCAGCAGCAGCAGCAGCAGCA
n pure stretch of GCAGCAGCAGCAGCAG
22 CAGs
GAGCCUAAAACAUACCAGAGAAAUCU
Tetracycline Tetracycline
GGAGAGGUGAAGAAUACGACCACCUA
Aptamer binding RNA
GGCUC
0.0.0 GGCACAAAUGCAACACUGCAUUACCA
_0.0. 38 n
Triptycene 3- UGCGGUUGUGCC
Way Junction
0.0.0 GGCACAAAUGCAACACUGCAUUACCA
Triptycene 3- UGCGGUUGUGCC
RNA3WJ_0.0. 5' Iowa Black; Way on
0_5|B_3FAM 3' 6FAM with
fluorophore &
quencher
0.0.0 GGCACAAAUGCAACACUGCAUUACCA
Triptycene 3- UGCGGUUGUGCC
RNA3WJ_0.0. Way Junction
3' 6FAM
0_3FAM with
fluorophore but
no quencher
1.0.0 GGCACACAAUGCAACACUGCAUUACC
Triptycene 3- AUGCGGUUGUGCC
RNA3WJ_1 .o. 5' Iowa Black; Way Junction
0_5|B_3FAM 3' 6FAM with
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quencher
_1 .1 .
' Iowa Black; 1.1.0 GGCACACAAUGCAACACUGCAUUGAC
0_5|B_3FAM 3' 6FAM Triptycene 3- CAUGCGGUUGUGCC
Way Junction
RNA Length
Modifications ce (5' to 3')
Designation (nt)
with
fluorophore &
quencher
2.0.0 GGCACACGAAUGCAACACUGCAUUAC
cene 3- CAUGCGGUUGUGCC
RNA3WJ_2.0. 5' Iowa Black; Way Junction
0_5|B_3FAM 3' 6FAM with
fluorophore &
quencher
1.1.1 GGCACACAAUGCAACACUGCAUUGAC
Triptycene 3- CAUGCGGUAUGUGCC
RNA3WJ_1 .1 .
' Iowa Black; Way Junction
3FAM 3' 6FAM with
fluorophore &
2.1.0 GGCACACGAAUGCAACACUGCAUUGA
Triptycene 3- CCAUGCGGUUGUGCC
RNA3WJ_2.1.
' Iowa Black; Way Junction
0_5|B_3FAM 3' 6FAM with
fluorophore &
quencher
3.0.0 GGCACACAGAAUGCAACACUGCAUUA
Triptycene 3- CCAUGCGGUUGUGCC
RNA3WJ_3.0. 5' Iowa Black; Way Junction
0_5|B_3FAM 3' 6FAM with
fluorophore &
quencher
0.0.0 GGCACAAAUGCAAC
Triptycene 3-
Split3WJ.1_up
' Iowa Black Way Junction
_5IB
split at first
Ioop;5'end
0.0.0 ACUGCAUUACCAUGCGGUUGUGCC
Triptycene 3-
WJ.1_do
3' 6FAM Way Junction
wn_3FAM
split at first
Ioop;3'end
0.0.0 GGCACAAAUGCAACACUGCAUUACCA
Triptycene 3- U
Split3WJ.2_up
' Iowa Black Way Junction
_5IB
split at second
loop; 5' end
RNA Length .. . . . , ,
0.0.0 GCGGUUGUGCC
prfvg’lflfh—Ado.
Triptycene 3-
11 3' 6FAM Way Junction
- split at second
loop;3'end
Example 18: Fluorescence Quenching g Assay
This assay will be used to test binding of compounds for RNA three way junction
(such as a 38 nt construct). This is a fluorescence quenching assay ing FAM as
fluorescence tag and Iowa Black as quencher. Tags are ed at the 3’ and 5’ end,
tively. Stable formation of 3WJ upon compound binding would lead to ing of the
FAM fluorescence due to close proximity of the Iowa Black tag. Assay readout: FAM (485 —
520 nm) Fluorescence Intensity.
Nucleic acid ons are ubiquitous structural motifs, occurring in both DNA and
RNA. They represent important and sometimes transient structures in biological processes, such
as replication and recombination, while also ing in triplet repeat expansions, which are
associated with a number of neurodegenerative diseases. Nucleic acid junctions are ubiquitous
in viral genomes and are important structural motifs in riboswitches. way junctions are
key building blocks present in many nanostructures, soft materials, multichromophore
assemblies, and aptamer-based sensors. In the case of aptamer based sensors, DNA three-way
junctions serve as an ant structural motif.
This assay can serve as a part of the toolkit for discovering RNA-binding small
molecules by testing binding to a 3WJ in the context of a controlled system with a readily
observable t. PEARL-seq or other methods disclosed herein may then be used to further
screen compounds.
Assay sample buffer used: 10 mM CacoK pH 7.2, 30 mM NaCl. Buffer preparation
in Dnase / Rnase Free distilled water (Gibco Life Technologies).
Compound Preparation
Tool compounds provided as dry powder are prepared as 50 mM stock solution in
100% dé-DMSO. Stock solutions of 50 mM concentration in ds-DMSO are stored at RT.
Hardware
Sample plate: Greiner cat# 784076, black, 384 (Dilution plate: Greiner REF 781101,
PS-Microplate, 384 well, . Fluorescence Intensity : Envision 1040285
Assay Protocol
Assay Buffer preparation
Daily fresh (10 ml): 1 ml 100 mM CacoK pH 7.2 and 0.3 ml 1 M NaCl filled up to 10
ml with Dnase / Rnase Free distilled water
RNA preparation [RNA sample homogenization]
Dilute the RNA 1:10 (final 10 uM) in Assay Buffer.
Heat up the diluted RNA up to 90 0C for 5 min (sealed Eppendorf Tube).
Cool down the RNA probe slowly to RT.
nd preparation
Dilute the compounds to 800 uM in DMSO (Assay: 8 uM).
Sample preparation
71.2-78.4 uL Assay Buffer are pipetted into Greiner REF 781101, PS-Microplate,
384 (each well needed).
Add 0.8-8 uL of the RNA-Solution (100 mM).
] Add 08 [LL nd—Solution (800 mM).
Mix gently with Multi-Channel Pipette.
Final trations in the sample: 1—10 uM RNA, 8 uM compound, 1 % DMSO
Thermal Shift measurement Li htC cler480
Pipet 25 uL Sample Solution into Greiner cat# 784076, black, 384
When sample transfer is finished put lid on top.
] Measure the 96 plate with the LightCycler480 (Channel: 485/520 nm).
Readout
Software used was PerkinElmer Envision Manager.
——-_-
——-_-
. Copy 2 of
Protocol Name. --
F|_picogreen_fi|ter_LV_opt
Picogreen_fi|ter_LV_opt _- 4000045
——-_
——-_
——-_
—-_—
——-_
——-_
Description. . X485 CWL=485nm BW=14nm
DELFIA - Time-resolved
Used with
scence
TRF Emission520 _—
Filter type -_—
. . M520 CWL=520nm BW=25nm
. DELFIA - Time-resolved
Results
Calibration of the expected cence signal at various RNA trations in
either CacoK or NaPO4 buffers was performed first. Experiments in buffers containing salt show
distinct fluorescence quenching behavior. A calibration experiment for the CacoK buffer is
shown in Figure 72. r s were ed for the NaPO4 buffer (results not shown).
