Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Multivalent Ligand Clusters with Diamine Scaffold for Targeted Delivery of
Therapeutic Agents
BACKGROUND
Due to their high molecular weight and polyanionic nature, oligonucleotides
generally
have low cell membrane permeability. Thus, target ligands are often conjugated
to
oligonucleotide compounds to enhance cell uptake and improve tissue
specificity of in vivo
delivery, through a well-known mechanism of receptor-mediated endocytosis. In
some cases,
multivalent ligand clusters have an advantage over single ligands in enhancing
delivery to
targeted tissues via specific receptors. Asialoglycoprotein receptor (ASGPR)
is one of such
receptors.
It has been demonstrated that N-acetylgalactosamine (GalNAc), a ligand for
ASGPR,
can facilitate delivery of oligonucleotide drugs into hepatocytes. It has also
been
demonstrated that multivalent GalNAc ligand clusters have higher binding
affinity to ASGPR
than individual GalNAc ligands, and thus higher efficiency in delivering
therapeutic
oligonucleotides into liver hepatocytes.
SUMMARY
One aspect of the present disclosure relates to a compound for targeted
delivery of
one or more pharmaceutical agents, where the compound has the formula:
linkerA H
TL-vv--u¨Nl linkerB
) i,(",
riN ~.)-% . , - \ rxhi W
0
0 11")rn
linkerA A.,(4,.. N ..(c)
TI:NINJACkt.4." r=N
H n n
,
TL--,-----",N u
linkerA H
,
where each TL is an independently selected targeting ligand, m is an integral
number
between 1 and 10, each n is an independently selected integral number between
1 and 10,
each linkerA is an independently selected spacer, linkerB is a spacer, and W
is either the one
or more pharmaceutical agents or a functional group capable of linking to the
one or more
pharmaceutical agents. In some embodiments, m is 1. In some embodiments, m is
2.
In some embodiments, n is 1. In some embodiments, n is 2.
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In some embodiments, at least one of the independently selected TLs is capable
of
binding to one or more cell receptors, cell channels, and cell transporters
capable of
facilitating endocytosis. In some embodiments, at least one of the
independently selected TLs
comprises at least one small molecule ligand. In some embodiments, at least
one small
molecule comprises at least one of N-acetylgalactosamine, galactose,
galactosamine, N-
formyl-galactosamine, N-propionylgalactosamine, N-butanoylgalactosamine, and N-
iso-
butanoylgalactosamine, a macrocycle, a folate molecule, a fatty acid, a bile
acid, and a
cholesterol. In some embodiments, at least one of the independently selected
TLs comprises
at least one peptide. In some embodiments, at least one of the independently
selected TLs
comprises at least one cyclic peptide. In some embodiments, at least one of
the independently
selected TLs comprises at least one aptamer. In some embodiments, at least one
of the
independently selected TLs is capable of binding to at least one
Asialoglycoprotein receptor
(ASGPR). In some embodiments, at least one of the independently selected TLs
is capable of
binding to at least one transferrin receptor. In some embodiments, at least
one of the
independently selected TLs is capable of binding to at least one integrin
receptor. In some
embodiments, at least one of the independently selected TLs is capable of
binding to at least
one folate receptor. In some embodiments, at least one of the independently
selected TLs is
capable of binding to at least one G-protein-coupled receptor (GPCR).
In some embodiments, at least one of the independently selected linkerAs
comprises
at least one of polyethylene glycol, an alkyl group, a cycloalkyl group, an
alkenyl group, a
cycloalkenyl group, an alkynyl group, an aryl group, an aralkyl group, an
aralkenyl group,
and an aralkynyl group. In some embodiments, at least one of the independently
selected
linkerAs comprises at least one heteroatom. In some embodiments, the at least
one
heteroatom comprises at least one of oxygen, nitrogen, sulfur, or phosphorous.
In some
embodiments, at least one of the independently selected linkerAs comprises at
least one
aliphatic heterocycle. In some embodiments, the at least one aliphatic
heterocycle comprises
at least one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine,
piperazine,
pyrrolidine, and azetidine. In some embodiments, at least one of the
independently selected
.. linkerAs comprises at least one heteroaryl group. In some embodiments, the
at least one
heteroaryl group comprises at least one of imidazole, pyrazole, pyridine,
pyrimidine, triazole,
and 1,2,3-triazole. In some embodiments, at least one of the independently
selected linkerAs
comprises at least one amino acid. In some embodiments, at least one of the
independently
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selected linkerAs comprises at least one nucleotide. In some embodiments, at
least one of the
independently selected linkerAs comprises at least one saccharide. In some
embodiments, the
at least one saccharide comprises at least one of glucose, fructose, mannose,
galactose, ribose,
and glucosamine. In some embodiments, at least one of the independently
selected linkerAs
comprises one or more of:
0 0
)f(,\,,o){.)ry,,
P q p q H PP p q H
PP
0
p q H p q H PP
, and
where p is an integral number between 0 and 12, pp is an integral number
between 0 and 12,
q is an integral number between 1 and 12, and qq is an integral number between
1 and 12.
In some embodiments, linkerB comprises at least one of a polyethylene glycol,
an
alkyl group, a cycloalkyl group, an alkenyl group, a cycloalkenyl group, an
alkynyl group, an
aryl group, an aralkyl group, an aralkenyl group, and an aralkynyl group. In
some
embodiments, linkerB comprises at least one heteroatom. In some embodiments,
the at least
one heteroatom comprises at least one of oxygen, nitrogen, sulfur, and
phosphorous. In some
.. embodiments, linkerB comprises at least one aliphatic heterocycle. In some
embodiments, the
at least one aliphatic heterocycle comprises at least one of tetrahydrofuran,
tetrahydropyran,
morpholine, piperidine, piperazine, pyrrolidine, and azetidine. In some
embodiments, linkerB
comprises at least one heteroaryl group. In some embodiments, the at least one
heteroaryl
group comprises at least one of imidazole, pyrazole, pyridine, pyrimidine,
triazole, and 1,2,3-
triazole. In some embodiments, linkerB comprises at least one amino acid. In
some
embodiments, linkerB comprises at least one nucleotide. In some embodiments,
the at least
one nucleotide comprises at least one of an abasic nucleotide and an inverted
abasic
nucleotide. In some embodiments, the abasic nucleotide is an abasic
deoxyribonucleic acid.
In some embodiments, the inverted abasic nucleotide is an inverted abasic
deoxyribonucleic
acid. In some embodiments, the abasic nucleotide is an abasic ribonucleic
acid. In some
embodiments, the inverted abasic nucleotide is an inverted abasic ribonucleic
acid. In some
embodiments, linkerB comprises at least one saccharide. In some embodiments,
the at least
one saccharide comprises at least one of glucose, fructose, mannose,
galactose, ribose, and
glucosamine. In some embodiments, linkerB comprises at least one of:
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,
H
ko i
, ,
H H 11
41-I ID cs
0
/3(
0 0 0
¨k 11
0 0
,
1YN
0 k
0
, 0 o
o
o o
, , ,
OH
0 Q( J0
(õ ?. 0
i H
N . q
0 H 1
0 '''OH 0 0 OX
, ,
4
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OH X
I -..,-1-
0 0 0 OH
xitt_rii 450
Ay' xli..(,,c1rni 0i.
0 o i
OH
0
0
'Ql1"r0 e- 0 (?';- =
, and ,
where j is an integral number between 1 and 12, and k is an integral number
between 0 and
12.
In some embodiments, linkerB-W is:
0
ki 0 0 \AI ODIVT
H 0 ...), õse CO H ,.,4)1,H---,iN-..../='--0)(1..., )I-'
,,,),4_1,õ____ N.,,,,,C
ODMT 0 -,0 DKr C 0
0
ODMT 0
õItHr.y.NH,,,,Co 0
9 k --OH
-r()H Qt.(Thniq
-_ , = crir.N
k , , 0 0 ODMT 0 ODMT
,
0,\
\fµ -OH
OH 0 - OH
0 . 0
ri--'
4i5-0DMT ,, T! ,,,, r___z -00MT
", (---r',,e'N
¨.1 II
Ar-ri N"--9-"ODMT
0 0 0
o
cAeThrOH 0
OFI
k II
µ5dL('-'Isi'- /LOMAT ODMT
i 'Xills--)i1N1
u ODMT
,
0
OH
H k
0
,a.õ 0
0
zõ,_,..0DM't ;Fl .rf')i**--'--0 ODMT
, ODMT j
,or
, ,
where j is an integral number between 0 and 12, and k is an integral number
between 0 and
12.
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In some embodiments, W is a hydroxy group. In some embodiments, W is a
protected
hydroxy group. In some embodiments, the protected hydroxy group is protected
using at least
one of 4,4'-dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-(p-
methoxyphenyl)xanthen-9-y1 (Mox), and 9-phenylxanthen-9-y1 (Px). In some
embodiments,
.. W is a phosphoramidite group having the formula:
Ra
0.
Rc
where:
Ra is a Cl to C6 alkyl, C3 to C6 cycloalkyl, an isopropyl group, or Ra joins
with Rb
through a nitrogen atom to form a cycle,
Rb is a Cl to C6 alkyl, C3 to C6 cycloalkyl, an isopropyl group, or Rb joins
with Ra
through a nitrogen atom to form a cycle, and
Itc is a phosphite protecting group, phosphate protecting group, or a 2-
cyanoethyl
group.
In some embodiments, the phosphite protecting group comprises at least one of
.. methyl, allyl, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethylethyl,
2-
(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivaloylthio)ethyl, 2-(4-
nitrophenyl)ethyl,
2,2,2-trichloroethyl, 2,2,2-trichloro-1, 1-dimethylethyl, 1,1,1,3,3,3-
hexafluoro-2-propyl,
fluoreny1-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2,4-dichlorophenyl. In
some
embodiments, the phosphate protecting group comprises at least one of methyl,
allyl, 2-
.. cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethylethyl, 2-
(trimethylsilyl)ethyl, 2-(S-
acetylthio)ethyl, 2-(S-pivaloylthio)ethyl, 2-(4-nitrophenyl)ethyl, 2,2,2-
trichloroethyl, 2,2,2-
trichloro-1,1- dimethylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl, fluoreny1-9-
methyl, 2-
chlorophenyl, 4-chlorophenyl, and 2,4-dichlorophenyl.
In some embodiments, W is a carboxyl group. In some embodiments, W is an
activated carboxyl group having the formula:
0
I I
1¨
s C
where X is a leaving group. In some embodiments, the leaving group is selected
from the
group consisting of carboxylate, sulfonate, chloride, phosphate, imidazole,
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hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (NETS), tetrafluorophenol,
pentafluorophenol, and para-nitrophenol.
In some embodiments, W is a Michael acceptor. In some embodiments, the Michael
acceptor has the formula:
E
Rd
where E is an electron withdrawing group, and Rd is hydrogen or a Cl-C6 alkyl
substitution
group on olefin. In some embodiments, the electron withdrawing group is
carboxamide or an
ester. In some embodiments, E and the carbon-carbon double bond are part of
maleimide.
In some embodiments, W is an oligonucleotide. In some embodiments, the
oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the
oligonucleotide is a double-stranded oligonucleotide. In some embodiments, the
oligonucleotide comprises at least 3 independently selected nucleotides. In
some
embodiments, the oligonucleotide comprises between 16 and 23 independently
selected
nucleotides. In some embodiments, the oligonucleotide comprises about 100
independently
selected nucleotides. In some embodiments, the oligonucleotide comprises up to
fourteen
thousand independently selected nucleotides.
In some embodiments, W is:
o X linkerC
II
where:
linkerC is absent or a spacer attached to a 3' or 5' end of an
oligonucleotide,
X is a methyl group, oxygen, sulfur, or an amino group, and
Y is oxygen, sulfur, or an amino group.
In some embodiments, linkerC comprises at least a heterocyclic compound. In
some
embodiments, the heterocyclic compound is an abasic nucleotide or an inverted
abasic
nucleotide.
In some embodiments, W is:
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H linkerC
0
where linkerC is a spacer attached to a 3' or 5' end of an oligonucleotide. In
some
embodiments, linkerC comprises at least one of polyethylene glycol (PEG), an
alkyl group,
and a cycloalkyl group. In some embodiments, linkerC comprises at least one
heteroatom. In
some embodiments, the at least one heteroatom comprises at least one of
oxygen, nitrogen,
sulfur, and phosphorous. In some embodiments, linkerC comprises at least one
aliphatic
heterocycle. In some embodiments, the at least one aliphatic heterocycle
comprises at least
one of tetrahydrofuran, tetrahydropyran, morpholine, piperidine, piperazine,
pyrrolidine, and
azetidine. In some embodiments, linkerC comprises at least one heteroaryl
group. In some
embodiments, the at least one heteroaryl group comprises at least one of
imidazole, pyrazole,
pyridine, pyrimidine, triazole, and 1,2,3-triazole. In some embodiments,
linkerC comprises at
least one amino acid. In some embodiments, linkerC comprises at least one
nucleotide. In
some embodiments, the at least one nucleotide comprises at least one of an
abasic nucleotide
and an inverted abasic nucleotide. In some embodiments, the abasic nucleotide
is an abasic
deoxyribonucleic acid (DNA). In some embodiments, the inverted abasic
nucleotide is an
inverted abasic deoxyribonucleic acid (DNA). In some embodiments, the abasic
nucleotide is
an abasic ribonucleic acid (RNA). In some embodiments, the inverted abasic
nucleotide is an
inverted abasic ribonucleic acid (RNA). In some embodiments, linkerC comprises
at least one
saccharide. In some embodiments, the at least one saccharide comprises at
least one of
glucose, fructose, mannose, galactose, ribose, and glucosamine. In some
embodiments,
linkerC comprises one or more of:
X /
X
= V''....e00, 1,014
k I I ik
ti
it
y
41-Gru. IX
.0A
it It it
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P II
II
Y ,and Y ,
where j is an integral number between 1 and 12, and k is an integral number
between 0 and
12.
In some embodiments, W is:
0 linkerC
N
0 ,
where linkerC is a spacer attached to a 3' or 5' end of an oligonucleotide. In
some
embodiments, linkerC comprises at least one of polyethylene glycol (PEG), an
alkyl group,
and a cycloalkyl group. In some embodiments, linkerC comprises one or more of:
X
>4.---8-0, 1,02( 1/2("-..-a)..'"oj 0 ?(Cli)-0.,),OA
P k P' P
= ii II
1 y Y Y , , ,
X X ,, X
0,, i ,0)õk ....-0-10, 1 õolk 4-0 u, I ,0 5
P P P I-
I 1 1 1 1 1
io Y Y Y , , ,
P I I
ii
Y , and Y ,
where j is an integral number between 1 and 12, and k is an integral number
between 0 and
12.