First, two compounds (i.e. Ark000007 & Ark000008) were tested in the fluorescence
quenching assay to assess concentration dependent influence on the cence signal. Only
Ark000007 showed an increase of quenching effect vs. 3WJ_0.0.0_SIB_3FAM construct at
conc. >5 uM (Figure 73). Remaining buffer and sample conditions did not show significant
influence of the compound on the fluorescence .
The fluorescence quenching experiment was repeated for compounds Ark0000013
and Ark0000014 to measure binding with:
A) RNA3WJ_1.0.0_SIB_3FAM (cis 3WJ with one unpaired nucleotide)
B) Split3WJ.1_up_SIB -- Split3WJ.1_down_3FAM (trans 3WJ as 1:1 mix)
C) Split3WJ.2_up_SIB -- Split3WJ.2_down_3FAM (trans 3W] as 1:1 mix)
Likely structures for the sequences are illustrated in Figure 74, and the results of the
experiments are shown in Figure 75. Both cmpds were tested at two concentration points in the
fluorescence quenching assays to assess effect upon RNA constructs utilized in the study.
Ark000013 (curves associated with de 13 in the Figure) shows a significant concentration
dependent effect upon all three RNA constructs used (least effect for cis 3W] and equal effects
for trans 3WJs). The data suggest specific interaction of Ark000013 with the 3WJ constructs.
Ark000014 (de14) shows a smaller effect on the RNA constructs (Split3WJ_2 shows larger
effect). The compound does appear to be interacting with the RNA target species.
Example 19: Thermal Shift Binding Assay
Purpose: Test binding of nds for RNA three way junction (for example, a
construct of 38 nt). Thermal shift assay based on established cence quenching assay
utilizing FAM as fluorescence tag and Iowa Black as quencher. Tags were attached at the 3’ and
’ end, respectively. Stable formation of 3WJ upon compound binding would lead to quenching
of the FAM fluorescence due to close proximity of the Iowa Black tag. l unfolding leads
to se of fluorescence emission. Assay readout: FAM (465 — 510 nm) Thermal Shift.
This assay can serve as a part of the toolkit for discovering RNA-binding small
molecules by testing binding to a 3WJ in the context of a controlled system with a readily
observable readout. PEARL-seq or other methods disclosed herein may then be used to r
screen compounds.
Assay sample buffer used: 10 mM CacoK pH 7.2, 30 mM NaCl. Buffer ation
in Dnase / Rnase Free distilled water (Gibco Life logies).
Compound Preparation
Tool compounds provided as dry powder are prepared as 50 mM stock solution in
100% ds-DMSO. Stock solutions of 50 mM tration in d6-DMSO are stored at RT.
Hardware
Sample plate: Roche, Light Cycler480 Multiwell 6, white, REF 04729692001.
(Dilution plate: Greiner REF 781101, PS-Microplate, 384 well, clear). Thermal Shift device:
Roche, Light Cycler480.
Assay Protocol
Assay Buffer preparation
Daily fresh (10 ml): 1 ml 100 mM CacoK pH 7.2 and 0.3 ml 1 M NaCl filled up to 10
ml with Dnase / Rnase Free distilled water.
RNA re aration NA sam le homo enization
Dilute the RNA 1:10 (final 10 uM) in Assay Buffer.
Heat up the diluted RNA up to 90 0C for 5 min (sealed Eppendorf Tube).
Cool down the RNA probe slowly to RT.
nd preparation
Dilute the compounds to 800 uM in DMSO (Assay: 8 uM).
Sample preparation
78.4 uL Assay Buffer are pipetted into Greiner REF 781101, PS-Microplate, 384
(each well ).
Add 0.8 uL of the RNA-Solution (100 mM).
Add 0.8 uL Compound-Solution (800 mM).
Mix gentle with Multi-Channel Pipette.
Final concentrations in the sample: 1 uM RNA, 8 uM compound, 1% DMSO
Thermal Shift measurement [LightCycler480]
Pipet 20 uL Sample Solution into Roche, Light Cycler480 Multiwell Plate96, white,
REF 04729692001.
When sample transfer is d, seal the plate with a clear l (part of REF
04729692001).
Centrifuge the plate with a table-top device to spin down the samples.
Measure the 96plate with the LightCycler480 el: 480/510 nm; ature:
41 — 91°C).
Analyse measurement-data with the MeltingCurveGenotyping Mode.
LightCycler480 LCS480 1.5.1.62 LightCycler ThermalShift Analysis
Settings: ition mode: continuous; Ramp rate: 0.1 C°/sec; Acquisition: 6/C°
Melt Curve Genotyping for All Samples
Channel 480/510 nm
Progam Name Program
Stds Settings Auto-Group
Sensitivity normal
Temp Range 41 — 91°C
Score 0.7
Res. 0.1
Curves are fitted with raw and normalized data.
Results
Melting curves is show melting ature (Tm) of ~51 °C. Range of RNA
concentrations was tested and assay window was determined (conc. range of 0.5 — 1 uM yields
best results). The choice of buffer also affected the Tm. RNA constructs were tested under
different buffer ions (especially in presence of salt) in the thermal shift assay. Increase of
salt tration shows a tendency to increase melting ature. However, as seen already
for the fluorescence quenching assay, this observation is strongly dependent on buffer
conditions. CacoK with 30 mM salt at 1 uM RNA conc. was used to assess compound effects on
3WJ stability. RNA ucts were tested under different buffer conditions (especially in
presence of salt) in the thermal shift assay. As expected, an increase of salt concentration shows
a tendency to increase melting temperature. However, as seen already for the fluorescence
quenching assay, this observation is strongly dependent on buffer conditions. The RNA
construct was folded in ce of higher salt concentration and had a melting temperature of
61 oC rather than the 51 0C at lower salt concentration. These conditions were used for
screening test compounds.
Compounds Ark000007 & Ark000008 were tested in the thermal shift assay with the
3WJ_0.0.0_5D3_3FAM RNA construct (Figure 76). Data analysis shows a significant effect for
Ark000007 with melting temperature shift of ~5°C (i.e. from 61.2 0C to 65.6 0C). In contrast,
only a very small effect for Ark000008 was observed. These data suggest that the presence of
Ark000007 increases stability of the 3WJ.
Compounds 0013 and 0014 were also tested in the thermal shift assay
against three RNA 3WJ constructs, A) RNA3WJ_1.0.0_5IB_3FAM (cis 3WJ with one unpaired
nucleotide); B) Split3WJ.1_up_5IB + Split3WJ.1_down_3FAM (trans 3WJ as 1:1 mix); and C)
Split3WJ.2_up_5IB + Split3WJ.2_down_3FAM (trans 3WJ as 1:1 mix).
When the compounds were tested with _1.0.0_5IB_3FAM, data analysis
showed a significant effect for Ark000013 in the melting curves with a significantly lower
fluorescence signal in presence of the nd (Figure 77).
Normalized data showed no proper melting curve in the presence of Ark000013 and
the algorithm of data analysis software was unable to determine a meaningful melting point. A
weaker effect was observed for 014, with a g temperature shift of ~3 °C (i.e. from
65.6 0C to 68.4 °C). The data suggest that the presence of Ark000013 increases stability of the
3W1 fold upon binding, whereas Ark000014 shows a much less pronounced effect. These results
are in line with the cence quenching assay.
In the ce of the B) RNA above,
Split3WJ.1_up_5IB+Split3WJ.1_down_3FAM, data analysis showed a significant effect for
Ark000013, with a g ature shift of ~21 °C (i.e. from 37.5 0C to 58.2 C’C) (Figure
78). Only a minor effect was ed for Ark000014 with a melting temperature shift of only
~1 °C (i.e. from 37.5 0C to 38.8 0C). The data suggested that the presence of Ark000013
increased the stability of the 3W1 fold upon binding, s Ark000014 showed a much less
pronounced effect. The 3WJ formed in trans from 2 RNA molecules shows a significantly lower
ity than the cis folded 3WJ (in absence and presence of cmpd). Especially in absence of a
compound, a stem-loop structure with a larger bulge is possibly the most populated
conformation.
In the presence of the C) RNA above,
Split3WJ.2_up_5IB+Split3WJ.2_down_3FAM, data analysis showed a significant effect for
Ark000013, with a melting temperature shift of ~13 °C (i.e. from 44.0 °C to 56.9 C’C) (Figure
79). Only a minor effect was observed for Ark000014, with melting temperature shift of only ~1
oC (i.e. from 44.0 0C to 44.7 0C). The data suggest that the presence of Ark000013 increases the
stability of the 3WJ fold upon g, whereas Ark000014 shows a much less pronounced
effect. The trans 3WJs studied seem to show lower stability than the cis 3WJs, however, the
Split_2 3WJ adopts a more stable conformation than Split_l (in absence of a compound). In the
presence of a compound, the melting temperature for both trans 3WJ Split_l & Split_2 is
similar, suggesting the formation of a 3WJ fold in the presence of the compound.
Ark0000013 and Ark0000014 were tested with a number of RNA constructs. The
results are shown below in Tables 8 and 9. nd Ark000039 was also tested in the thermal
shift assay vs. the cis folded RNA 3WJs at different RNA:ligand ratios (i.e. 1:1, 1:3). For
construct 3WJ_0.0.0_5IB_3FAM the raw data shows no cant effect for Ark000039 in the
melting curves (neither at equimolar trations nor 3x molar excess). Also, normalized data
show no significant effect for cmpd Ark000039. It appears that Ark000039 does not
significantly influence stability of the 3WJ fold and hence no indication of binding for
Ark000039 was observed. The same lack of effect was noted in tests with sequences
RNA3WJ_3.0.0_SIB_3FAM and RNA3WJ_1.0.0_5IB_3FAM.
Table 8: 0013 Thermal Shift Data
3WJ construct Meltinimitp [°C] ' Meltingciiigidp [°C] + Temp. Shift [°C]
RNA3WJ_0.0.0_5|B_3FAM 61.2 84.1 24.2
RNA3WJ_1.0.0_5|B_3FAM 65.6 87.0 21.4
RNA3WJ_1.1.0_5|B_3FAM 63.3 85.5 22.2
RNA3WJ_1.1.1_5|B_3FAM 62.2 82.9 20.7
RNA3WJ_2.0.0_5|B_3FAM 62.2 84.3 22.1
RNA3WJ_2.1.0_5|B_3FAM 41.9 45.7 3.8
RNA3WJ_3.0.0_5|B_3FAM 62.0 83.7 21.7
Split3WJ_1 37.8 58.2 20.4
Splil3WJ_2 44.7 56.9 12.2
Table 9: Ark0000014 Thermal Shift Data
3WJ construct Me'tinixgap [°C] - Meltingctlfigdp [°C] + Temp. Shift [°C]
RNA3WJ_0.0.0_5|B_3FAM 59.9 61.5 1.6
RNA3WJ_1.0.0_5|B_3FAM 65.6 68.1 2.5
RNA3WJ_1.1.0_5|B_3FAM 63.3 65.1 1.8
_1.1.1_5|B_3FAM 62.2 64.3 2.1
_2.0.0_5|B_3FAM 62.2 64.4 2.2
RNA3WJ_2.1.0_5|B_3FAM 41.9 42.0 0.1
RNA3WJ_3.0.0_5|B_3FAM 62.0 63.9 1 .9
Split3WJ_1 37.5 37.8 0.3
Split3WJ_2 44.3 44.0 -o.3
Table 10: Thermal Shift Data for onal Compounds Tested with RNA Sequence
0.0_SIB_FAM
Compound Collaboration Remarks
007 ARK-1 61.2 65.6
ARK000008
ARK000009 ARK-3 62.3 62.9
ARK000010 ARK-4 61.6 61.4
ARK000011
ARK000012 ARK-6 N/A N/A N/A
ARK000013 ARK-7 59.9 84.1 +24.2
ARK000014 ARK-8 59.9 61 5 +1.6
Enantiomer of target
ARK-10, TFA Salt of
ARK000015- same material is
ARKO1 +17.6
registered as
ARK000015-2 (ARK
Enantiomer of target
ARK-10, HCI Salt of
ARK000015- same material is
ARK02 +19.0
registered as
ARK000015-1 (ARK
Enantiomer of target
ARK000016 ARK-10 62.5 83.8 +21.3
ARK000015 (ARK-9)
Enantiomer of target
ARK000017
ARK000018-1 (ARK-12)
Enantiomer of target
ARK000018
ARK000017-1 (ARK-11)
Enantiomer of target
ARK000022 ARKD 59.8 60.2 +0.4 ARK000023-1 (ARK
Enantiomer of target
ARK000023 ARK000022-1 (ARK
ARK000019 ARK-14 60.3 60.0
ARK000021 ARK-16 59.5 40.5
LCMS carried out in a
long 16 min run time
ARK000024 ARK-80 60.4 60.7 +0.3
method hence HPLC is
not ed separately.
025 ARK-81 60.6 50.4 LCMS carried out in a
2017/040514
Compound Collaboration Remarks
.0 Code
long 16 min run time
method hence HPLC is
LCMS carried out in a
long 16 min run time
ARK000026 ARK-82 59.7 60.9 I _\ N
. method hence HPLC is
not recorded tely.
LCMS carried out in a
long 16 min run time
ARK000027 ARK-89 59.7 83.4 +23.7
method hence HPLC is
not recorded tely.
LCMS carried out in a
long 16 min run time
ARK000028 ARK-90 60.3 82.3 +22.0
method hence HPLC is
not recorded separately.
LCMS carried out in a
long 16 min run time
ARK000029 ARK-91 60.2 63.2 + (A) O
. method hence HPLC is
not recorded separately.
LCMS carried out in a
long 16 min run time
ARK000030 ARK-125 60.0 64.8 +A
. G) method hence HPLC is
not ed separately.
LCMS carried out in a
long 16 min run time
ARK000031 ARK-126 60.0 84.1 +24.1
method hence HPLC is
not ed separately.
LCMS carried out in a
long 16 min run time
ARK000032 ARK-127 60.1 75.1 +15.0
method hence HPLC is
not recorded separately.
LCMS carried out in a
long 16 min run time
ARK000033 ARK-77 61.5 62.4 +O
. (O method hence HPLC is
not recorded separately.
LCMS carried out in a
long 16 min run time
ARK000034 ARK-77A 59.9 60.2 +0.3
method hence HPLC is
not recorded separately.
ARK000039 ARK-138 61.5 60.3
Interestingly, hook and click compounds (PEARL-seq compounds) bearing ligand,
tether, warhead, and click-ready group, such as ARKOOOO31 and ARKOOOO32, showed large
thermal shift values of +24] and +150 C’C, indicating strong binding to the RNA target
sequence.