In some embodiments, the compound is selected from the group consisting of:
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OAc
Ac0\_..\,......
0 ,
Ac0 k_),.z.----,.0
NHAc NJH 0,,.=
H
1\1-1N
N
OAc
(S
Ac0
0 , 0
NHAc N CN
H
Ac0 OAc / '0
Ac0\.0 HN0)
NHAc Compound 1;
OAc
Ac0
0
AGO HN 0
NHAc H
Y
NIThrN
0¨, N
0 P-
OAc 0
Ac0 0
0 )-N CN
NHAc H
"D
Ac0 OAc HN
0 ,
Ac0 ll cy0 ,....)
NHAc Compound 2;
OAc
Ac0
0
Ac0 HN }D
NHAc Y
al
OAc
Ac0 I0
\,_ 0
Ac0 õ ,_/0õ----,,,_õ-0.---N,N N CN
NHAc H ,.
/ -0
OAc
Ac0 HN
Ac0\___.\..:___\) Oc)0)
NHAc Compound 3;
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OAc
Ac0
Ac0 HN,CD
y
NHAc
N 0 N
P-
O
OAc
AcO____\._. 0 < CN
0
Ac0 00C) N)-
NHAc H
"D
Ac0 OAc
HN
NHAc Compound 4;
OAc
Ac0
Ac0-.
NHAc (:)
7
NH o
0
Y
N 0-..õ N
P-
OAc 0
Ac0
0
AcO400 Z ).NN CN
NHAc
N
H
AGO OAc / -0
Ac0 HN
______________________ 00..)
NHAc Compound 5;
OAc
Ac0
'--------.
0 , ,-, 0 0
Ac0 k-)
µ..,N...--k,..,,--.N 0, N
P-
NHAc
0
Ac0 H
Ac0
0 (:)0N ( CN
Ac0 N
NHAc H
0
Ac0 OAc 0
0
Ac0 0 NH
0
NHAc Compound 6;
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OAc
Ac0 j
u AcOk-,,,
,,,.õ.õõ...--...,N1-1 0
NHAc
0 0
1\1 0
OAc F F
Ac0\___\_s_...
0 0
Ac0 00 AN7 N F F
NHAc \/N
H
Ac0 OAc
/0
Ac0\..E.)_.\,00 HN
..)
NHAc Compound 7;
OAc
Ac0\.\
0 ,
Ac0 k-)
0
NHAc NH 0
0 0
N )N
OAc /
Ac0\._.. 0
0 0 Z Ac0 00 ).N N
NHAc \/N
H
Ac0 OAc
/0
0 HN
Ac0 00)
NHAc Compound 8;
OAc
Ac0\__\_.,
0
Ac0 0(:)
NHAc NJH 0
,.
Y
N
0P
õ ,N
OAc
Ac0\...0___ 0
0
Ac0 0
0.õ
CN .õ....,,,,,
NHAc N N
H
O
AcO\ cAc "D 0
AcOsZ__ v0 HN0.)
NHAc Compound 9;
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OAc
AcO____\_._
H N 0
NHAc Y
N (:)1:'-N
OAc 0
Ac0 0
CN
NHAc H
Ac0 OAc
HN
O
0 r,
Ac0_ \____\_,L,,.,....õ---,...,0õ---...,..Øõ.....,..-=
NHAc Compound 10;
OAc
AcO_____7___\
0 r,
Ac0 ,._,.,....,0
NHAc NI-10
\/
N (i--- p'N
OAc 0
Ac0
AcO0 0 CN
0 )N
NHAc N
Ac0 OAc H
/ -0
0 Ac0 0 HN
NHAc Compound
11;
OAc
Ac0j
Ac0 __________ 0
_....\_,, _
\ 0_,....----.....õNH 0
NHAc \/
0-, N
N p'
OAc
AcO\ r, 0
0
Ac0 .._,õ,---, CN
NHAc N
Ac0 OAc H
"D
0 HN
Ac0,-,,-, -..õ,..--.Ø..,.,,.,)
NHAc Compound 12;
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OAc
Ac0
Ac00
_ 0
NH0
NHAc \/
=-==--
N. _ N
N Pi -
OAc
L-,... 0õõ
Ac0\......
0 0
Ac0 Or..1
NHAc CN
`-'NV"\ N
H A.
Ac0 OAc
/0
Ac00-.õ....--*--.0 HN
,,)
NHAc Compound 13;
OAc
Ac0.__..\____\
0
Ac0 00,-0-..._.--------HN
NHAc
N(:IP-N
OAc
Ac0\__________\. 0
0 CN
Ac0 0c)ONN
NHAc H
f 0
Ac0 OAc
HN
Ac0 uo0-,,_)
NHAc Compound 14;
OAc
Ac0
0
Ac0 FIN 0 \/
NHAc
-,.
OAc
Ac0 0
0 r% )- < CN
NHAc H
A.
Ac0 OAc
HN
NHAc Compound 15;
14
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OAc
AcO\
µ_.-0
AcOA _____ ___\.0,,
\ l_l N H
.,.........., 0
NHAc \/
N
P-
OAc
Ac00____\ 0
0
Ac0 0 0 N ).N N CN
NHAc
H
Ac0 OAc
/(:)
Ac00 HN
,)
NHAc Compound 16;
OAc
Ac0\____\._...\
0 r,
Ac0 1._,0
N NI-1
HAc 0
Y
P
OAc
Ac0 0
0
N CN
7.
NHAc N
H
Ac0 OAc
/ '0
0 HN
Ac0 0 0.)
NHAc Compound 17;
OAc
AcO\
HN 0
NHAc Y
N 0, N
P-
OAc (S
Ac0._ _____ .\_____ 0
0 Ac0
N CN 0( j(DNA
NHAc H
"D
Ac0 OAc
HN
NHAc Compound 18;
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OAc
Ac0
Ac0 0 HN
NHAc
N
P
OAc
AcO
0
N CN
Ac0 0 0 N
NHAc
Ac0 KOAc
HN
Ack4_.\O
NHAc
Compound 19;
OAc
Ac0
0 ,-, 0
Ac0 N p, N
iii-
NHAc
Ac0
Ac0
0 0
Ac0 N N
NHAc CN
AcO\ OAc 0 (D
AcOO 0
N H
\ 0
NHAc
Compound 20;
OAc
AcO\
0 AcO 0
0õ N N P
NHAc
(S
Ac0
Ac0
0 0
Ac0 N N CN
NHAc
Ac0 OAc 0 O-
N H
NHAc Compound 21;
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OAc
Ac0 0
Y
Ac0000NNC)--,p-N/
NHAc H
(S
Ac0
Ac0 0,7\ N_Z/N \ CN
Ac0 0 H
0
NHAc 0
Ac0
OAc NH
NHAc Compound 22;
OAc
Ac0 0
Y
Ac0000NNO,p,1\J
NHAc H
6
Ac0
Ac0 CN
0 0 /
Ac0 (D(2 HN ,/\N
NHAc
0 C)
Ac0 OAc HN
0 r,
Ac0
NHAc Compound 23;
OAc
AcO_____\....
N,I-L,,___.--,..
NHAc N P
(S
Ac0 H
Ac0
0 Ac0 0_,_7---0,,,,,zõ,,
N _N
NHAc H CN
Ac0
OAc 0 C)
0
Ac0 0 NH
NHAc Compound 24;
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OAc
Ac0
0 0
Y Ac0 0õ
N
NHAc P-
C)
Ac0 H
Ac0
0 0___7----0õrõ,,,, /
Ac0 N-" CN
NHAc H
Ac0 OAc 0 0
0
Ac0 Oo NH
NHAc Compound 25;
OAc
Ac0 0
0
AGO N,J-N 0, P-
N
iii
NHAc H
(S.
Ac0
Ac0\____\_____\7 N _,7/N CN
Ac0 0 H
0
NHAc 0
Ac0 OAc NH
NHAc Compound 26;
OAc
Ac0 0
Y
0
AGO N)-N 0, ,N
P
NHAc H
(S
Ac0
AGO N CN
---,7---Fi __.õ--...õõ,õ IN
NHAc
0 C)
Ac0 OAc HN
0 n
NHAc Compound 27;
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OAc
Ac0.__\_.
0 r,
NI-1 0
NHAc
Y
0
0, N
N OAc
AcO\_.(E)._ 0
0 H
Ac0 00 ).N
N
CN ..,..,_,õ---N,
NHAc N N
H
Ac0 OAc / -0
HN
Ac000.,)
NHAc
Compound 28;
OAc
Ac0
0
AGO 1-IN 0 0
y
NHAc
N 0,, N
P-
OAc 6
AGO 0
CN
0 , )-1\1
NHAc H
"21
Ac0 OAc HN
0 ,
NHAc
Compound 29;
OAc
AcO\
\/
r, 0 0
AcO _________ L) \ vN_...--t.õ_,-----õN -
N
P
NHAc
Ac0 H 0
Ac0
0 Ac0 00õ,,y--,N--_N CN
NHAc H
Ac0 OAc 0 (D
0
Ac0 0 NH
0
NHAc Compound 30;
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OAc
Ac0\_..\,..... 0 0
Y
0
Ac0 0c)ONIJ-N 0,N
P
NHAc H
cS
Ac0
Ac0
Ac0 ON_7/N \
0 0,,,7----.0/'-,,
H CN
NHAc 0 0
Ac0 OAc NH
0 r,
NHAc Compound 31;
OAc
Ac0 0 0
Y
Ac0 N.,õ...--õN 0,N
P
NHAc H
C)
Ac0
Ac0
0 /
Ac0 00 0 HN-,/\N CN
NHAc
0 C)/
Ac0
0Ac HN
0 Ac0 L,
, 0
NHAc Compound 32;
OAc
Ac0
0
Ac0 00
NHAc NH 0
H \/
1\1-1N
OAc P
Ac0\__..\.._\ 0
0 0 0
Ac0 0c)
)-NN
CN
NHAc N
H i=
Ac0
/ '0
0 HN
Ac0 00,)
NHAc Compound 33;
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OAc
Ac0\___\...._.
0
H N 0
NHAc H
Y
NNI
N
OAc 0 P
6
Ac0 0
0 N AN cN
Ac0 0 00
NHAc H
"D
Ac0OAc ._..\,._\ HN
0
Ac0 0 00
NHAc
Compound 34;
OAc
Ac0
Ac0 %-,0
NHAc NH 0
0 0
N OH
OAc
AcO\...
0 H
Ac0 00 ),
-..õ-----õN '"i
NHAc
Ac0 OAc H"21
0 HN
Ac0 ,,,)
NHAc
Compound 35;
OAc
Ac0
Ac000
NI-1,(D o
NHAc
0
N OH
OAc
Ac0\_____
0 0
Ac0 0() 7N N/
NHAc -...........õ---N,N
H
AGO OAc / -0
0 HN
Ac0 00)
NHAc Compound 36;
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OAc
Ac0
AcO00C).----HN O 0 0
NHAc
' N (DH
OAc
Ac0\___\._.__\ 0
0
Ac0 0 00 N zN
NHAc H
/o
Ac0
\OAc
HN
0
NHAc Compound 37;
OAc
Ac0
Ac0 00,-----0,...õ------HN 0 0
0
NHAc
N OH
OAc
Ac0\____\___\ 0
0 N N ),
Ac0 000
NHAc H
Ac0 OAc HN
0 r,
NHAc Compound 38;
OAc
Ac0\_____\_______
0 Ac0 0
0NH 0
NHAc
0 0
N 0
OAcAc0 F F
0 Ac0 ,-, 0
,_,..0
),NN F F
NHAc N
Ac0 OAc H
"D
0 Ac0 0 HN
0
NHAc Compound
39;
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OAc
Ac0\___.
0
0 0
NHAc
N 0
OAc F F
Ac0 0
0 N F F
Ac0 0 0C) N
NHAc H
AcO\ ,OAc /o
HN
AcOA_____s000)
NHAc Compound 40;
OAc
Ac0
Ac0 HN O 0 0
NHAc
N 0
OAc F F
Ac0\_....\____ 0
Ac0 0 / F F N7--.. N ,,,
NHAc H
Ac0 OAc /(:)
HN
0 r,
NHAc Compound 41;
OAc
Ac0\_____\_____
0 0 0 0
Ac0 0
µ-'N N OH
NHAc
Ac0 H
Ac0
0 0,õz----0,__,,,_z,,,
Ac0 N ..//N \
NHAc H
0 (D
OAc
Ac0
0
Ac0 0 o N H
NHAc Compound 42;
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OAc
Ac0
0 0 0 0
Ac0
NHAc OH
Ac0
Ac0
0
AcO00 N
NHAc
Ac0 OAc 0
0
Ac0 OoNH
NHAc Compound 43;
OAc
AcO
0 0 0
0
Ac0 OH
NHAc
Ac0
Ac0 0 N
0
Ac0
NHAc 0
Ac0 OAc NH
0
Ac0 0c)(D/
NHAc Compound 44;
OAc
Ac0 0 0 0
0 AcOOO N NOH
NHAc
Ac0
AcO
Ac0 HN
NHAc
0 (D
Ac0 OAc HN
0
Ac0
NHAc Compound 45;
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OAc
AcO\
AcO lin _,.,.....õ----,
\ 0 N
N_J---,....
NHAc 0
Ac0 H
F F
Ac0
0 Ac0 0 13____7----...õ,,,,,
NHAc H
Ac0 OAc 0 C)
AcO00 N H
NHAc
Compound 46;
OAc
AcO\
AcOA___ ,-N LI =,.,....õ/- \
\ NHAc 0.....õ..õ..----õNN
0
F F
Ac0 H
Ac0
0 00 K./ F F
Ac0--------- N --_,...õ------,-- IN -....,.
NHAc H
Ac0 OAc 0 CD
Ac00o NH
NHAc
Compound 47;
OAc
Ac0\___\_..._\ 0 0 0
0
Ac0 0(j(DN N 0
NHAc H
F F
Ac0
Ac0\__\____\zN F F
0 0/"--,7
Ac0 H
NHAc 0 0
Ac0 OAc NH
0 r,
NHAc Compound 48;
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OAc
Ac0 0 0 0
Ac0N õ..---,õ_,..,...N.-------....õ....õ----,...}-.0
NHAc H
F F
Ac0
Ac0\_,4.)_.\ õ./ F F
0
Ac0 C)(:) 1-1N.___/\"
NHAc
0 0
Ac0 OAc HN
0 r,
NHAc Compound 49;
OAc
Ac0 j
AcOA___ 0,,
NHAc
--\ u...,õ,------_,NH 0
0 0
N-N
OAc /
Ac0\___\..._..
0 Ac0 0 0 0 0 )m
NHAc
Ac0 OAc H"D
0 Ac0 0 HN
0
NHAc Compound 50;
OAc
AcO____\..