Example 20: Ligand Observed NMR g Assay
Purpose: Test direct binding of compounds for RNA three way junction (3WJ). This
ligand observed NMR assay is used to test direct binding of compounds to an RNA target, for
example a 38 nt synthetic RNA 3WJ and others as described below. Ligand observed assay was
used for hit validation studies of single nds. Established experiments were eventually
used to m group epitope mapping, described below.
Assay Reagents and Hardware
Sample buffer: 10 mM Cacodylate, pH 7.1; 0.68 g [MW: 137.99 g/mol]; fill up to 500
ml with Millipore H20.
Compound Preparation
Compound Stocks: Tool compounds provided as dry powder were prepared as 50
mM stock solution in 100% d6—DMSO. Test compounds ed as dry powder were ed
as 50 mM stock solution in 100% ds-DMSO. Stock solutions of 50 mM concentration in d6'
DMSO were stored at 4 oC.
Hardware
Sample tube: NMR tube; Norell, article# ST500-7 for NMR sample ement
NMR spectrometer: Bruker AVANCE6OO spectrometer ing at 600.0 1Vle for
1H. 5-mm z-gradient TXI Cryoprobe.
Assay Procedure
RNA preparation [RNA sample homogenization]
Dried RNA pellet is solubilized in sample buffer 10 mM Cacodylate pH 7.1.
] RNA aliquot at 200 uM (stock concentration) is denatured at 95 0C for 3 min and
ooled on ice for 3 min.
Sample preparation
23 pL d6-DMSO are pipetted into a 1.5 mL eppendorf tube to ensure 5% ds-DMSO
present in the sample as a locking agent.
Add 2 pL of each fragment (50 mM stock solution).
Add 450 pL assay buffer.
] Add 25 uL homogenized RNA stock solution of the RNA 3WJ (200 uM stock
on).
] Sample is vortexed to ensure proper mixing and placed into NMR spectrometer to
start measurement of the sample.
Final concentrations in the : 200uM each compound and 10 uM RNA target
molecule.
NMR measurement
Sample is placed into magnet and temperature adjusted to 288 K. Spectrometer
frequency at 600 MHz is matched and tuned. Magnetic field is shimmed to homogenize
magnetic field around the sample.
Proton 90° pulse is determined and water resonance ncy is adjusted to ensure
maximal water suppression. The determined values are transferred to the NMR experiments that
will be recorded for the respective sample.
Sequence of experiments includes a Proton lD experiment with a Watergate sequence
for water suppression, a WaterLOGSY Y) and a 1D Saturation transfer difference
(STD) experiment to test for direct binding of the compounds to the RNA.
Details ID Watergate experiment: For each lD WATERGATE spectrum a total of
8192 complex points in fl (1H) with 128 scans were acquired (experiment time 4 min.). The
spectral width was set to 16.66 ppm.
s WLOGSY experiment: For the WLOGSY-spectrum, a total of 1024 complex
points in fl (1H) with 256 scans were acquired (experiment time 25 min.) The carrier frequency
for 1H was set at the water resonance (~4.7 ppm). The spectral width was set to 16.66 ppm in the
direct dimension (1H).
Details STD experiment: For the STD-spectrum, a total of 1024 complex points in fl
(H) with 1024 scans were acquired iment time 65 min). The carrier frequency for 1H was
set at the water resonance (~4.7 ppm). The al width was set to 16.66 ppm in the direct
dimension (1H). For the on-resonance experiments saturation is set to 2.0 sec at a saturation
frequency of -2500 Hz. For the off-resonance experiment tion frequency is set at 10200
Readout
Software: TopspinTM version: 2.1 (October 24, 2007)
Measurement mode: 1D
Python scripts are used to s all recorded spectra in the assay setup, ing
and deconvolution s.
Spectra were analyzed for direct binding signals of the compounds. Identified single
compound hits were reported.
Ligand Observed NMR Binding Assay for CAG Repeat RNA
Following the above procedure, various tool and test compounds were assayed for
binding. In a first series of experiments, nds were tested for binding to 17CAG or
41CAG (sample was 3 uM in RNA). Compounds HP-AC008001-A08, HP-AC008002-A06,
HP-AC008002-D10, and most of an l screen of 41 small molecule fragments did not show
significant difference in binding signals for both RNA target species 17CAG and 41CAG.
However, several of the compounds did show significant changes in their signals in the presence
of the two RNA target species.
Structure ID STD signal
/ | H
HP-AT005003-C03 ct
O N\/L
QM/N\/N HP-AC008001-E02 distinct
/ HP-AC008002-E01 distinct
N/ HP-AC008004-C07 weak
N/fi /
K/N '
N CPNQ d'Istlnct
ARK0000013 was also tested in the NMR binding assay. Test Sample: 10 uM
RNA3WJ_0.0.0_5IB_3FAM +/- 200 Ark000013. 1H 1D Watergate & WaterLOGSY spectra
recorded of Ark000013 were used as a reference (Note: aromatic signals observed between 7.4 —
7.9 ppm and due to symmetry of the center triptycene scaffold all 9 protons are magnetically
equivalent). In the presence of RNA a clear reduction of negative sign signals occured for the
Ark000013 resonances. Data suggested binding of Ark000013 to the 3WJ RNA as the target
s. STD experiments showed small signals that were sufficient to qualitatively confirm
binding.
Epitope Mapping
Epitope mapping was performed on a number of compounds. As a first e,
compound CPNQ was analyzed at a concentration of 50 uM. A 1H 1D Watergate um with
zoom to aromatic region of the spectrum was obtained. Preliminary ment of 1H
resonances for this and the following examples was based on al shift distribution,
coupling pattern and simulation of NMR spectrum (www.nmrdb.org). The structure of CPNQ,
assigned proton resonances, NlVIR spectrum, and epitope mapping results are shown in Figure
80. Due to signal overlap no individual assignment of the piperazine ring system was possible.
ions: 10 mM Tris pH 8.0, 5 mM DTT, 5% DMSO-ds; T = 288.1 K. Epitope mapping
experiments were performed in the presence of 4lCAG and l7CAG ces using the STD
mental conditions described above. In the case of CPNQ, data suggests for both RNA
constructs the tendency that protons of the chlorophenyl moiety are in closer proximity to RNA
than the nitroquinoline.
The same experiment was performed for compound 08002-E01 under similar
conditions (see Figure 81). The scaled STD effect was plotted onto the molecule according to
the preliminary assignments. The data suggests for both RNA constructs that protons of the
pyridine ring are in closer proximity to RNA than the benzene ring. The aliphatic CH2 group
could not be observed due to buffer signal overlap in that region.
The same experiment was performed for compound HP-AC00800l-E02 under similar
conditions (see Figure 82). The scaled STD effect was plotted onto the molecule according to
the preliminary assignments. The data suggest for both RNA constructs that ic protons
t to the heterocycle are in closer proximity to RNA protons. Aliphatic proton resonances
could not be assessed by STD due to direct saturation artifacts/buffer signal overlap in that
region (epitope mapping by WaterLOGSY).
The same experiment was performed for compound HP-AT005003-C03 under similar
conditions (see Figure 83). The scaled STD effect was plotted onto the molecule according to
the preliminary assignments. Due to signal overlap no individual assignment of the CH2 groups
was possible. The data suggest for both RNA constructs that protons of the furan moiety are in
closer proximity to RNA protons than the phenyl.