0
HN O 0
0
NHAc
N N
OAc /
Ac0\_____\._ 0 0
0
Ac0 0 00 N ) N
NHAc H
"21
Ac0 OAc
HN
Ac0000)
NHAc Compound 51;
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OAc
Ac0
0 AcOOOOHNO0
0
NHAc
)N
OAc 0
0
Ac0
Ac0 0 0(D
NHAc
AcO OAc /
HN
0 ,
Ac0
NHAc
Compound 52;
OAc
AcO
0 0 0
Ac0 0c)
N N
NHAc
Ac0
0
Ac0
0 (:) Th
/z\NN
Ac0
AcO
NHAc
OAc (D
Ac0 0 (D NH
0
NHAc
Compound 53;
OAc
Ac0
0 0 0
0 r,
Ac0
NHAc
Ac0 0
Ac0
0 (:)(Dz\
Ac0
NHAc
0
Ac0OAc
0
Ac0 NH
0
NHAc
Compound 54;
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OAc
Ac0,___\_....., 0 0 0
0
N ...,_õ..N .,N
NHAc H
/
Ac0 0
Ac0 Ac0 N
0 0_, 0
,,
H
NHAc 0 0
Ac0 OAc NH
NHAc
Compound 55;
OAc
Ac0\__\____\, 0 0 0
Ac0N,K,__.-----..N.----..õ------.N
NHAc H
/
Ac0 0
Ac0
õ,/
HN-õ.., IN
NHAc
0 0
Ac0 OAc HN
NHAc
Compound 56;
OAc
Ac0
0
Ac0 oONI-1 0
NHAc
0 0
N NQ, 'OHOAc
Ac0\ n
..
0 0 H ODMT
Ac0,,,...,.,,..---...,,,
NHAc
H
Ac0 OAc
"21
0 , HN
Ac0 ,..,.---,,,,,,, j
NHAc Compound 57;
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OAc
Ac0
Ac00 _ 0
NI-1 0
NHAc
0 0
N Nq.10H
OAc
Ac0
0 ODMT
Ac00 / _ ....,...õ..---.. N )NN
NHAc
Ac0 OAc H":)
0 HN
Ac0 0 0,)
NHAc
Compound 58;
OAc
Ac0
0
0
NHAc
N NQ 10H
OAc
Ac0 0 ODMT
0 r,
Ac0 N7---....õ- N,,
NHAc H
Ac0 zOAc /O
HN
NHAc
Compound 59;
OAc
Ac0
0 r,
Ac0 HN, ,0
0
NHAc
--I--.õ---..õ...---,
N NQ 01H
OAc
AcO 0 ODMT
0 , )N
Ac0 k..,o,,ON
NHAc H
Ac0 OAc /(:)
HN
NHAc Compound 60;
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OAc
Ac0
AcO0
NHAc
0
0 0
1\1
r\p.i0H
AcO
OAc
AcO\O
0 ODMT
NHAc
Ac0 OAc
/(:)
0 HN
Ac0
NHAc
Compound 61;
OAc
Ac0
0 Ac0 _________ r,
Th\IH 0
NHAc
0 0
1\1 N ..10H
OAc
Ac0
0 ODMT
AcO\O
),NN
NHAc
Ac0 OAc / -0
0 Ac0 HN
NHAc
Compound 62;
OAc
Ac0
0
AcOOOOHNO 0
0
NHAc
N IC)H
OAc
AcO
0
0
N) N ODMT
Ac0 0c)O
NHAc
Ac0 OAc HN
0
Ac0
NHAc Compound 63;
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OAc
AcO_____\...Ø_
Ac0 00C)/---1-IN O 0 0
NHAc
1\1 N ..10H
OAc
Ac0 0
O
0 7- DMT
Ac0 0c)C)N
NHAc H
"21
AcOOAc HN
NHAc
Compound 64;
OAc
AcO___\,._ 0 0 0
Ac0 0
N -1.,..----.N N. 'OH
NHAc
Ac0 H
Ac0
0
ODMT (:)Th ODMT
---/\N
Ac0
NHAc H
AcO\ OAc 0 (3
AcO___ /\/\NH
\ u
NHAc
Compound 65;
OAc
Ac0 0 0 0
Ac0 u 0
0N----lt,õ-----....N Q..10H
NHAc
H
Ac0
ODMT
Ac0
Ac0
0 (30NN( NHAc H
0
Ac0 OAc 0
Ac0 0 (:)oNH
NHAc Compound 66;
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OAc
Ac0 0 0 0
0
Ac0 OcI(DN-J-N Q. 'OH
NHAc H
Ac0 ODMT
Ac0\7____\, N /N \
0 cr---.7
Ac0 0 H
0
NHAc 0
Ac0 OAc NH
NHAc
Compound 67;
OAc
Ac0 ._._ 0 0 0
Ack4)._0o0
N N N, 'OH
NHAc H
Ac0
ODMT
Ac0_,...7c2._\r m/
Ac0 C) (:) 07 HNõ/\"
NHAc
0 0
Ac0 OAc HN
0 Ac0 ,n ,,,,_,----Ø----0..õ---
NHAc
Compound 68;
OAc
Ac0
0 0 0 0
Ac0 n L.,,..õ..õ,--,õ,,
N
,I0H
NHAc
H
Ac0
Ac0
0 OZ'O ODMT
Ac0 __N \
NHAc H
0 0
AcR OAc
0
Ac0\--- 0o NH
\
NHAc Compound 69;
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OAc
AcO____\___\
0 0 0 0
Ac0 0
N
0 N ...._.õ_õ...-,..
NHAc N . 'OH
H
Ac0
Ac0
ODMT
0 Ac0 0______õ-------0 N--
m/
" \
NHAc H
Ac0 OAc 0 CD
0
Ac0 Oo NH
NHAc Compound 70;
OAc
AcO_____7____ 0 0 0
0
Ac0 0c)C)N .-N
p, IC)H
NHAc H
Ac0
ODMT
Ac0
Ac00 oH N
0
NHAc 0
Ac0 OAc NH
NHAc Compound 71;
OAc
AcO___..v__\. 0 0 0
0
Ac0 0(DON
N
NIOH
NHAc H
Ac0
Ac0
0
N m
ODMT
/
.,/\" \
NHAc
0 C)
Ac0 OAc HN
0 Ac0 n .,0
,....õ---
NHAc Compound 72;
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OAc
Ac0
Ac00
NHAc C)NH 0
0 0
N N-10H
OAc
Ac0
0 0 ODMT
Ac0 r) .._,..---,,N)-N
NHAc
H
Ac0 OAc
/0
AcO 0 _
a HN_
NHAc Compound 73;
OAc
Ac0
Ac00 _ ..,.õ,.....õ.."...,0
NHAc NI-1 0
0 0
N ill ---.0H
OAc
Ac0
0 0 ODMT
Ac0n µ_..õ.s........---,...Nz-N
NHAc
Ac0 OAc H
"2,
0 Ac0 k..) , HN
0
-------------
NHAc Compound 74;
and
OAc
Ac0
0
Ac0:_" 0
0 NH 0
NHAc
0 0
N N /
N¨
OAc
Ac0\.____\_, 0 \
0
0 n 0
Ac0,._,,....._,,--,..., )N C N
0
NHAc
N
Ac0 OAc H /0
Ac0\ 0\(:) ......\,,_0 HN
..,)
NHAc Compound 75.
In some embodiments, the compound is a stereoisomer of one of Compound 1-75.
In some embodiments, W is the one or more pharmaceutical agents. In some
embodiments, the one or more pharmaceutical agents comprises at least one of a
small
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interfering RNA (siRNA), a single strand siRNA, a double stranded siRNA, a
small
activating RNA, an RNAi, a microRNA (miRNA), an antisense oligonucleotide, a
short guide
RNA (gRNA), a single guide RNA (sgRNA), a messenger RNA (mRNA), a ribozyme, a
plasmid, an immune-stimulating nucleic acid, an antagomir, and an aptamer. In
some
embodiments, the double stranded siRNA comprises at least one modified
ribonucleotide. In
some embodiments, the double stranded siRNA each strand is 19-23 nucleotides
in length. In
some embodiments, substantially all ribonucleotides of the double stranded
siRNA are
modified. In some embodiments, all ribonucleotides of the double stranded
siRNA are
modified. In some embodiments, the modified ribonucleotide comprises a 2'-0-
methyl
nucleotide,2'-Fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide
mimic, locked
nucleotide, 2'-F-Arabino nucleotide, 2'-methoyxyethyl nucleotide, abasic
nucleotide, ribitol,
inverted nucleotide, inverted abasic nucleotide, inverted 2'-0Me nucleotide,
inverted 2'deoxy
nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide,
mopholino
nucleotide, and 3'-0Me nucleotide, a nucleotide comprising a 5'-
phosphorothioate group, or a
5'-(E)-vinyl phosphonate nucleotide (antisense strand only), or a terminal
nucleotide linked to
a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2'-amino-
modified
nucleotide, 2'-alkyl-modified nucleotide, a phosphoramidate, or a non-natural
base
comprising nucleotide. In some embodiments, at least one strand of the double-
stranded
siRNA comprises at least one phosphorothioate linkage. In some embodiments, at
least one
strand of the double-stranded siRNA comprises up to 6 phosphorothioate
linkages. In some
embodiments, the double-stranded siRNA comprises at least one locked nucleic
acid. In some
embodiments, the double-stranded siRNA comprises at least one unlocked nucleic
acid. In
some embodiments, the double-stranded siRNA comprises at least one glycerol
nucleic acid.
Another aspect of the present disclosure relates to a pharmaceutical
composition
comprising the any one of the compounds detailed above. In some embodiments,
the
pharmaceutical composition comprises one or more pharmaceutical agents. In
some
embodiments, the pharmaceutical composition comprises one or more therapeutic
agents. In
some embodiments, the pharmaceutical composition comprises a pharmaceutically
acceptable carrier.
A further aspect of the present disclosure relates to a composition for
targeted
delivery of one or more pharmaceutical agents, where the composition comprises
any one of
the compounds described above, and where W is the one or more pharmaceutical
agents. In
some embodiments, the one or more pharmaceutical agents comprises at least one
of a small
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interfering RNA (siRNA), a single strand siRNA, a double stranded siRNA, a
small
activating RNA, a microRNA (miRNA), an antisense oligonucleotide, a short
guide RNA
(gRNA), a single guide RNA (sgRNA), a messenger RNA (mRNA), a ribozyme, a
plasmid,
an immune stimulating nucleic acid, an antagomir, and an aptamer. In some
embodiments,
the double-stranded siRNA comprises at least one modified ribonucleotide in
one or both
strands of the siRNA. In some embodiments, the double stranded siRNA each
strand is 19-
23 nucleotides in length.In some embodiments, substantially all
ribonucleotides of the
double-stranded siRNA are modified. In some embodiments, all ribonucleotides
of the
double-stranded siRNA are modified. In some embodiments, the modified
ribonucleotide
comprises: a 2'-0-methyl nucleotide,2'-Fluoro nucleotide, 2'-deoxy nucleotide,
2'3'-seco
nucleotide mimic, locked nucleotide, 2'-F-Arabino nucleotide, 2'-methoyxyethyl
nucleotide,
abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide,
inverted T-OMe
nucleotide, inverted 2'deoxy nucleotide, 2'-amino-modified nucleotide, 2'-
alkyl-modified
nucleotide, mopholino nucleotide, and 3'-0Me nucleotide, a nucleotide
comprising a 5'-
phosphorothioate group, or a 5'-(E)-vinyl phosphonate nucleotide (antisense
strand only), or a
terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid
bisdecylamide group,
a 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, a
phosphoramidate, or a non-
natural base comprising nucleotide. In some embodiments, at least one strand
of the-double
stranded siRNA comprises at least one phosphorothioate linkage. In some
embodiments, at
least one strand of the double-stranded siRNA comprises up to 6
phosphorothioate linkages.
In some embodiments, the double-stranded siRNA comprises at least one locked
nucleic acid.
In some embodiments, the double-stranded siRNA comprises at least one unlocked
nucleic
acid. In some embodiments, the double-stranded siRNA comprises at least one
glycerol
nucleic acid.
Another aspect of the present disclosure relates to a pharmaceutical
composition
comprising any one of the compositions described above. In some embodiments,
the
pharmaceutical composition comprises one or more therapeutic agents. In some
embodiments,
the pharmaceutical composition comprises a pharmaceutically acceptable
carrier.
A further aspect of the present disclosure relates to a method for making a
compound
for targeted delivery of one or more pharmaceutical agents, where the method
comprises:
receiving a first compound comprising a diamine, the diamine comprises a first
nitrogen and
a second nitrogen, the first nitrogen being a primary amine, the second
nitrogen being a
secondary amine comprising a protecting group; producing a second compound by
coupling a
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plurality of protected carboxylic acids to the first compound, the first
nitrogen in the second
compound being a tertiary amine comprising a first protected carboxylic acid
and a second
protected carboxylic acid, the second nitrogen of the second compound being a
tertiary amine
comprising the protecting group and a third protected carboxylic acid;
producing a third
compound by deprotecting the second nitrogen of the second compound, resulting
in the
second nitrogen becoming a secondary amine comprising the third protected
carboxylic acid;
producing a fourth compound by attached a moiety comprising a hydroxy group to
the second
nitrogen of the third compound, resulting in the second nitrogen becoming a
tertiary amine or
an amide comprising the third protected carboxylic acid and the moiety
comprising the
hydroxy group; producing a fifth compound by converting the protected
carboxylic acids of
the fourth compound into carboxylic acids; and producing a sixth compound by
performing
an amide coupling reaction using the fifth compound, the first nitrogen in the
sixth compound
being a tertiary amine comprising a first amide and a second amide, the second
nitrogen in
the sixth compound being a tertiary amine comprising the moiety comprising the
hydroxy
group and a third amide, wherein the first amide, the second amide, and the
third amide are
each coupled to an independently selected targeting ligand.
In some embodiments, the protecting group is selected from the group
consisting of a
benzyl group and a triphenylmethyl group. In some embodiments, producing the
second
compound comprises performing a SN2 substitution reaction using the first
compound. In
some embodiments, producing the second compound comprises performing a
reductive
amination reaction using the first compound. In some embodiments, producing
the second
compound comprises performing a Michael addition reaction using the first
compound. In
some embodiments, the protecting group is a benzyl group, and producing the
third
compound comprises performing a hydrogenation reaction using the second
compound. In
some embodiments, the protecting group is a triphenylmethyl group, and
producing the third
compound comprises reacting the second component with at least one acid. In
some
embodiments, producing the fourth compound comprises performing a SN2
substitution
reaction using the third compound. In some embodiments, producing the fourth
compound
comprises performing a reductive amination reaction using the third compound.