NMR Competition ments
Competition experiments were also performed. Test Sample: 2.5 uM 4lCAG RNA
(476 nt) was combined with the following: 100 uM HP—AC008002-E01 (A); +/— 200 — 400 uM
HP-ACOOSOOl—EOZ (B); and +/- 200 — 400 uM 05003-C03 (C). 1H lD Watergate &
OGSY a ed of HP-AC008002—E01 are used as a reference. In the presence
of itor (i.e. either HP-AT005003-CO3 or HP-ACOOSOOl—EOZ) the WaterLOGSY signals
of HP-AC008002-E01 were still observed, even at a 1:4 ratio of compound vs. competitor. The
experiments did not reveal any indication of itive behavior in the utilized compound
mixtures. Data suggests that compounds do not compete for the same single binding site.
In a further experiment, as Test Sample 2.5 uM 4lCAG RNA (476 nt) was used in
the presence of: 100 uM I-IP-ACOOSOOl-EOZ (B) or 100 uM HP-AT005003-C03 (C); +/- 200 —
400 uM HP-AC008002-E01 (A). 1H 1D Watergate and OGSY spectra recorded single
cmpds were used as a reference. In the presence of a competitor (i.e. I-IP-AC008002-E01 (A))
the WaterLOGSY signals of HP-ACOOSOOl-EOZ (B) or HP-AT005003-CO3 (C) were still
observed even at a 1:4 ratio of cmpd vs. competitor. Experiments did not reveal any indication
of itive or in the utilized compound es. The data suggest that compounds do
not compete for the same single binding site.
In a r experiment, as Test Sample: 2.5 pM 41CAG RNA (476 nt) was used in
the presence of: 100 [AM HP-ACOOSOOl—EO2 (B) +/- 200 — 400 pM HP-ATOO5003-CO3 (C) 1H
1D Watergate & WaterLOGSY spectra recorded of single cmpd HP-ACOOSOOl-EO2 were used
as a reference, In the presence of competitor (i.e. HP-AT005003-C03 (C)) the WaterLOGSY
signals of HP-ACOOSOOl-EO2 (B) were still observed even at a 1:4 ratio of cmpd vs. competitor.
The experiments did not reveal any indication of competitive behavior in the utilized compound
mixture. The data suggest that compounds do not compete for the same single binding site.
Example 21: Ligand Observed NMR Binding Assay for CAG Repeat RNA
Purpose: Test direct binding of compounds for httmRNA (construct with 41 CAG
repeats 474 nt) and others as described below. Ligand observed NMR assay to test direct
binding of fragments to RNA target (e.g. construct with 41 CAG repeats 474 nt). Single
compound hits were identified for further characterization by orthogonal assay (eg. surface
plasmon resonance, SPR). Ligand observed assay was used for primary screen and
deconvolution into single fragment hits. ished ments were eventually used for group
epitope mapping.
CAG repeat expansions in protein coding portions of specific genes are fied as
Category I repeat expansion diseases. Currently, nine ogic ers are known to be
caused by an increased number of CAG repeats, typically in coding s of otherwise
unrelated ns. During protein synthesis, the expanded CAG repeats are translated into a
series of uninterrupted glutamine residues forming what is known as a utamine tract
(“polyQ”).
This assay tests for direct binding of compounds to httmRNA and may be adapted for
other repeat RNAs. Compounds are tested in pools (i.e. pool size of 12 fragments in each sample
in the primary screen and smaller pool sizes during deconvolution and eventually single
nd measurements).
Assay Reagents and Hardware
Sample buffer: 10mM Tris-HCl, pH 8.0, 0.78g [MW: 157.56 g/mol]; 75 mM KCl,
2.79g [MW: 74.55 g/mol], 3 mM MgC12, 0.14g [MW: 95.21 g/mol], fill up to 500 mL with
Millipore H20.
Compound Preparation
Compound Stocks: nt library stock solutions are provided at 100 mM
concentration in 100% d6-DMSO. Tool compounds provided as dry powder are ed as 100
mM stock on in 100% d6-DMSO. Stock solutions of 100 mM concentration in ds—DMSO
are stored at 4°C.
Hardware
Sample tube: NMR tube; Norell, e# ST500-7 for N1V[R sample measurement.
NMR spectrometer: Bruker AVANCE6OO spectrometer ing at 600.0 1Vle for
1H. 5-mm z-gradient TXI Cryoprobe.
Assay Procedure
RNA preparation [RNA sample homogenization]
Dried RNA pellet is solubilized in sample buffer 10 mM Tris-HCl pH 8.0, 75 mM
KCl, 3 mM MgClz. RNA aliquot at 13.9 uM (stock concentration) is denatured at 95 °C for 3
min and snap-cooled on ice for 3 min, and refolded at 37 °C for 30 min.
Sample preparation
13-24 uL d6—DMSO are ed into an 1.5 mL orf tube to ensure 5% d6-
DMSO t in the sample as a locking agent (depending on pool size of the prepared sample).
Add 1 “L of each fragment (100 mM stock solution).
Add 367 uL assay buffer.
Add 108 uL homogenized RNA stock solution of the httmRNA (13.9 [1M stock
solution).
Sample is vortexed to ensure proper mixing and placed into NMR spectrometer to
start measurement of the sample.
Final concentrations in the sample: 200 [1M each fragment and 3 [1M RNA target
molecule.
NMR measurement
Sample is placed into magnet and temperature adjusted to 288 K. Spectrometer
frequency at 600 1Vle is matched and tuned. Magnetic field is shimmed to homogenize
magnetic field around the sample.
Proton 90° pulse is determined and water resonance frequency is adjusted to ensure
maximal water suppression. The determined values are transferred to the NMR ments that
will be recorded for the tive sample.
Sequence of experiments includes a Proton 1D experiment with a Watergate sequence
for water suppression, a WaterLOGSY (WLOGSY) and a 1D Saturation transfer ence
(STD) experiment to test for direct binding of the compounds to the RNA.
Details for 1D Watergate experiment: For each 1D WATERGATE spectrum a total of
8192 x points in fl (1H) with 128 scans were acquired (experiment time 4 min.) The
spectral width was set to 16.66 ppm.
s WLOGSY experiment: For the WLOGSY-spectrum, a total of 1024 complex
points in f1 (1H) with 256 scans were acquired (experiment time 25 min.) The carrier frequency
for 1H was set at the water resonance (~4.7 ppm). The spectral width was set to 16.66 ppm in the
direct dimension (1H).
Details STD experiment: For the STD—spectrum, a total of 1024 complex points in f1
(H) with 1024 scans were acquired (experiment time 65 min.) The carrier frequency for 1H was
set at the water nce (~47 ppm). The spectral width was set to 16.66 ppm in the direct
dimension (1H). For the on-resonance experiments saturation is set to 2.0 sec at a saturation
frequency of -2500 Hz. For the off-resonance experiment saturation frequency is set at 10200
Software: TopspinTM version: 2.1 er 24, 2007)
Measurement mode: 1D
Python scripts were used to process all recorded spectra in the assay setup, screening
and olution process. Spectra were analyzed for direct binding signals of the compounds.
fied single compound hits were reported.
Example 22: Illumina Small RNA-Seq Library Preparation Using T4 RNA Ligase 1
Adenylated Adapters
Purpose: Enable deep sequencing of a short synthetic RNA after treatment with
SHAPE reagents or PEARL-seq compounds. The herein-described library preparation protocol
describes a method to generate next generation sequencing libraries from small synthetic RNAs
by ligating adapters to both ends. The ligation is required in order to allow cDNA synthesis from
the ligated adapters — hence sequencing the whole target RNA. The technique represents one
step in the process of SHAPE sequencing. SHAPE sequencing aims at analysing RNA
secondary structure by ination of a mutation frequency after treatment with conformation
selective SHAPE reagents.
Target name for this example: targetRNA ucleotide “RNA3WJ_0.0.0_noLa ”,
sequence rGrGrCrArCrArArArUrGrCrArArCrArCrUrGrCrArUrUrArCrCrArUrGrCrGrGrUrU
rGrUrGrCrC. Physiological role: Synthetic RNA oligonucleotide capable of g a three
way junction secondary ure. Assay principle: 1) Ligation of 3’-adapter to target RNA; 2)
Phosphorylation of 5’ end of target RNA; 3) lst and 2nd strand cDNA synthesis from ligated
adapters; 4) oration and amplification of barcoded Illumina primers by PCR. Assay
readout: Agarose Gel Electrophoresis, Sanger-sequencing.
Assay Reagents and Hardware
— T4 RNA Ligase 2, truncated KQ (NEB #M0373 S)
— 50% PEG8000 (supplied with NEB #M0373 S)
— UT (Invitrogen)
— T4 RNA Ligase 1 (ssRNA Ligase) (NEB #MO204 S)
— 10 mM ATP (supplied with NEB #MO204S)
— SuperScriptIII e Transcriptase (Invitrogen)
— Phusion® Hot Start Flex DNA rase (NEB M0535)
— MinElute Gel Extraction kit (Qiagen)
— Quant-iT HS DNA assay kit (Inivtrogen)
— 0.2 M Cacodylic acid
0.1 M Potassium Cacodylate pH 7.2 (CacoK-stock) stock final
AdJusttosomIMImporeHo________________________________________________________________________________________________________
Storage4C
_______________________________________________________________________ _____________________________________________________________________
mM CacoK pH 7.2 stock final
1 ml ECacoK pH 7.2 100 mM 10 mM
eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee
Buffer1 (10 mM CacoK pH 7.2; 30 mM NaCl) stock final
.1..................mICacoKpH72....................................100mM .................................................
03ml
...............................................................ENE?!....................................................
....§7mIMIIIIp0reH20
ucleotides
targetRNA oligonucleotide “RNA3WJ_0.0.0_noLab” (IDT custom synthese)
’ rGrGrCrArCrArArArUrGrCrArArCrArCrUrGrCrArUrUrArCrCrArUrGrCrGrGrUrUrGrU
rGrCrC 3’
3’ adapter DNA oligo rsal miRNA Cloning ” (NEB S1315S)
’ rAppCTGTAGGCACCATCAAT—NHz 3’
’ adapter RNA oligo
’ rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC 3 ’
Reverse transcription primers: (NNNNNN indicates an 8 base “unique molecular
identifier” tag)
1st strand synthesis Primer (P7 RT-Anti UCL)
’ GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNATTGATGGTGC
CTACAG 3’
2nd strand synthesis Primer (P5 2nd strand)
’ TCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNNNGTTCAGAGTTCTACAGTCC
GACGATC 3’
Library PCR amplification primers: All primers contain specific 8 nt index
sequence tag (INDEX) required for library olution.
Several forward PCR primers
’ AATGATACGGCGACCACCGAGATCTACAC(INDEX)TCTTTCCCTACACGACGCTC
TTCCGATCT 3’
l reverse PCR primers
’ CAAGCAGAAGACGGCATACGAGAT(INDEX)GTGACTGGAGTTCAGACGTGTGCTC
TTCCGATCT 3 ’
qPCR/ Sequencing Primers:
Quanti qPCR 1_fw 5’ GATACGGCGACCACCGAG 3’
Quanti qPCR 1_rv 5’ GACGGCATACGAGAT 3’
Assay Procedure
Preparation
Dissolve target RNA with RNase free water to 100 uM.
Pipette 3 aliquots a 180 [.11 and additional small volume aliquots (5 ul). Storage: -80
] For ligation, resuspend the lyophilized Universal miRNA Cloning Linker (UCL) in
RNAse—free water to 100 uM stock concentration. In] UCL has a concentration of 100 pmol
(100 uM).
] Adjust the adapter concentration to 10 pmol/pl (10 uM) with RNase-free water (1:10
dilution).
RNA Folding
] Dilute the dissolved target RNA 1:10 with Buffer l to get a 10 iiM solution for
ligation.
Incubate at 90 0C for 5 min, cool down slowly to RT and store on ice.
3’ adapter ligation
Denature the 3’ adapter (UCL) at 65 °C for 30 sec, immediately chill on ice.
Ligations are carried out with T4 RNA Ligase 2 in the absence of ATP.
] Setup the ligation reaction with:
1 in RNA
4 [1| 3’ adapter “Universal miRNA Cloning Linker”
2 “L 10x T4 RNA ligase buffer without ATP
4 pl PEG8000
0.5 “L RNase tor
0.5 “L T4 RNA ligase 2, truncated
8.5 pl RNase-free H20 —
Ad 20 in —
1 in RNA
2 pl 3’ r “Universal miRNA Cloning Linker”
2 pL 10x T4 RNA ligase buffer without ATP
4 pl PEG8000 10 % (w/v)
0.5 pL RNase inhibitor
0.5 pL T4 RNA ligase 2, truncated
.5 pl free H20 —
Ad 20 in —
2 l“ RNA
2 pl 3’ adapter “Universal miRNA Cloning Linker”
2 pL 10x T4 RNA ligase buffer without ATP
4 pl 0 10 % (w/v)
0.5 pL RNase inhibitor
0.5 pL T4 RNA ligase 2, truncated
95 pl RNase-free H20 —
Ad 20 iii —
4 in RNA
2 pl 3’ adapter “Universal miRNA Cloning Linker”
2 pL 10x T4 RNA ligase buffer without ATP
4 pl PEG8000 10 % (WM
0.5 pL RNase inhibitor
0.5 pL T4 RNA ligase 2, ted
75 pl RNase-free H20 —
Ad 20 in —
The reaction is incubated at 25 0C for 4 h or 18 OC overnight. Note: ligation reaction
must be performed in the absence ofA TP. Heat inactivation: 65 °C 20 min.
’ adapter ligation
Denature the 5’ adapter RNA oligo (10 pM, in RNase—free water) at 65°C for 30 sec,
immediately chill on ice.
Add to 20 pl 3’ Adapter-RNA—Mix to:
4 bzw. 2 pl 5’ Adapter RNA oligo 20 pM
1 pL 10x T4 RNA ligase buffer
3 iii 10 mM ATP
% (w/v)
2017/040514
] The on is incubated at 25°C for 4 h or 18°C overnight. Heat inactivation: 65°C
for 15 minutes. Note: the 3’ end of the small RNA has already been ligated to the 3’ adapter that
has an amine group at the 3’ end, and could no longer take part in the ligation reaction; thus its 5’
end could be ligated to an RNA oligo in the presence of ATP.
Reverse transcription 11st strand cDNA synthesis]
Mix and briefly centrifuge each component before use.