In some
embodiments, producing the fourth compound comprises performing a Michael
addition
reaction using the third compound. In some embodiments, producing the fourth
compound
comprises performing an amide coupling reaction using the third compound. In
some
embodiments, producing the fourth compound comprises performing a nucleophilic
addition
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reaction using the third compound. In some embodiments, the moiety comprising
the hydroxy
group is attached to the second nitrogen using any linkerB described above. In
some
embodiments, producing the fifth compound comprises reacting the fourth
compound with at
least one acid. In some embodiments, the at least one acid comprises at least
one of
hydrochloric acid, hydrobromic acid, trifluoroacetic acid, and formic acid. In
some
embodiments, producing the fifth compound comprises performing a hydrogenation
reaction
using the fourth compound. In some embodiments, producing the fifth compound
comprises
performing a hydrolysis reaction using the fourth compound. In some
embodiments, the first
amide, the second amide, and the third amide are each coupled to an
independently selected
targeting ligand using any independently selected linkerA described above. In
some
embodiments, the independently selected targeting ligand is an independently
selected
targeting ligand described above. In some embodiments, the method further
comprises
converting the hydroxy group to a phosphoramidite group using a
phosphitylation reaction. In
some embodiments, converting the hydroxy group to the phosphoramidite group is
performed
after performing the amide coupling reaction to produce the sixth compound.
Another aspect of the present disclosure relates to a method for making a
compound
for targeted delivery of one or more pharmaceutical agents, where the method
comprises:
receiving a first compound comprising a diamine, the diamine comprising a
first nitrogen and
a second nitrogen, the first nitrogen being a secondary amine comprising a
first protecting
group, the second nitrogen being an amine comprising a second protecting
group; producing
a second compound by coupling a first protected carboxylic acid to the first
nitrogen of the
first compound, resulting in the first nitrogen becoming a tertiary amine;
removing the first
protecting group from the first nitrogen of the second compound to produce a
third compound
comprising the first nitrogen and the second nitrogen, the first nitrogen
being a secondary
amine comprising the first protected carboxylic acid, the second nitrogen
being an amine
comprising the second protecting group; producing a fourth compound by
coupling a second
protected carboxylic acid to the first nitrogen of the third compound,
resulting in the first
nitrogen becoming a tertiary amine; removing the second protecting group from
the fourth
compound to produce a fifth compound comprising the first nitrogen and the
second nitrogen,
the first nitrogen being a tertiary amine comprising the first protected
carboxylic acid and the
second protected carboxylic acid, the second nitrogen being a primary amine;
producing a
sixth compound by coupling a third protected carboxylic acid to the second
nitrogen of the
fifth compound, resulting in the second nitrogen becoming a secondary amine;
producing a
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seventh compound by attaching a moiety comprising a hydroxy group to the
second nitrogen
of sixth compound, resulting in the second nitrogen becoming a tertiary amine;
producing an
eighth compound by converting the third protected carboxylic acid of the
seventh compound
into a first carboxylic acid; producing a ninth compound by performing an
amide coupling
reaction using the eighth compound, the first nitrogen of the ninth compound
comprising the
first protected carboxylic acid and the second protected carboxylic acid, the
second nitrogen
of the ninth compound comprising the a first amide having a first targeting
ligand coupled
thereto and the moiety comprising the hydroxy group; producing a tenth
compound by
converting the second protected carboxylic acid of the ninth compound into a
second
carboxylic acid; producing an eleventh compound by performing an amide
coupling reaction
using the tenth compound, the first nitrogen of the eleventh compound
comprising the first
protected carboxylic acid and a second amide having a second targeting ligand
coupled
thereto, the second nitrogen of the eleventh compound comprising the first
amide having the
first targeting ligand coupled thereto and the moiety comprising the hydroxy
group;
producing a twelfth compound by converting the first protected carboxylic acid
of the
eleventh compound into a third carboxylic acid; and producing a thirteenth
compound by
performing an amide coupling reaction using the twelfth compound, the first
nitrogen of the
thirteenth compound comprising the second amide having the second targeting
ligand
coupled thereto and a third amide having a third targeting ligand coupled
thereto, the second
nitrogen of the thirteenth compound comprising the first amide having the
first targeting
ligand coupled thereto and the moiety comprising the hydroxy group.
In some embodiments, the first protecting group is a benzyl group and the
second
protecting group is a tert-butyloxycarbonyl (Boc) group. In some embodiments,
producing
the second compound comprises performing a SN2 substitution reaction using the
first
compound. In some embodiments, producing the second compound comprises
performing a
reductive amination reaction using the first compound. In some embodiments,
producing the
second compound comprises performing a Michael addition reaction using the
first
compound. In some embodiments, producing the third compound comprises
performing a
hydrogenation reaction using the second compound. In some embodiments,
producing the
fourth compound comprises performing a SN2 substitution reaction using the
third compound.
In some embodiments, producing the fourth compound comprises performing a
reductive
amination reaction using the third compound. In some embodiments, producing
the fourth
compound comprises performing a Michael addition reaction using the third
compound. In
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some embodiments, producing the fourth compound comprises performing an amide
coupling reaction using the third compound. In some embodiments, producing the
fourth
compound comprises performing a nucleophilic addition reaction using the third
compound.
In some embodiments, producing the fifth compound comprises reacting the
fourth
compound with at least one acid. In some embodiments, the at least one acid
comprises at
least one of hydrochloric acid and trifluoroacetic acid. In some embodiments,
producing the
sixth compound comprises performing a SN2 substitution reaction using the
fifth compound.
In some embodiments, producing the sixth compound comprises performing a
reductive
amination reaction using the fifth compound. In some embodiments, producing
the sixth
compound comprises performing a Michael addition reaction using the fifth
compound. In
some embodiments, producing the seventh compound comprises performing a SN2
substitution reaction using the sixth compound. In some embodiments, producing
the seventh
compound comprises performing a reductive amination reaction using the sixth
compound. In
some embodiments, producing the seventh compound comprises performing a
Michael
addition reaction using the sixth compound. In some embodiments, producing the
seventh
compound comprises performing an amide coupling reaction using the sixth
compound. In
some embodiments, producing the seventh compound comprises performing a
nucleophilic
addition reaction using the sixth compound. In some embodiments, the first
amide is coupled
to the first targeting ligand using an independently selected linkerA
described above. In some
embodiments, the second amide is coupled to the second targeting ligand using
an
independently selected linkerA described above. In some embodiments, the third
amide is
coupled to the third targeting ligand using an independently selected linkerA
described above.
In some embodiments, the first targeting ligand, the second targeting ligand,
and the third
targeting ligand are independently selected to be one or more of the targeting
ligands
described above. In some embodiments, the hydroxy group is coupled to the
second nitrogen
using a linkerB described above. In some embodiments, the method further
comprises
converting the hydroxy group to a phosphoramidite group using a
phosphitylation reaction. In
some embodiments, converting the hydroxy group to the phosphoramidite group is
performed
after producing the thirteenth compound.
A further aspect of the present disclosure relates to a method for delivering
a
pharmaceutical agent to a subject, the method comprising administering, to the
subject, (a) a
compound described above, where W is the one or more pharmaceutical agents, or
(b) a
composition described above. In some embodiments, the subject is a vertebrate.
In some
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embodiments, the subject is a mammal. In some embodiments, the mammal is a
human. In
some embodiments, the compound is administered in a pharmaceutically
acceptable carrier.
Another aspect of the present disclosure relates to a method for delivering a
pharmaceutical agent to a subject, the method comprising administering, to the
subject, a
pharmaceutical composition described above. In some embodiments, the subject
is a
vertebrate. In some embodiments, the subject is a mammal, optionally the
mammal is a
human. In some embodiments, the one or more pharmaceutical agents comprises at
least one
of a small interfering RNA (siRNA), a single strand siRNA, a double stranded
siRNA, a
small activating RNA, a microRNA (miRNA), an antisense oligonucleotide, a
short guide
RNA (gRNA), a single guide RNA (sgRNA), a messenger RNA (mRNA), a ribozyme, a
plasmid, an immune stimulating nucleic acid, an antagomir, and an aptamer. In
some
embodiments, the double-stranded siRNA comprises at least one modified
ribonucleotide in
one or both strands of the siRNA. In some embodiments, substantially all
ribonucleotides of
the double-stranded siRNA are modified. In some embodiments, all
ribonucleotides of the
double-stranded siRNA are modified. In some embodiments, the modified
ribonucleotide
comprises: a 2'-0-methyl nucleotide,2'-Fluoro nucleotide, 2'-deoxy nucleotide,
2'3'-seco
nucleotide mimic, locked nucleotide, 2'-F-Arabino nucleotide, 2'-methoyxyethyl
nucleotide,
abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide,
inverted T-OMe
nucleotide, inverted 2'deoxy nucleotide, 2'-amino-modified nucleotide, 2'-
alkyl-modified
nucleotide, mopholino nucleotide, and 3'-0Me nucleotide, a nucleotide
comprising a 5'-
phosphorothioate group, or a 5'-(E)-vinyl phosphonate nucleotide (antisense
strand only), or
a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid
bisdecylamide
group, a 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, a
phosphoramidate, or a
non-natural base comprising nucleotide. In some embodiments, at least one
strand of the-
double stranded siRNA comprises at least one phosphorothioate linkage. In some
embodiments, at least one strand of the double-stranded siRNA comprises up to
6
phosphorothioate linkages. In some embodiments, the double-stranded siRNA
comprises at
least one locked nucleic acid. In some embodiments, the double-stranded siRNA
comprises at
least one unlocked nucleic acid. In some embodiments, the double-stranded
siRNA comprises
at least one glycerol nucleic acid. In some embodiments, the pharmaceutical
composition
further comprises one or more therapeutic agents.
Another aspect of the present disclosure the compounds for use to deliver a
pharmaceutical agent to a subject. In some embodiments, the subject is a
vertebrate. In some
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embodiments, the subject is a mammal. In some embodiments, the mammal is a
human. In
some embodiments, the compound is administered in a pharmaceutically
acceptable carrier.
Another aspect of the present disclosure, the dsRNA agent comprises 2'-fluoro
modified nucleotides at positions 2, 7, 12, 14 and 16 of the antisense strand
(counting from
the first paired nucleotide from the 5' end of the antisense strand), and/or
2'- fluorine-
modified nucleotides at positions 9, 11 and 13 of the sense strand (counting
from the first
paired nucleotide from the 3' end of the sense strand).
DETAILED DESCRIPTION
Overview
The present disclosure provides multivalent ligand clusters, having a diamine
scaffold,
for targeted delivery of pharmaceutical agents conjugated thereto. In some
embodiments, the
multivalent ligand cluster may comprise one or more N-acetylgalactosamine
(GalNAc)
targeting ligands. In some embodiments, the multivalent ligand cluster may be
conjugated to
one or more small interfering ribonucleic acids (siRNAs), with siRNA being an
example of a
pharmaceutical agent. The present disclosure also provides compositions
comprising the
multivalent ligand clusters of the present disclosure, and methods of making
and using the
multivalent ligand clusters of the present disclosure.
Definitions
Before further description of the invention, and in order that the invention
may be
more readily understand, certain terms are first defined and collected herein
for convenience.
As used herein, the term "treat," "treating," or "treatment" may include
prophylaxis
and means to ameliorate, alleviate symptoms, eliminate the causation of the
symptoms either
on a temporary or permanent basis, or to prevent or slow the appearance of
symptoms of the
named disorder or condition.
As used herein, the term "about," in connection with a measured quantity,
refers to
the normal variations in that measured quantity, as expected by the skilled
artisan making the
measurement and exercising a level of care commensurate with the objective of
measurement
and the precision of the measuring equipment.
As used herein, the term "conjugate" or "conjugate group" means an atom or
group of
atoms bound to an oligonucleotide or other oligomer. In general, conjugate
groups modify
one or more properties of the compound to which they are attached, including,
but not limited
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to pharmacodynamics, pharmacokinetic, binding, absorption, cellular
distribution, cellular
uptake, charge, and/or clearance properties.
As used herein, the term "linked," when referring to the connection between
two
molecules, means the two molecules are joined, directly or indirectly, by a
covalent bond or
that the two molecules are associated via noncovalent bonds (e.g., hydrogen
bonds or ionic
bonds). An example of a Compound A being directly joined to a Compound B may
be
represented as A-B. An example of a Compound A being indirectly joined to a
Compound B
may be represented as A-C-B, where Compound A is indirectly joined to Compound
B
through Compound C. It will be appreciated that more than one intermediary
compound may
be present in situations of indirect joining of compounds.
As used herein, the term "nucleic acid" refers to molecules composed of
monomeric
nucleotides. A nucleic acid includes ribonucleic acids (RNAs),
deoxyribonucleic acids
(DNAs), single-stranded nucleic acids (ssDNAs), double-stranded nucleic acids
(dsDNAs),
small interfering ribonucleic acids (siRNAs) and microRNAs (miRNAs). A nucleic
acid may
also comprise any combination of these elements in a single molecule. A
nucleic acid may
include natural nucleic acids, non-natural nucleic acids, or a combination of
natural and non-
natural nucleic acids. A nucleic acid may also be referred to herein as a
nucleotide sequence,
or as a polynucleotide.
As used herein, the term "oligomer" refers to nucleotide sequence containing
up to 5,
up to 10, up to 15, up to 20, or more than 20 nucleotides or nucleotide base
pairs. In some
embodiments, an oligomer has a nucleobase sequence that is at least partially
complementary
to a coding sequence in an expressed target nucleic acid or target gene within
a cell. In some
embodiments, the oligomers, upon delivery to a cell expressing a gene, are
able to inhibit the
expression of the underlying gene. The gene expression can be inhibited in
vitro or in vivo.
Non-limiting examples of oligomers that may be included in methods and
complexes of the
invention are oligonucleotides, single-stranded oligonucleotides, single-
stranded antisense
oligonucleotides, short interfering RNAs (siRNAs), single-stranded siRNA,
double-strand
RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), ribozymes,
interfering RNA molecules, and dicer substrates.
As used herein, the term "oligonucleotide" refers to a polymer of linked
nucleotides
each of which can be independently modified or unmodified.
As used herein, the term "single-stranded oligonucleotide" refers to a single-
stranded
oligomer and in certain embodiments a single-stranded oligonucleotide may
comprise a
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sequence at least partially complementary to a target mRNA, that is capable of
hybridizing to
a target mRNA through hydrogen bonding under mammalian physiological
conditions (or
comparable conditions in vitro). In some embodiments, a single-stranded
oligonucleotide is a
single stranded antisense oligonucleotide.