Combine the following in a 0.2-ml PCR tube:
Adaooter-liated tar etRNA
P7 RT-Anti UCL rimer2 M
mM dNTP mix
DEPC-treated waterto 20 pl
Incubate at 65 °C for 5 min, then place on ice for at least 1 min.
Prepare the following cDNA Synthesis Mix, adding each ent in the indicated
order.
10X RT buffer
mM MgCIz
0-1 M DTT
RNaseOUT (40 Uml)
SuperScript ||| RT (200 U/pl)
Add 20 ul of cDNA Synthesis Mix to each RNA/primer mixture, mix , and
collect by brief centn'fugation. Incubate at: 50 min at 50 °C. Terminate the reactions at 85 °C
for 5 min. Chill on ice. Collect the ons by brief centrifugation. cDNA synthesis reaction
can be stored at —20 °C or used for PCR immediately.
2nd strand cDNA synthesis
Prepare the following PCR Mix:
1st strand cDNA _—
10X PCR Buffer, -Mg
P5 2nd strand primer 2 HM
Taq DNA Polymerase (5 U/pL)
RNase-free H20 ad 30 pl 4.9 ul—
] Place samples in PCR analyzer and execute the following cycling program:
re: 95 CC, 3 min
Annealing: 65°C 10 sec, Decrease 65°C—55°C at 041°C /sec
Elongation: 72 °C 3 min
Cool to 4 CC 00
Store at —20 0C until PCR enrichment.
PCR enrichment
Prepare the following PCR Mix:
—m-—
Place samples in PCR analyzer and execute the following cycling program
Initiation: Denature 98 °C, 30 Seconds
Cycles:
1. Denature 98 °C, 10 Seconds
2. Annealing 72 0C, 20 Seconds*
3. Elongation 72 °C, 15 Seconds
Final Extension 72 0C, 3 s
Hold 4-10°C
*To determine the optimal annealing temperature for a given set ers, use of
the NEB Tm Calculator is highly recommended.
The remaining RT product can be stored at —20°C.
Readout
Separate the PCR product on a 2% e gel using an appropriate molecular weight
marker. Note: The accurate ligated and amplified Library has a size of 233 bases. Cut the band
and gel-purify the product with Qiagen MinElute kit.
Subject the purified fragment to direct Sanger Sequencing (at a Provider of Choice)
using either i qPCR 1_fw” or “Quanti qPCR 1_rv” primers. The steps and ces
involved are shown in Figure 84.
Example 23: Alternate Procedure for Producing Illumina Small q Library
An ate procedure for ing the desired RNA library was developed that
included the further step of ligating the 5’ adapter to the target RNA. The principal steps of the
alternate method were: 1) Ligation of 3’-adapter to target RNA; 2) Phosphorylation of 5’ end of
target RNA; 3) Ligation of 5’-adapter to target RNA; 4) lst and 2nd strand cDNA synthesis from
ligated adapters; 5) Incorporation and amplification of barcoded Illumina primers by PCR.
To effect this additional step, T4 Polynucleotide Kinase (NEB) was ed among
the reagents. The additional phosphorylation step was performed as s:
Phosphorylation with T4 Polynucleotide Kinase
For non-radioactive phosphorylation, use up to 300 pmol of 5' termini
pl 3' Adapter-RNA-Mix 200/400/800 pmol
4 pL 10x T4 RNA ligase buffer 1x (1mM DTT)
4 pl 10mMATP 1mM
3,6 pl DTT 0,1 M 9mM
1 pl T4 Polynucleotide Kinase 10 U
7,4 pl RNase-free H20
Ad 40 pl
Incubate at 37 0C for 30 s. Fresh buffer is required for optimal activity (loss of
DTT due to oxidation lowers activity).
Also, during the subsequent 5’ r ligation step, 40 pl phosphorylated 3’
Adapter-RNA-Mix instead of 20 pl was used.
The steps and sequences involved in two methods of production of the library are
shown in Figures 85 and 86.
Example 24: ation and Immobilization of DELs (DNA-Encoded Libraries)
Sequences HTT41CAG and HTTl7CAG were successfully synthesized and refolded
after incubation for 2 h in the selection buffer described below. This was confirmed by native
PAGE (results not shown). Native PAGE: Denatured at 95 0C for 3 min, snap cooled on ice for
3 min and refolded at 37 °C for 30 min (10 mM Tris-HCl, pH 8.0, 75 mM KCl, and 3 mM
MgClz). About 50% of the RNA targets were immobilized on vidin resin. The RNA
targets were stable under selection conditions after the following improvements: apply stain after
gel electrophoresis. Decreasing the tration of ssDNA and Rnase inhibitor during
immobilization also helped.
Selection Conditions
DEL specif1cs: DEL Set 1 = 610 DEL libraries, 5.521 billion compounds in total;
DEL Set 2 = 205 DEL libraries, 70 million compounds in total (sets screened separately)
Selection rounds: 3-4
ion mode: Target immobilized
] e resin: Neutravidin resin
Target amount: 100 pmol
Immobilization buffer composition: NMR buffer, 0.1% Tween-20, 0.03mg/ml
ssDNA, 2mM Vanadyl ribonucleoside complexes.
Selection buffer ition: 50 mM Tris-HCl (pH 8), 75 mM KCl, 3 mM or 10 mM
MgClz, 0.1% Tween-20, 0.3mg/ml ssDNA, 20 mM Vanadyl ribonucleoside complexes.
Volume, temperature, and time: 100 uL, RT, 1 hour
Wash Conditions
Buffer composition: 50 mM Tris-HCl (pH 8), 75 mM KCl, 3 mM or 10 mM MgClz.
Number and volume: 2 X 200uL
Temperature and time: RT, fast
Elution Conditions:
Elution mode: Heat elution
Buffer composition: 50 mM Tris-HCl (pH 8), 75 mM KCl, 3 mM or 10 mM MgClz.
Volume, temperature, and time: 80 uL; 80 °C; 15 s.
Stability of the RNA complexes was confirmed by tion in the selection buffer
for 2 h at room temperature. The refolded RNA was successfully immobilized on the resin.
RNA Flow . % of total
Sample RNA Input (ng) RNA on resm (ng)
Through (ng) lized
HTT17CAG 2000 802.5 1197.5 60%
HTT41CAG 500 138.5 361.5 72%
sions:
After decreasing the concentration of ssDNA and Rnase inhibitor during
immobilization: 50% refolded HTTl7CAG was ed on vidin resin; after incubation
with DEL compounds, refolded HTT17CAG was recovered from Neutravidin resin; the target is
now ready for affinity selection.
Example 25: Surface n Resonance Experiments
Figures 87 and 88 show possible methods of employing surface plasmon resonance
(SPR) to screen ligands and hook and click constructs for binding to a target RNA of st.
SPR is especially useful for monitoring biomolecular interactions in real time. Typically, target
species and unrelated control are immobilized to a sensor chip, then analytes
unds/fragments) are flowed over the surface. Binding of the compound to target species
results in increase of SPR
signal (association . Washing away bound compound with buffer s in a decrease
of SPR signal (dissociation phase). Fitting of sensorgrams recorded at ent compound
concentrations is med to an appropriate interaction model. The method allows extraction
of kinetic parameters (ka, kd 9 K13). Requirements/limitations e that the ka / kd values be
in reasonable ranges; and the target size must not be too large (< 100 kDa). It is an excellent
method to screen fragments and profile or validate hits. BC4000 may be used for primary
screening (up to 4,000 data pts/week). Biacore T200 is suitable for hit profiling and validation.