As used herein, the term "siRNA" refers to a short interfering RNA or
silencing RNA.
siRNAs are a class of double-stranded RNA molecules, that may be 20-25 (or
shorter) base
pairs in length, similar to microRNA (miRNA) that operate within the RNA
interference
(RNAi) pathway. siRNAs interfere with the expression of specific genes with
complementary
nucleotide sequences to the siRNA by degrading mRNA after transcription,
preventing
translation. siRNAs act in cells to silence gene expression by inducing the
RNA-induced
silencing complex (RISC) to cleave messenger RNA (mRNA).
As used herein, the term "effective amount," "therapeutically effective
amount," or
"effective dose" refers to an amount sufficient to elicit the desired
pharmacological or
therapeutic effects, thus resulting in effective prevention or treatment of
the disorder.
Prevention of the disorder is manifested by delaying the onset of the symptoms
of the
disorder to a medically significant extent. Treatment of the disorder is
manifested by a
decrease in the symptoms associated with the disorder or an amelioration of
the reoccurrence
of the symptoms of the disorder.
As used herein, the term "pharmaceutical composition" or "composition" refers
to a
mixture of substances suitable for administering to an individual. For
example, though not
intended to be limiting, a pharmaceutical composition can comprise one or more
active
agents and a pharmaceutical carrier, also referred to herein as a
"pharmaceutically acceptable
carrier" (e.g. a sterile aqueous solution). In some embodiments, a
pharmaceutical
composition is sterile.
As used herein, the term "alkyl" as used by itself or as part of another group
refers to
a straight- or branched-chain aliphatic hydrocarbon containing one to twelve
carbon atoms
(i.e., C1_12 alkyl) or the number of carbon atoms designated (i.e., a C1 alkyl
such as methyl, a
C2 alkyl such as ethyl, a C3 alkyl such as propyl or isopropyl, etc.). Non-
limiting illustrative
C1_10 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,
tert-butyl, iso-
butyl, 3-pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like.
As used herein, the term "substituted alkyl" by itself or as part of another
group
means that the alkyl as defined herein is substituted with one or more (e.g.,
one, two, or three)
independently selected substituents. A non-limiting list of independently
selected substituents
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includes amino, (alkyl)amino, (alkyl)carbonyl, (aryl)carbonyl,
(alkoxy)carbonyl,
[(alkoxy)carbonyl]amino, carboxy, aryl, heteroaryl, ureido, guanidino,
halogen, sulfonamido,
hydroxyl, (alkyl)sulfanyl, nitro, haloalkoxy, aryloxy, aralkyloxy,
(alkyl)sulfonyl,
(cycloalkyl)sulfonyl, (aryl)sulfonyl, cycloalkyl, sulfanyl, caboxamido,
heterocyclyl, and
(heterocyclyl)sulfonyl.
As used herein, the term "cycloalkyl" by itself or as part of another group
refers to
saturated and partially unsaturated (containing one or two double bonds)
cyclic aliphatic
hydrocarbons containing one to three rings having from three to twelve carbon
atoms (i.e.,
C3_12 cycloalkyl) or the number of carbons designated. Non-limiting
illustrative cycloalkyl
groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
cyclooctyl,
norbornyl, decalin, adamantyl, cyclohexenyl, cyclopentenyl, cyclohexenyl, and
the like.
As used herein, the term "substituted cycloalkyl" by itself or as part of
another group
means that the cycloalkyl as defined herein is substituted with one, two, or
three
independently selected substituents. A non-limiting list of independently
selected substituents
includes halo, nitro, cyano, hydroxyl, amino, (alkyl)amino, (dialkyl)amino,
haloalkyl,
(hydroxyl)alkyl, (dihydroxy)alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy,
alkylthio,
carboxamido, sulfonamido, (alkyl)carbonyl, (aryl)carbonyl, (alkyl)sulfonyl,
arylsulfonyl,
ureido, guanidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl,
alkynyl, aryl,
heteroaryl, heterocyclyl, (alkoxy)alkyl, (amino)alkyl, (hydroxyl)alkylamino,
(alkylamino)alkyl, (dialkylamino)alkyl, (cyano)alkyl, (carboxamido)alkyl,
(alkyl)sulfanyl,
(heterocyclo)alkyl, (heteroaryl)alkyl, (alkoxy)carbonyl, and mercaptoalkyl.
As used herein, the term "alkenyl" by itself or as part of another group
refers to an
alkyl group as defined herein containing one, two or three carbon-to-carbon
double bonds.
Non-limiting illustrative alkenyl groups include ethenyl, propenyl,
isopropenyl, butenyl, sec-
butenyl, pentenyl, and hexenyl.
As used herein, the term "substituted alkenyl" by itself or as part of another
group
means the alkenyl as defined herein is substituted with one, two, or three
independently
selected substituents. A non-limiting list of independently selected
substituents includes halo,
nitro, cyano, hydroxyl, amino, (alkyl)amino, (dialkyl)amino, haloalkyl,
(hydroxy)alkyl,
(dihydroxy)alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, (alkyl)sulfanyl,
carboxamido,
sulfonamido, (alkyl)carbonyl, (aryl)carbonyl, (alkyl)sulfonyl, (aryl)sulfonyl,
ureido,
guanidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,
heteroaryl, and
heterocyclyl.
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As used herein, the term "cycloalkenyl" by itself or as part of another group
refers to
non-aromatic cyclic alkyl groups of from 4 to 10 carbon atoms having single or
multiple
cyclic rings and having at least one >C=C< ring unsaturation and preferably
from 1 to 2 sites
of >C=C< ring unsaturation.
As used herein, the term "substituted cycloalkenyl" by itself or as part of
another
group refers to a cycloalkenyl as defined herein having from 1 to 5
independently selected
sub stituents. A non-limiting list of independently selected sub stituents
includes oxo, thione,
alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted
alkynyl, alkoxy,
substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino,
aminocarbonyl,
aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino,
aminocarbonyloxy,
aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, aryl,
substituted aryl,
aryloxy, substituted aryloxy, arylthio, substituted arylthio, azido, carboxyl,
carboxyl ester,
(carboxyl ester)amino, (carboxyl ester)oxy, cyano, cyanate, cycloalkyl,
substituted cycloalkyl,
cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted
cycloalkylthio,
cycloalkenyl, substituted cycloalkenyl, cycloalkenyloxy, substituted
cycloalkenyloxy,
cycloalkenylthio, substituted cycloalkenylthio, guanidino, substituted
guanidino, halo,
hydroxy, hydroxyamino, alkoxyamino, hydrazino, substituted hydrazino,
heteroaryl,
substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy,
heteroarylthio, substituted
heteroarylthio, heterocyclic, substituted heterocyclic, heterocyclyloxy,
substituted
heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SO3H,
substituted
sulfonyl, sulfonyloxy, thioacyl, thiocyanate, thiol, alkylthio, and
substituted alkylthio.
As used herein, the term "alkynyl" by itself or as part of another group
refers to an
alkyl group as defined herein containing one to three carbon-to-carbon triple
bonds. Non-
limiting illustrative alkynyl groups include ethynyl, propynyl, butynyl, 2-
butynyl, pentynyl,
and hexynyl groups.
As used herein, the term "substituted alkynyl" by itself or as part of another
group
means the alkynyl as defined herein is substituted with one, two, or three
independently
selected substituents. A non-limiting list of independently selected
substituents includes halo,
nitro, cyano, hydroxyl, amino, alkylamino, dialkylamino, haloalkyl,
(hydroxy)alkyl,
(dihydroxy)alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, (alkyl)sulfanyl,
carboxamido,
sulfonamido, alkylcarbonyl, arylcarbonyl, alkylsulfonyl, arylsulfonyl, ureido,
guanidino,
carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,
heteroaryl, and heterocyclyl.
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As used herein, the term "haloalkyl" by itself or as part of another group
refers to an
alkyl group substituted by one or more fluorine, chlorine, bromine and/or
iodine atoms. Non-
limiting illustrative haloalkyl groups include fluoromethyl, difluoromethyl,
trifluoromethyl,
pentafluoroethyl, 1,1-difluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl,
3,3,3 -
trifluoropropyl, 4,4,4-trifluorobutyl, and trichloromethyl groups.
As used herein, the term "alkoxy" by itself or as part of another group refers
to an
alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,
substituted alkenyl,
alkynyl, or substituted alkynyl attached to a terminal oxygen atom.
As used herein, the term "haloalkoxy" by itself or as part of another group
refers to a
haloalkyl attached to a terminal oxygen atom. Non-limiting illustrative
haloalkoxy groups
include fluoromethoxy, difluoromethoxy, trifluoromethoxy, and 2,2,2 -
trifluoroethoxy.
As used herein, the term "aryl" by itself or as part of another group refers
to a
monocyclic or bicyclic aromatic ring system having from six to fourteen carbon
atoms (i.e.,
C6-C14 aryl). Non-limiting illustrative aryl groups include phenyl, naphthyl,
phenanthryl,
anthracyl, indenyl, azulenyl, biphenyl, biphenylenyl, and fluorenyl groups.
As used herein, the term "substituted aryl" by itself or as part of another
group means
that the aryl as defined herein is substituted with one to five independently
selected
sub stituents. A non-limiting list of independently selected sub stituents
includes halo, nitro,
cyano, hydroxyl, amino, alkylamino, dialkylamino, haloalkyl, (hydroxy)alkyl,
(dihydroxy)alkyl, alkoxy, haloalkoxy, aryloxy, heteroaryloxy, aralkyloxy,
alkylthio,
carboxamido, sulfonamido, (alkyl)carbonyl, (aryl)carbonyl, (alkyl)sulfonyl,
(aryl)sulfonyl,
ureido, guanidino, carboxy, carboxyalkyl, alkyl, cycloalkyl, alkenyl, alkynyl,
aryl, heteroaryl,
heterocyclo, (alkoxy)alkyl, (amino)alkyl, [(hydroxyl)alkyl]amino,
[(alkyl)amino]alkyl,
[(dialkyl)amino)alkyl, (cyano)alkyl, (carboxamido)alkyl, mercaptoalkyl,
(heterocyclo)alkyl,
(cycloalkylamino)alkyl, (halo(Ci-C4)alkoxy)alkyl, (heteroaryl)alkyl, and the
like. Non-
limiting illustrative substituted aryl groups include 2-methylphenyl, 2-
methoxyphenyl, 2-
fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl,
3-
fluorophenyl, 3-chlorophenyl, 4-methylphenyl, 4-ethylphenyl, 4-methoxyphenyl,
4-
fluorophenyl, 4-chlorophenyl, 2,6-di-fluorophenyl, 2,6-di-chlorophenyl, 2-
methyl, 3-
methoxyphenyl, 2-ethyl, 3-methoxyphenyl, 3,4-di-methoxyphenyl, 3,5-di-
fluorophenyl 3,5-
di-methylphenyl, 3,5-dimethoxy, 4-methylphenyl, 2-fluoro-3-chlorophenyl, and 3-
chloro-4-
fluorophenyl. The term substituted aryl is meant to include groups having
fused substituted
cycloalkyl and fused substituted heterocyclo rings.
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As used herein, the term "aryloxy" by itself or as part of another group
refers to an
aryl or substituted aryl attached to a terminal oxygen atom. A non-limiting
illustrative
aryloxy group is Ph0¨.
As used herein, the term "heteroaryloxy" by itself or as part of another group
refers to
a heteroaryl or substituted heteroaryl attached to a terminal oxygen atom.
As used herein, the term "aralkyloxy" by itself or as part of another group
refers to an
aralkyl group attached to a terminal oxygen atom. A non-limiting illustrative
aralkyloxy
group is PhCH20¨.
As used herein, the term "heteroaryl" refers to monocyclic and bicyclic
aromatic ring
systems having 5 to 14 ring atoms (i.e., C5-C14 heteroaryl) and 1, 2, 3, or 4
heteroatoms
independently chosen from oxygen (0), nitrogen (N), and sulfur (S). Non-
limiting illustrative
heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl,
thianthrenyl, furyl,
benzofuryl, pyranyl, isobenzofuranyl, benzooxazonyl, chromenyl, xanthenyl, 2H-
pyrrolyl,
pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,
isoindolyl, 3H-
indolyl, indolyl, indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl,
naphthyridinyl,
cinnolinyl, quinazolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, 13-
carbolinyl, phenanthridinyl,
acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, thiazolyl, isothiazolyl,
phenothiazolyl,
isoxazolyl, furazanyl, and phenoxazinyl. The term "heteroaryl" is also meant
to include
possible N-oxides. Illustrative N-oxides include pyridyl N-oxide and the like.
As used herein, the term "substituted heteroaryl" by itself or as part of
another group
means that the heteroaryl as defined herein is substituted with one to four
independently
selected substituents. A non-limiting list of independently selected
substituents includes halo,
nitro, cyano, hydroxy, amino, (alkyl)amino, (dialkyl)amino, haloalkyl,
(hydroxy)alkyl,
(dihydroxy)alkyl, alkoxy, haloalkoxy, aryloxy, aralkyloxy, alkylthio,
carboxamido,
sulfonamido, (alkyl)carbonyl, (aryl)carbonyl, (alkyl) sulfonyl,
(aryl)sulfonyl, ureido,
guanidino, carboxy, (carboxy)alkyl, alkyl, cycloalkyl, alkenyl, alkynyl, aryl,
heteroaryl,
heterocyclo, (alkoxy)alkyl, (amino)alkyl, [(hydroxyl)alkyl]amino,
[(alkyl)amino]alkyl,
[(dialkyl)amino]alkyl, (cyano)alkyl, (carboxamido)alkyl, mereaptoalkyl,
(heterocyclo)alkyl,
and (heteroaryl)alkyl. Any available carbon or nitrogen atom can be
substituted.
As used herein, the term "heterocyclo" or "heterocycly1" by itself or as part
of another
group refers to saturated and partially unsaturated (e.g., containing one or
two double bonds)
cyclic groups containing one, two, or three rings having from three to
fourteen ring members
(i.e., a 3- to 14-membered heterocyclo) and at least one heteroatom. Each
heteroatom is
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independently selected. The term "heterocyclo" or "heterocyclyl" is meant to
include cyclic
ureido groups, such as, 2-imidazolidinone, and cyclic amide groups, such as,
13-lactam, y-
lactam, 6-lactam and c-lactam. The term "heterocyclo" or "heterocyclyl" is
also meant to
include groups having fused aryl or substituted aryl groups, e.g., indolinyl.
The heterocyclo
.. or heterocyclyl can be linked to the rest of the molecule through a carbon
or nitrogen atom.
Non-limiting illustrative heterocyclo (or heterocyclyl) groups include 2-
oxopyrrolidin-3-yl,
2-imidazolidinone, piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl, and
indolinyl.
As used herein, the term "substituted heterocyclo" or "substituted
heterocyclyl" by
itself or part of another group means the heterocyclo or heterocyclyl group as
defined above
is substituted with one to four independently selected substituents. A non-
limiting list of
independently selected substituents includes halo, nitro, cyano, hydroxyl,
amino,
(alkyl)amino, (dialkyl)amino, haloalkyl, (hydroxy)alkyl, (dihydroxy)alkyl,
alkoxy,
haloalkoxy, aryloxy, aralkyloxy, alkylthio, carboxamido, sulfonamido,
(alkyl)carbonyl,
(aryl)carbonyl, (alkyl)sulfonyl, (aryl)sulfonyl, ureido, guanidino, carboxy,
carboxyalkyl,
alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl,
alkoxyalkyl, (amino)alkyl,
[(hydroxyl)alkyl]amino, [(alkyl)amino] alkyl, [(dialkyl)amino] alkyl,
(cyano)alkyl,
(carboxamido)alkyl, mercaptoalkyl, (heterocyclyl)alkyl, and (heteroaryl)alkyl.
Substitution
may occur on any available carbon or nitrogen atom, and may form a spirocycle.
As used herein, the term "amino" by itself or as part of another group refers
to ¨NH2.
As used herein, the term "alkylamino" or "(alkyl)amino" by itself or as part
of another
group refers to ¨NHR, wherein R is alkyl.
As used herein, the term "dialkylamino" or "(dialkyl)amino" by itself or as
part of
another group refers to ¨NRR-, wherein It' and R- are each independently alkyl
or It' and
R- are taken together to form a 3- to 8-membered heterocyclo or substituted
heterocyclo.
As used herein, the term "cycloalkylamino" by itself or as part of another
group refers
to ¨NRR-, wherein It' is cycloalkyl or substituted cycloalkyl, and R- is
hydrogen or alkyl.
As used herein, the term "(amino)alkyl" by itself or as part of another group
refers to
an alkyl group substituted with an amino group. Non-limiting illustrative
(amino)alkyl groups
include ¨CH2CH2NH2, ¨CH2CH2CH2NH2, and ¨CH2CH2CH2CH2NH2.
As used herein, the term "(alkylamino)alkyl" or "[(alkyl)amino]alkyl" by
itself or as
part of another group refers to an alkyl group substituted with an alkylamino
group. A non-
limiting illustrative (alkylamino)alkyl group is ¨CH2CH2N(H)CH3.
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As used herein, the term "(dialkylamino)alkyl" by itself or as part of another
group
refers to an alkyl group substituted by a dialkylamino group. Non-limiting
illustriative
(dialkylamino)alkyl groups include ¨CH2N(CH3)2 and ¨CH2CH2N(CH-3)2.
As used herein, the term "(cycloalkylamino)alkyl" by itself or as part of
another group
refers to an alkyl group substituted by a cycloalkylamino group. Non-limiting
illustrative
(cycloalkylamino)alkyl groups include ¨CH2N(H)cyclopropyl, ¨CH2N(H)cyclobutyl,
and
¨CH2N(H)cyclohexyl.
As used herein, the term "carboxamido" by itself or as part of another group
refers to
a radical of formula ¨C(=0)NRit-, where It' and R- are each independently
hydrogen,
alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, substituted
aryl, heteroaryl, or
substituted heteroaryl, or It' and R- taken together with the nitrogen to
which they are
attached form a 3- to 8-membered heterocyclo group. Non-limiting illustrative
carboxamido
groups include ¨CONH2, ¨CON(H)CH3, CON(CH3)2, and CON(H)Ph.
As used herein, the term "sulfonamido" by itself or as part of another group
refers to a
radical of the formula ¨SO2NRit-, where It' and R- are each independently
hydrogen, alkyl,
substituted alkyl, aryl, or substituted aryl, or It' and R- taken together
with the nitrogen to
which they are attached form a 3- to 8-membered heterocyclo group. Non-
limiting illustrative
sulfonamido groups include ¨SO2NH2, ¨SO2N(H)CH3, and ¨SO2N(H)Ph.
As used herein, the term "(alkyl)carbonyl" by itself or as part of another
group refers
to a carbonyl group, i.e., ¨C(=0)¨, substituted by an alkyl group. A non-
limiting
illustrative alkylcarbonyl group is ¨COCH3.
As used herein, the term "(alkoxy)carbonyl" (or "ester") by itself or as part
of another
group refers to a carbonyl group, i.e., ¨C(=0)¨, substituted by an alkoxy
group. A non-
limiting illustrative (alkoxy)carbonyl group is ¨C(0)0CH3.
As used herein, the term "(aryl)carbonyl" by itself or as part of another
group refers to
a carbonyl group, i.e., ¨C(=0)¨, substituted by an aryl or substituted aryl
group. A non-
limiting illustrative arylcarbonyl group is ¨COPh.
As used herein, the term "sulfanyl" by itself or as part of another group
refers to a ¨
SH group.
As used herein, the term "(alkyl)sulfanyl" or "alkylthio" by itself or as part
of another
group refers to a sulfur atom substituted by an alkyl or substituted alkyl
group. Non-limiting
illustrative alkylthio groups include ¨SCH3, and ¨SCH2CH3.
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As used herein, the term "mercaptoalkyl" by itself or as part of another group
refers to
an alkyl group substituted by a ¨SH group.
As used herein, the term "alkylsulfonyl" or "(alkyl)sulfonyl" by itself or as
part of
another group refers to a sulfonyl group, i.e., ¨SO2¨, substituted by an alkyl
or substituted
alkyl group. A non-limiting illustrative alkylsulfonyl group is ¨S02CH3.
As used herein, the term "arylsulfonyl" or "(aryl)sulfonyl" by itself or as
part of
another group refers to a sulfonyl group, i.e., ¨SO2¨, substituted by an aryl
or substituted
aryl group. A non-limiting illustrative arylsulfonyl group is ¨SO2Ph.
As used herein, the term "carboxy" by itself or as part of another group
refers to a
radical of the formula ¨COOH.
As used herein, the term "(carboxy)alkyl" by itself or as part of another
group refers
to an alkyl group substituted with a ¨COOH. A non-limiting illustrative
carboxyalkyl group
is ¨CH2CO2H.
As used herein, the term "aralkyl" by itself or as part of another group
refers to a
residue in which an aryl moiety is attached to an alkyl residue. The aralkyl
group may be
attached to the parent structure at either the aryl or the alkyl residue.
As used herein, the term "substituted aralkyl" by itself or as part of another
group
refers to a residue in which an aryl moiety is attached to a substituted alkyl
residue.
As used herein, the term "aralkenyl" by itself or as part of another group
refers to a
radical of the formula ¨Rd---R c where Rd is an alkenylene chain and Itc is
one or more aryl
radicals.
As used herein, the term "substituted aralkenyl" by itself or as part of
another group
refers to an aralkenyl radical where the alkenylene chain of the aralkenyl
radical is an
optionally substituted alkenylene chain, and each aryl radical of the
aralkenyl radical is an
optionally substituted aryl radical.
As used herein, the term "aralkynyl" by itself or as part of another group
refer to a
radical of the formula ¨RR, where Re is an alkynylene chain and Rc is one or
more aryl
radicals.
As used herein, the term "substituted aralkynyl" by itself or as part of
another group
refers to an aralkynyl radical where the alkynylene chain of the aralkynyl
radical is an
optionally substituted alkynylene chain, and each aryl radical of the
aralkynyl radical is an
optionally substituted aryl radical.
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As used herein, the term "aliphatic heterocycle" by itself or as part of
another group
refers to a non-aromatic ring in which one or more of the ring-forming atoms
is a heteroatom.
As used herein, the term "heteroatom" refers to an atom inserted between a
carbon
atom and its parent molecule (i.e., between the points of attachment). Non-
limiting
illustrative heteroatoms include oxygen, nitrogen, sulfur (including sulfoxide
and sulfone),
and phosphorous (P).
As used herein, the term "saccharide" refers to a single sugar moiety or
monosaccharide unit as well as combinations of two or more single sugar
moieties or
monosaccharide units covalently linked to form disaccharides,
oligosaccharides, and
polysaccharides. The polysaccharide may be linear or branched.
As used herein, the term "monosaccharide" refers to a single sugar residue in
an
oligosaccharide.
As used herein, the term "disaccharide" refers to a polysaccharide composed of
two
monosaccharide units or moieties linked together by a glycosidic bond.
As used herein, an "oligosaccharide" refers to a compound containing two or
more
monosaccharide units or moieties. Within the context of an oligosaccharide, an
individual
monomer unit or moiety is a monosaccharide which is, or can be, bound through
a hydroxy
group to another monosaccharide unit or moiety. Oligosaccharides can be
prepared by either
chemical synthesis from protected single residue sugars or by chemical
degradation of
biologically produced polysaccharides. Alternatively, oligosaccharides may be
prepared by in
vitro enzymatic methods.
As used herein, the term "ureido" by itself or as part of another group refers
to a
radical of the formula ¨NR'¨C(=0)¨NR-R-, wherein 11' is hydrogen, alkyl, aryl,
or
substituted aryl, and R- and R¨ are each independently hydrogen, alkyl, aryl
or substituted
aryl, or R and Rõ, taken together with the nitrogen to which they are attached
form a 4- to 8-
membered heterocyclo group. Non-limiting illustrative ureido groups include
¨NH¨
C(=0)¨NH2 and ¨NH¨C(=0)¨NHCH3.
As used herein, the term "guanidino" by itself or as part of another group
refers to a
radical of the formula ¨NR'¨C(=NR-)¨NR¨R--, wherein It', R¨, and R¨ are each
independently hydrogen, alkyl, aryl, or substituted aryl, and R- is hydrogen,
alkyl, cyano,
alkylsulfonyl, alkylcarbonyl, carboxamido, or sulfonamido. Non-limiting
illustrative
guanidino groups include ¨NH¨C(=NH)¨NH2, ¨NH¨C(=NCN)¨NH2, and ¨NH¨
C(=NH)¨NHCH3.
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As used herein, the term "(heteroaryl)alkyl" by itself or as part of another
group refers
to an alkyl group substituted with one, two, or three heteroaryl or
substituted heteroaryl
groups.
As used herein, the term "heteroalkyl" by itself or part of another group
refers to a
stable straight or branched chain hydrocarbon radical containing at least one
heteroatom,
which can be the same or different. The heteroatom(s) can be placed at any
interior position
or terminal position of the heteroalkyl group, or at a position at which the
heteroalkyl
group is attached to the remainder of the molecule. Non-limiting illustrative
heteroalkyl
groups include ¨CH2N(H)CH2CH2N(CH3)2, ¨CH2N(CH3)CH2CH2N(CH3)2, ¨
CH2N(H)CH2CH2CH2N(CH3)2, ¨CH2N(H)CH2CH2OH, ¨CH2N(CH3)CH2CH2OH, ¨
CH2OCH2CH2OCH3, ¨OCH2CH2OCH2CH2OCH3, ¨CH2NHCH2CH2OCH2, ¨
OCH2CH2NH2, and ¨NHCH2CH2N(H)CH3.
As used herein, the term "(heterocyclo)alkyl" or "(heterocyclyl)alkyl" by
itself or as
part of another group refers to an alkyl group substituted with one
heterocyclyl or substituted
heterocyclyl group, and optionally one hydroxy group.
As used herein, the term "(carboxamido)alkyl" by itself or as part of another
group
refers to an alkyl group substituted with one carboxamido group, and
optionally one
heterocyclo, amino, alkylamino, or dialkylamino group.
As used herein, the term "N-oxide" refers to a compound that contains a
functional
group, wherein N is further connected to H and/or the rest of the compound
structure.
As used herein, the term "integral number" refers to an integer including, but
not
limited to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.
Compound
General Structure
A multivalent ligand cluster, having a diamine scaffold, may have the general
structure of Formula 1:
TL "-'N H
N finkerB11 w
LH
TL )4õ.
1,1.0 m
linkerA N
n
linker A H Formula 1
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where:
each TL is an independently selected targeting ligand,
m is an integral number between 1 and 10,
each n is an independently selected integral number between 1 and 10,
each linkerA is an independently selected spacer, with one end attached to a
TL and
the other end attached to the nitrogen of an alkylcarboxamide,
linkerB is a spacer, with one end attached to a pharmaceutical agent or a
functional
group capable of linking to one or more pharmaceutical agents, and the other
end attached to
a diamine nitrogen, and
W is either one or more pharmaceutical agents, or a functional group capable
of
linking to one or more pharmaceutical agents.
In some embodiments, m may be configured based on a starting material used to
synthesize the multivalent ligand cluster. For example, m may be 1 due to
ethylenediamine
being used as a starting material, m may be 2 due to 1,3-propanediamine being
used as a
starting material, m may be 3 due to 1,4-butanediamine being used as a
starting material, etc.
As used herein, a "spacer" refers to a compound or molecule that links other
groups
together. Example linkerA and linkerB spacers are described in detail herein
below.
As used herein in reference to elements of multivalent ligand clusters of the
invention,
the term "independently selected" means that each of a given type of element
may differ from
one or more others of the same type of element in a multivalent ligand
cluster. For example, a
multivalent ligand cluster may include more than one TLs, where each may be
selected to be
different from or the same as one or more other TLs in that multivalent ligand
cluster. For
further example, a multivalent ligand cluster may include more than one "n,"
where each may
be selected to be different from or the same as one or more other "n" in that
multivalent
ligand cluster. In another example, a multivalent ligand cluster may include
more than one
linkerA, where each may be selected to be different from or the same as one or
more other
linkerAs in that multivalent ligand cluster.
Targeting Ligands
As mentioned above with respect to Formula 1, a multivalent ligand cluster of
the
present disclosure may include multiple (e.g., three) independently selected
targeting ligands.
In this context, the term "independently selected" means that each targeting
ligand may be
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selected to be different from or the same as one or more other targeting
ligands in the same
multivalent ligand cluster.
At least one of the independently selected targeting ligands of Formula 1 may
be
capable of binding to one or more cell receptors, cell channels, and/or cell
transporters
capable of facilitating endocytosis.
In some embodiments, at least one of the independently selected targeting
ligands of
Formula 1 may comprise at least one small molecule ligand. As used herein, a
"small
molecule ligand" refers to a ligand smaller than a protein. In some
embodiments, at least one
small molecular ligand may comprise at least one of N-acetylgalactosamine
(GalNAc),
.. galactose, galactosamine, N-formyl-galactosamine, N-propionylgalactosamine,
N-
butanoylgalactosamine, and N-iso-butanoylgalactosamine, a macrocycle, a folate
molecule, a
fatty acid, a bile acid, a cholesterol, and derivatives thereof
A macrocycle is a molecule or ion containing a twelve or more membered ring.
The
present disclosure is not limited to any particular macrocycle. A non-limiting
list of
macrocycles within the scope of the present disclosure include terpenoid
macrocycles,
porphyrins, and cyclodextrins.