In the PEARL-seq context, SPR allows monitoring binding of “hooks” to DNA/RNA
aptamers. The target species is immobilized to sensor chip, analytes (i.e. hooks) are flowed over
surface (association phase), DNA/RNA aptamer is flowed over surface (plateau phase),
competitor compound is washed over surface (dissociation phase), thus ng binding data.
The requirements/limitations are that, again, ka/kd values must be in reasonable ranges and fitting
for their respective purpose. Furthermore, the target size must be < 100 kDa. In addition, steps 1
and 2 need to be in place (tested first) in order to enable setup. A competitor with fitting affinity
will also be needed.
With the goal of identifying interaction partners (RNA/DNA) that bind to capture
RNA (3WJ), the ing steps are contemplated:
Use biotinylated capture-RNA (bi03WJ) to fold into secondary structure;
Allow binding of warhead triptycene ligands;
Fish cting RNA/DNA‘s by covalent linking to warhead;
Precipitate complexes via binding of bi03WJ to streptavidin beads;
Wash and elute; and
Library generation from eluate and sequencing.
] A protocol for smooth generation of cell lysates or RNA preps will be required. One
exemplary protocol would involve the following steps:
Preparation of RT-qPCR-ready cell lysates:
MDCK-London cells in 24-well plates were washed once with PBS (1 mL/well). Cell
lysatesare prepared by exposing cell monolayers to 200 mL/well of Cell-Lysis (CL) Buffer. The
final formulation of CL Buffer consist of 10 mM Tris-HCl pH 7.4; 0.25% Igepal CA-630; and
150 mM NaCl. CL Buffer is freshly ed from appropriate stock solutions. All reagents are
molecular biology grade and dilutions are made with DEPC-treated water (351721; Quality
Biological; Inc). For certain experiments; CL Buffer also includes MgClz (M1028; Sigma) or
RNasin Plus RNase Inhibitor (N2615; Promega). Cells are exposed for appropriate times
(typically 5 min for CL Buffer). The resulting lysates are carefully collected without disturbing
the cell monolayer ts and either analyzed ately or stored frozen. See, e. g.
es et al.; “A simple; inexpensive method for preparing cell lysates suitable for downstream
reverse transcription quantitative PCR,” Scientific Reports 4, Article : 4659 .
Simple Lysis buffer: using Igepal CA-63O and lSOmM NaCl; tes crude cell
lysate, ns still everything (no polyA-enrichment or protein removal).
Different Protocols possible: smallRNA work/low: Adapter ligation; cDNA-synthesis;
Library (small rs); or total RNA workflow: random primed w/wo RiboZero, standard
library prep (normal clusters).
] While we have described a number of embodiments of this invention; it is apparent
that our basic examples may be altered to provide other embodiments that utilize the compounds
and methods of this invention. Therefore; it will be appreciated that the scope of this invention is
to be defined by the ed claims rather than by the specific embodiments that have been
represented by way of example.
Claims (10)
1. A method of modulating the ream protein sion associated with a target mRNA to treat a disease or disorder caused by the target mRNA, comprising the step of contacting the target mRNA with a compound that binds to a nucleic acid 3-way junction (3WJ) in the target mRNA, wherein the compound is of Formula I: (Rom or a ceutically acceptable salt thereof, wherein: Rings A, B, and C are each, independently, a 3—8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, an 8-10 membered bicyclic aromatic carbocyclic ring, a 4—8 membered ted or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms ndently selected from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered ic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each Y is independently CR or N, each R1 is independently -R, halogen, -CN, -OR, -N(R)2, -NOz, -N3, -SR, or ; each R2 is independently -R, halogen, -CN, -OR, -N(R)2, -N02, -N3, -SR, —L2-R6, or two R2 groups on the same carbon are optionally taken together to form =NR6, =NOR6, =O, or =S; each R3 is independently -R, n, -CN, -OR, -N(R)2, -N02, -N3, -SR, or —L3-R6, each R6 is independently hydrogen or C1-6 alkyl optionally substituted with 1, 2, 3, 4, 5, or 6 halogens, each R is independently hydrogen or an optionally substituted group selected from C14, aliphatic, a 3—8 membered saturated or partially unsaturated monocyclic carbocyclic ring, , an 8-10 membered bicyclic aromatic carbocyclic ring, a 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently ed from nitrogen, oxygen, or sulfur, a 5-6 membered monocyclic heteroaromatic ring having 1— 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or an 8-10 membered ic heteroaromatic ring having 1-5 heteroatoms ndently selected from nitrogen, , or ; each L1, L2, and L3 is independently a covalent bond or a C1-8 bivalent ht or branched hydrocarbon chain wherein l, 2, or 3 methylene units of the chain are independently and optionally replaced with -O-, -C(O)—, -C(O)O-, -OC(O)—, -N(R)-, -C(O)N(R)—, -(R)NC(O)-, - OC(O)N(R)—, -(R)NC(O)O—, -N(R)C(O)N(R)—, -S-, —SO-, -SOz-, -SOzN(R)—, -(R)NSOz-, - C(S)—, -, -OC(S)-, -C(S)N(R)—, -(R)NC(S)-, -(R)NC(S)N(R)-, or -Cy-; m is 0,1, 2, 3, or 4; n is 0,], 2, 3, or 4, and p is 0,1, 2, 3, or4.
2. The method of claim 1, wherein Ring A is pyridyl, Ring B is phenyl, and Ring C is phenyl.
3. The method of claim 1 or 2, wherein the target mRNA forms a 3WJ in the presence of an effector miRNA RNA and the nd of Formula I binds to a trans 3WJ formed between the effector RNA and the target mRNA.
4. The method of any one of claims 1-3, wherein the compound of Formula I binds to a cis 3WJ formed between portions of the target mRNA,
5. A compound of Formulae XXXIII or XXXIV: XXXIII XXXIV or a pharmaceutically acceptable salt thereof; wherein: T1 is a bivalent ing group; and Rm"d is a difying moiety that reacts selectively with a 2’—OH group of a target RNA to which the compound binds.
6. The compound of claim 5 or a pharmaceutically acceptable salt thereof, further comprising a click-ready group covalently bound to Rm”l or any other substitutable hydrogen.
7. The compound of claim 6, wherein the ready group is an azide.
8. The compound of claim 5 or 6, wherein Rm"d is selected from N-methylisatoic anhydride, l-methylnitroisatoic ide, or l-methylnitroisatoic anhydride.
9. The method of any one of claims 1-4, wherein the disease or disorder is selected from selected from Huntington’s disease (HD), dentatorubral-pallidoluysian atrophy ), spinal- bulbar muscular atrophy (SBMA), or a spinocerebellar ataxia (SCA) selected from SCAl, SCAZ, SCA3, SCA6, SCA7, SCA17, Fragile X Syndrome, myotonic dystrophy (DMl or dystrophia myotonica), Friedreich’s Ataxia (FRDA), a spinocerebellar ataxia (SCA) selected from SCA8, SCAlO, or SCA12, or C9FTD (amyotrophic lateral sclerosis or ALS).
10. The method of any one of claims 1-4, wherein the disease or disorder is selected from a viral, microbial, or fungal infection. WO 06074
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Application Number | Priority Date | Filing Date | Title |
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US62/357,654 | 2016-07-01 | ||
US62/453,487 | 2017-02-01 |
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