Folate, also known as vitamin B9 and folacin, is used by the human body to
make
DNA and RNA, and metabolize amino acids necessary for cell division. Folate
receptors bind
folate, and reduced folic acid derivatives. Thus, in some embodiments, at
least one of the
independently selected targeting ligands may comprise a reduced folic acid
derivative.
A fatty acid is a carboxylic acid with a long aliphatic chain. In some
embodiments,
the fatty acid may be saturated, meaning the aliphatic chain has all single
carbon-to-carbon
bonds. In some embodiments, the fatty acid may be unsaturated, meaning the
aliphatic chain
includes at least one double or triple carbon-to-carbon bond. In some
embodiments, the fatty
.. acid may include a branched chain. In some embodiments, the fatty acid may
include a ring
structure. Fatty acids are known to assist in the update of pharmaceutical
agents into cells.
See Prakash et at. "Fatty acid conjugation enhances potency of antisense
oligonucleotides in
muscle." Nucleic Acids Res. 2019;47(12):6029-6044. doi:10.1093/nar/gkz354;
Raouane et at.
"Lipid Conjugated Oligonucleotides: A Useful Strategy for Delivery."
Bioconjugate
Chemistry 2012 23 (6), 1091-1104, DOT: 10.1021/bc200422w; and Osborn et at.
"Improving
siRNA Delivery In Vivo Through Lipid Conjugation." Nucleic Acid Ther.
2018;28(3):128-
136. doi:10.1089/nat.2018.0725.
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A bile acid is a steroidal acid found in bile. Bile acid is a known ligand for
the
farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (GPBAR1)
(TGR5).
In some embodiments, the bile acid may be a primary bile acid synthesized in
the liver. In
some embodiments, the bile acid may be a secondary bile acid resulting from
bacterial
actions in the colon. Bile acids are known to be beneficial in inhibiting RNA
translation. See
Gonzalez-Carmona et at. "Inhibition of hepatitis C virus RNA translation by
antisense bile
acid conjugated phosphorothioate modified oligodeoxynucleotides (ODN)."
Antiviral Res.
2013, 97, 49-59. doi:10.1089/nat.2018.0725.
In some embodiments, at least one of the independently selected targeting
ligands of
Formula 1 may comprise at least one peptide. Various peptides and
corresponding peptide
receptors are known to those skilled in the art. The present disclosure is not
limited to any
particular peptide. Peptides known and not yet discovered are within the scope
of the present
disclosure.
In some embodiments, at least one of the independently selected targeting
ligands of
Formula 1 may comprise at least one cyclic peptide. As known to those skilled
in the art, a
cyclic peptide is a polypeptide chain having a cyclic ring structure. In some
embodiments, the
cyclic ring structure may be formed by linking one end of the peptide to the
other with an
amide bond, or other chemically stable bond such as lactone, ether, thioether,
disulfide, etc.
In some embodiments, a cyclic peptide of the present disclosure may be a
biologically active
cyclic peptide where a head-to-tail (or N-to-C) cyclization is formed by an
amide bond
between amino and carboxyl termini. In some embodiments, a cyclic peptide of
the present
disclosure may be a biologically active cyclic peptide where cyclization is
formed by "click
chemistry." See Rashad A.A. (2019) Click Chemistry for Cyclic Peptide Drug
Design. In:
Goetz G. (eds) Cyclic Peptide Design. Methods in Molecular Biology, vol 2001.
Humana,
New York, NY. https://doi.org/10.1007/978-1-4939-9504-2 8. The present
disclosure is not
limited to any particular cyclic peptide. A cyclic peptide of the present
disclosure may be
naturally occurring or synthetically produced.
In some embodiments, at least one of the independently selected targeting
ligands of
Formula 1 may comprise at least one aptamer. An aptamer is a short, single-
stranded DNA or
RNA molecule that can selectively bind to a specific target, such as a
protein, peptide,
carbohydrate, small molecule, toxin, or live cell. Aptamers assume a variety
of shapes, as
they tend to form helices and single-stranded loops. The present disclosure is
not limited to
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any particular aptamer. Aptamers known and not yet discovered are within the
scope of the
present disclosure.
In some embodiments, at least one of the independently selected targeting
ligands of
Formula 1 may be capable of binding to at least one Asialoglycoprotein
receptor (ASGPR).
ASGPRs are lectins, located on liver cells, that bind galactose residues.
ASGPR has been
demonstrated to have high expression on the surface of hepatocytes, human
carcinoma cell
lines, and liver cancers. ASGPR is also weakly expressed by glandular cells of
the
gallbladder and stomach.
In some embodiments, at least one of the independently selected targeting
ligands of
Formula 1 may be capable of binding to at least one transferrin receptor. A
transferrin
receptor is a membrane glycoprotein that mediates cellular uptake of
transferrin, a protein in
the blood that binds to iron and transports it through the body. The
transferrin receptor-
mediated endocytosis pathway is known to those skilled in the art. See Qian et
at. "Targeted
drug delivery via the transferrin receptor-mediated endocytosis pathway."
Pharmacol Rev.
2002 Dec; 54(4):561-87. doi: 10.1124/pr.54.4.561. PMID: 12429868. The present
disclosure
is not limited to any particular ligand capable of binding to at least one
transferring receptor.
Transferrin receptor ligands known and not yet developed are within the scope
of the present
disclosure.
In some embodiments, at least one of the independently selected targeting
ligands of
Formula 1 may be capable of binding to at least one integrin receptor. An
integrin receptor is
a transmembrane receptor that facilitates cell-cell and cell-extracellular
matrix (ECM)
adhesion. Upon ligand binding, integrin receptors activate signal transduction
pathways that
mediate cellular signals such as regulation of the cell cycle, organization of
the intracellular
cytoskeleton, and movement of new receptors to the cell membrane. Integrin
targeted
delivery of gene therapeutics is known to those skilled in the art. See
Juliano et at. "Integrin
targeted delivery of gene therapeutics." Theranostics vol. 1 211-9. 2 Mar.
2011,
doi:10.7150/thno/vOlp0211. The present disclosure is not limited to any
particular ligand
capable of binding to at least one integrin receptor. Integrin receptor
ligands known and not
yet developed are within the scope of the present disclosure.
In some embodiments, at least one of the independently selected targeting
ligands of
Formula 1 may be capable of binding to at least one folate receptor. A folate
receptor binds
folate and reduced folic acid derivatives, and mediates delivery of
tetrahydrofolate to the
interior of cells. Targeted drug delivery via folate receptors is known to
those skilled in the
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art. See Zhao et at. "Targeted drug delivery via folate receptors." Expert
Opin Drug Deliv.
2008 Mar; 5(3):309-19. doi: 10.1517/17425247.5.3.309. PMID: 18318652. The
present
disclosure is not limited to any particular ligand capable of binding to at
least one folate
receptor. Folate receptor ligands known and not yet developed are within the
scope of the
present disclosure.
In some embodiments, at least one of the independently selected targeting
ligands of
Formula 1 may be capable of binding to at least one G-protein-coupled receptor
(GPCR).
GPCRs are cell surface receptors that bind, among other things, peptides,
lipids, sugars, and
proteins. GPCRs interact with G proteins in the plasma membrane. When an
external
signaling molecule binds to a GPCR, it causes a conformational change in the
GPCR, which
triggers the interaction between the GPCR and a nearby G protein. GPCRs
consist of a single
polypeptide that is folded into a globular shape and embedded in a cell's
plasma membrane.
GPCR targeted delivery of oligonucleotide therapeutics is known to those
skilled in the art.
See Knerr et al. "Glucagon Like Peptide 1 Receptor Agonists for Targeted
Delivery of
Antisense Oligonucleotides to Pancreatic Beta Cell" J. Am. Chem. Soc., 2021
143 (9), 3416-
3429. DOT: 10.1021/jacs.0c12043.
LinkerAs
As mentioned above with respect to Formula 1, a multivalent ligand cluster of
the
present disclosure may include multiple (e.g., three) independently selected
linkerAs. In this
context, the term "independently selected" means that each linkerA may be
selected to be
different from or the same as one or more other linkerAs in the same
multivalent ligand
cluster.
Each linkerA is an independently selected spacer, with one end attached to a
targeting
ligand (TL in Formula 1) and the other end attached to the nitrogen of an
alkylcarboxamide
of the multivalent ligand cluster.
In some embodiments, at least one of the independently selected linkerAs may
comprise polythethylene glycol (PEG). The PEG may have any number of repeating
0-CH2-
CH2 units. For example, the PEG may be PEG1, PEG2, PEG3, PEG4, PEGS, PEG6,
PEG7,
PEG8, PEG9, PEG10, PEG11, PEG12, PEG13, PEG14, PEG15, PEG16, PEG17, PEG18,
PEG19, PEG20, PEG21, PEG22, PEG23, PEG24, PEG25, PEG26, PEG27, PEG28, PEG29,
PEG30, PEG31, PEG32, PEG33, PEG34, PEG35, PEG36, PEG37, PEG38, PEG39, PEG40,
PEG41, PEG42, PEG43, PEG44, PEG45, PEG46, PEG47, PEG48, PEG49, PEG50, PEG51,
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PEG52, PEG53, PEG54, PEG55, PEG56, PEG57, PEG58, PEG59, PEG60, PEG61, PEG62,
PEG63, PEG64, PEG65, PEG66, PEG67, PEG68, PEG69, PEG70, PEG71, PEG72, PEG73,
PEG74, PEG75, PEG76, PEG77, PEG78, PEG79, PEG80, PEG81, PEG82, PEG83, PEG84,
PEG85, PEG86, PEG87, PEG88, PEG89, PEG90, PEG91, PEG92, PEG93, PEG94, PEG95,
PEG96, PEG97, PEG98, PEG99, PEG100, or larger.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one alkyl group. In some embodiments, an alkyl group may
have 2 carbons,
3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons,
10 carbons, 11
carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17
carbons, 18 carbons,
19 carbons, or 20 carbons.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one substituted alkyl group. In some embodiments, a
substituted alkyl group
may have 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8
carbons, 9
carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15
carbons, 16 carbons,
17 carbons, 18 carbons, 19 carbons, or 20 carbons. In some embodiments, a
substituted alkyl
group may include one or more of the following substitution groups: alkyl,
cycloalkyl,
hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one cycloalkyl group. In some embodiments, a cycloalkyl
group may be a
C3 cycloalkyl (i.e., cyclopropane), C4 cycloalkyl (i.e., cyclobutene), C5
cycloalkyl (i.e.,
cyclopentane), C6 cycloalkyl (i.e., cyclohexane), C7 cycloalkyl (i.e.,
cycloheptane), C8
cycloalkyl (i.e., cyclooctane), C9 cycloalkyl (i.e., cyclononane), or C10
cycloalkyl (i.e.,
cyclodecane).
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one substituted cycloalkyl group. In some embodiments, a
substituted
cycloalkyl group may be a C3 substituted cycloalkyl, C4 substituted
cycloalkyl, C5
substituted cycloalkyl, C6 substituted cycloalkyl, C7 substituted cycloalkyl,
C8 substituted
cycloalkyl, C9 substituted cycloalkyl, or C10 substituted cycloalkyl. In some
embodiments, a
substituted cycloalkyl group may include one or more of the following
substitution groups:
alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide,
sulfonyl, and
sulfonamide.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one alkenyl group. In some embodiments, an alkenyl group may
have 4
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carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11
carbons, 12
carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18
carbons, 19 carbons,
or 20 carbons.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one substituted alkenyl group. In some embodiments, an
alkenyl group may
have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10
carbons, 11 carbons,
12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18
carbons, 19
carbons, or 20 carbons. In some embodiments, a substituted alkenyl group may
include one
or more of the following substitution groups: alkyl, cycloalkyl, hydroxy,
alkoxide, carboxyl,
amine, amide, halide, sulfonyl, and sulfonamide.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one cycloalkenyl group. In some embodiments, a cycloalkenyl
group may
be a C5 cycloalkenyl, C6 cycloalkenyl, C7 cycloalkenyl, C8 cycloalkenyl, C9
cycloalkenyl,
or C10 cycloalkenyl.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one substituted cycloalkenyl group. In some embodiments, a
cycloalkenyl
group may be a C5 cycloalkenyl, C6 cycloalkenyl, C7 cycloalkenyl, C8
cycloalkenyl, C9
cycloalkenyl, or C10 cycloalkenyl. In some embodiments, a substituted
cycloalkenyl group
may include one or more of the following substitution groups: alkyl,
cycloalkyl, hydroxy,
alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one alkynyl group. In some embodiments, an alkynyl group may
have 4
carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11
carbons, 12
carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18
carbons, 19 carbons,
or 20 carbons.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one substituted alkynyl group. In some embodiments, a
substituted alkynyl
group may have 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9
carbons, 10 carbons,
11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17
carbons, 18
carbons, 19 carbons, or 20 carbons. In some embodiments, a substituted alkynyl
group may
include one or more of the following substitution groups: alkyl, cycloalkyl,
hydroxy, alkoxide,
carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
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In some embodiments, at least one of the independently selected linkerAs may
comprise at least one aryl or heteroaryl group. Examples include phenyl,
naphthyl, and
pyridinyl, although it is noted that other aryl and heteroaryl groups, that
fall within the
definitions provided herein, may be used.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one substituted aryl group. In some embodiments, a
substituted aryl group
may include one or more of the following substitution groups: alkyl,
cycloalkyl, hydroxy,
alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one aralkyl group. Example aralkyl groups include, but are
not limited to,
phenylmethyl, phenylethyl, and phenylpropyl.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one substituted aralkyl group. In some embodiments, a
substituted aralkyl
group may include one or more of the following substitution groups: alkyl,
cycloalkyl,
hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one aralkenyl group. Example aralkenyl groups include, but
are not limited
to ethenylbenzene and propenylbenzene.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one substituted aralkenyl group. In some embodiments, a
substituted
aralkenyl group may include one or more of the following substitution groups:
alkyl,
cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and
sulfonamide.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one aralkynyl group. Example aralkynyl groups include, but
are not limited
to ethynylbenzene and propynylbenzene.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one substituted aralkynyl group. In some embodiments, a
substituted
aralkynyl group may include one or more of the following substitution groups:
alkyl,
cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and
sulfonamide.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one heteroatom. In some embodiments, at least one of the
independently
selected linkerAs may comprise one or more oxygen (0) heteroatoms, one or more
nitrogen
(N) heteroatoms, one or more sulfur (S) heteroatoms, and/or one or more
phosphorous (P)
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heteroatoms. In some embodiments, at least one of the independently selected
linkerAs may
comprise at least one heteroalkyl group.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one aliphatic heterocycle. In some embodiments, at least one
of the
independently selected linkerAs may comprise at least one of tetrahydrofuran
(THE),
tetrahydropyran (THP), morpholine, piperidine, piperazine, pyrrolidine, and/or
azetidine.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one heteroaryl group. In some embodiments, at least one of
the
independently selected linkerAs may comprise one or more of imidazole,
pyrazole, pyridine,
pyrimidine, triazole, and.or 1,2,3-triazole.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one substituted heteroaryl group. In some embodiments, a
substituted
heteroaryl group may include one or more of the following substitution groups:
alkyl,
cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and
sulfonamide.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one amino acid. Various amino acids are known to those
skilled in the art.
An independently selected linkerA is not limited to including one or more
specific amino
acids. For example, an independently selected linkerA may comprise one or more
arginine
(Arg) amino acids, one or more histidine (His) amino acids, one or more lysine
(Lys) amino
acids, one or more aspartic acid (Asp) amino acids, one or more glutamic acid
(Glu) amino
acids, one or more serine (Ser) amino acids, one or more threonine (Thr) amino
acids, one or
more asparagine (Asn) amino acids, one or more glutamine (Gin) amino acids,
one or more
cysteine (Cys) amino acids, one or more selenocysteine (Sec) amino acids, one
or more
glycine (Gly) amino acids, one or more proline (Pro) amino acids, one or more
alanine (Ala)
amino acids, one or more valine (Val) amino acids, one or more isoleucine
(Ile) amino acids,
one or more leucine (Leu) amino acids, one or more methionine (Met) amino
acids, one or
more phenylalanine (Phe) amino acids, one or more tyrosine (Tyr) amino acids,
and/or one or
more tryptophan (Trp) amino acids.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one nucleotide. Various nucleotides are known to those
skilled in the art. An
independently selected linkerA is not limited to including one or more
specific nucleotides.
For example, an independently selected linkerA may comprise one or more
nucleotides
comprising a guanine nucleobase, one or more nucleotides comprising an adenine
nucleobase,
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one or more nucleotides comprising a cytosine nucleobase, one or more
nucleotides
comprising a thymine nucleobase, and/or one or more nucleotides comprising a
uracil
nucleobase.
In some embodiments, at least one independently selected linkerA may comprise
at
least one abasic nucleotide. As known in the art, an abasic nucleotide is a
nucleotide having
an abasic site, which is a location that has neither a purine nor a pyrimidine
base. For
example, at least one independently selected linkerA may comprise one or more
abasic DNAs
and/or one or more abasic RNAs. In some embodiments, at least one
independently selected
linkerA may comprise at least one inverted abasis nucleotide. As known in the
art, an
inverted abasis nucleotide is an abasic nucleotide whose 5' end connects to a
5' end of a next
nucleotide, and whose 3' end connects to a 3' end of a next nucleotide. For
example, at least
one independently selected linkerA may comprise one or more inverted abasic
DNAs and/or
one or more inverted abasic RNAs.
In some embodiments, at least one of the independently selected linkerAs may
comprise at least one saccharide. In some embodiments, at least one of the
independently
selected linkerAs may comprise at least one glucose monosaccharide unit, at
least one
fructose monosaccharide unit, at least one mannose monosaccharide unit, at
least one
galactose monosaccharide unit, at least one ribose monosaccharide unit, and/or
at least one
glucosamine monosaccharide unit.
In some embodiments, at least one of the independently selected linkerAs may
comprise one or more of:
0 0
P q p q H PP p q H
PP
0 0
p q H p q H PP
,and
where:
p is an integral number between 0 and 12,
pp is an integral number between 0 and 12,
q is an integral number between 1 and 12, and
qq is an integral number between 1 and 12.
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In some embodiments, p is an integral number independently selected from 0, 1,
2, 3,
4, 5, 6, 7, 8, 9, 10, 11 and 12. In some embodiments, pp is an integral number
independently
selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. In some
embodiments, q is an integral
number independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12.
In some
embodiments, qq is an integral number independently selected from 1, 2, 3, 4,
5, 6, 7, 8, 9, 10,
11 and 12.
LinkerB
LinkerB is a spacer, with one end attached to a pharmaceutical agent or a
functional
group capable of linking to one or more pharmaceutical agents, and the other
end attached to
a diamine nitrogen of the multivalent ligand cluster.
In some embodiments, linkerB may comprise polythethylene glycol (PEG). The PEG
may have any number of repeating O-CH2-CH2 units. For example, the PEG may be
PEG1,
PEG2, PEG3, PEG4, PEGS, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12, PEG13,
PEG14, PEG15, PEG16, PEG17, PEG18, PEG19, PEG20, PEG21, PEG22, PEG23, PEG24,
PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31, PEG32, PEG33, PEG34, PEG35,
PEG36, PEG37, PEG38, PEG39, PEG40, PEG41, PEG42, PEG43, PEG44, PEG45, PEG46,
PEG47, PEG48, PEG49, PEG50, PEG51, PEG52, PEG53, PEG54, PEG55, PEG56, PEG57,
PEG58, PEG59, PEG60, PEG61, PEG62, PEG63, PEG64, PEG65, PEG66, PEG67, PEG68,
PEG69, PEG70, PEG71, PEG72, PEG73, PEG74, PEG75, PEG76, PEG77, PEG78, PEG79,
PEG80, PEG81, PEG82, PEG83, PEG84, PEG85, PEG86, PEG87, PEG88, PEG89, PEG90,
PEG91, PEG92, PEG93, PEG94, PEG95, PEG96, PEG97, PEG98, PEG99, PEG100, or
larger. In some embodiments, it may be beneficial for the PEG to be PEG10 or
less.
In some embodiments, linkerB may comprise at least one alkyl group. In some
embodiments, linkerB may comprise at least one substituted alkyl group. In
some
embodiments, an alkyl group may have 2 carbons, 3 carbons, 4 carbons, 5
carbons, 6 carbons,
7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13
carbons, 14 carbons,
15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
In some embodiments, linkerB may comprise at least one substituted alkyl
group. In
some embodiments, a substituted alkyl group may have 2 carbons, 3 carbons, 4
carbons, 5
carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons,
12 carbons, 13
carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19
carbons, or 20
carbons. In some embodiments, a substituted alkyl group may include one or
more of the
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following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl,
amine, amide,
halide, sulfonyl, and sulfonamide.
In some embodiments, linkerB may comprise at least one cycloalkyl group. In
some
embodiments, a cycloalkyl group may be a C3 cycloalkyl (i.e., cyclopropane),
C4 cycloalkyl
(i.e., cyclobutene), C5 cycloalkyl (i.e., cyclopentane), C6 cycloalkyl (i.e.,
cyclohexane), C7
cycloalkyl (i.e., cycloheptane), C8 cycloalkyl (i.e., cyclooctane), C9
cycloalkyl (i.e.,
cyclononane), or C10 cycloalkyl (i.e., cyclodecane).
In some embodiments, linkerB may comprise at least one at least one
substituted
cycloalkyl group. In some embodiments, a substituted cycloalkyl group may be a
C3
substituted cycloalkyl, C4 substituted cycloalkyl, C5 substituted cycloalkyl,
C6 substituted
cycloalkyl, C7 substituted cycloalkyl, C8 substituted cycloalkyl, C9
substituted cycloalkyl, or
C10 substituted cycloalkyl. In some embodiments, a substituted cycloalkyl
group may
include one or more of the following substitution groups: alkyl, cycloalkyl,
hydroxy, alkoxide,
carboxyl, amine, amide, halide, sulfonyl, and sulfonamide.
In some embodiments, linkerB may comprise at least one alkenyl group. In some
embodiments, an alkenyl group may have 4 carbons, 5 carbons, 6 carbons, 7
carbons, 8
carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14
carbons, 15 carbons,
16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
In some embodiments, linkerB may comprise at least one substituted alkenyl
group.
In some embodiments, an alkenyl group may have 4 carbons, 5 carbons, 6
carbons, 7 carbons,
8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14
carbons, 15 carbons,
16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons. In some
embodiments, a
substituted alkenyl group may include one or more of the following
substitution groups: alkyl,
cycloalkyl, hydroxy, alkoxide, carboxyl, amine, amide, halide, sulfonyl, and
sulfonamide.
In some embodiments, linkerB may comprise at least one cycloalkenyl group. In
some
embodiments, a cycloalkenyl group may be a C5 cycloalkenyl, C6 cycloalkenyl,
C7
cycloalkenyl, C8 cycloalkenyl, C9 cycloalkenyl, or C10 cycloalkenyl.
In some embodiments, linkerB may comprise at least one substituted
cycloalkenyl
group. In some embodiments, a cycloalkenyl group may be a C5 cycloalkenyl, C6
cycloalkenyl, C7 cycloalkenyl, C8 cycloalkenyl, C9 cycloalkenyl, or C10
cycloalkenyl. In
some embodiments, a substituted cycloalkenyl group may include one or more of
the
following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl,
amine, amide,
halide, sulfonyl, and sulfonamide.
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In some embodiments, linkerB may comprise at least one alkynyl group. In some
embodiments, an alkynyl group may have 4 carbons, 5 carbons, 6 carbons, 7
carbons, 8
carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14
carbons, 15 carbons,
16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons.
In some embodiments, linkerB may comprise at least one substituted alkynyl
group.
In some embodiments, a substituted alkynyl group may have 4 carbons, 5
carbons, 6 carbons,
7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13
carbons, 14 carbons,
carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons. In
some
embodiments, a substituted alkynyl group may include one or more of the
following
10 substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl,
amine, amide, halide,
sulfonyl, and sulfonamide.
In some embodiments, linkerB may comprise at least one aryl group or
heteroaryl
group. Examples include phenyl, naphthyl, and pyridinyl, although it is noted
that other aryl
and heteroaryl groups, that fall within the definitions provided herein, may
be used.
15 In some embodiments, linkerB may comprise at least one substituted aryl
group. In
some embodiments, a substituted aryl group may include one or more of the
following
substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine,
amide, halide,
sulfonyl, and sulfonamide.
In some embodiments, linkerB may comprise at least one aralkyl group. Example
aralkyl groups include, but are not limited to, phenylmethyl, phenylethyl, and
phenylpropyl.
In some embodiments, linkerB may comprise at least one substituted aralkyl
group. In
some embodiments, a substituted aralkyl group may include one or more of the
following
substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl, amine,
amide, halide,
sulfonyl, and sulfonamide.
In some embodiments, linkerB may comprise at least one aralkenyl group.
Example
aralkenyl groups include, but are not limited to ethenylbenzene and
propenylbenzene.
In some embodiments, linkerB may comprise at least one substituted aralkenyl
group.
In some embodiments, a substituted aralkenyl group may include one or more of
the
following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl,
amine, amide,
halide, sulfonyl, and sulfonamide.
In some embodiments, linkerB may comprise at least one aralkynyl group.
Example
aralkynyl groups include, but are not limited to ethynylbenzene and
propynylbenzene.
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In some embodiments, linkerB may comprise at least one substituted aralkynyl
group.
In some embodiments, a substituted aralkynyl group may include one or more of
the
following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl,
amine, amide,
halide, sulfonyl, and sulfonamide.
In some embodiments, linkerB may comprise at least one heteroatom. In some
embodiments, linkerB may comprise one or more oxygen (0) heteroatoms, one or
more
nitrogen (N) heteroatoms, one or more sulfur (S) heteroatoms, and/or one or
more
phosphorous (P) heteroatoms. In some embodiments, at least one of the
independently
selected linkerAs may comprise at least one heteroalkyl group.
In some embodiments, linkerB may comprise at least one aliphatic heterocycle.
In
some embodiments, linkerB may comprise at least one of tetrahydrofuran (THE),
tetrahydropyran (THP), morpholine, piperidine, piperazine, pyrrolidine, and/or
azetidine.
In some embodiments, linkerB may comprise at least one heteroaryl group. In
some
embodiments, linkerB may comprise one or more of imidazole, pyrazole,
pyridine,
pyrimidine, triazole, and.or 1,2,3-triazole.
In some embodiments, linkerB may comprise at least one substituted heteroaryl
group.
In some embodiments, a substituted heteroaryl group may include one or more of
the
following substitution groups: alkyl, cycloalkyl, hydroxy, alkoxide, carboxyl,
amine, amide,
halide, sulfonyl, and sulfonamide.
In some embodiments, linkerB may comprise at least one amino acid. Various
amino
acids are known to those skilled in the art. LinkerB is not limited to
including one or more
specific amino acids. For example, linkerB may comprise one or more arginine
(Arg) amino
acids, one or more histidine (His) amino acids, one or more lysine (Lys) amino
acids, one or
more aspartic acid (Asp) amino acids, one or more glutamic acid (Glu) amino
acids, one or
more serine (Ser) amino acids, one or more threonine (Thr) amino acids, one or
more
asparagine (Asn) amino acids, one or more glutamine (Gin) amino acids, one or
more
cysteine (Cys) amino acids, one or more selenocysteine (Sec) amino acids, one
or more
glycine (Gly) amino acids, one or more proline (Pro) amino acids, one or more
alanine (Ala)
amino acids, one or more valine (Val) amino acids, one or more isoleucine
(Ile) amino acids,
one or more leucine (Leu) amino acids, one or more methionine (Met) amino
acids, one or
more phenylalanine (Phe) amino acids, one or more tyrosine (Tyr) amino acids,
and/or one or
more tryptophan (Trp) amino acids.
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In some embodiments, linkerB may comprise at least one nucleotide. Various
nucleotides are known to those skilled in the art. LinkerB is not limited to
including one or
more specific nucleotides. For example, linkerB may comprise one or more
nucleotides
comprising a guanine nucleobase, one or more nucleotides comprising an adenine
nucleobase,
one or more nucleotides comprising a cytosine nucleobase, one or more
nucleotides
comprising a thymine nucleobase, and/or one or more nucleotides comprising a
uracil
nucleobase. In some embodiments, linkerB may comprise at least one abasic
nucleotide. As
known in the art, an abasic nucleotide is a nucleotide having an abasic site,
which is a
location that has neither a purine nor a pyrimidine base. For example, linkerB
may comprise
.. one or more abasic DNAs and/or one or more abasic RNAs. In some
embodiments, linkerB
may comprise at least one inverted abasis nucleotide. For example, linkerB may
comprise
one or more inverted abasic DNAs and/or one or more inverted abasic RNAs.
In some embodiments, linkerB may comprise at least one saccharide. In some
embodiments, linkerB may comprise at least one glucose monosaccharide unit, at
least one
fructose monosaccharide unit, at least one mannose monosaccharide unit, at
least one
galactose monosaccharide unit, at least one ribose monosaccharide unit, and/or
at least one
glucosamine monosaccharide unit.
In some embodiments, linkerB may comprise one or more of:
.11
ik
-
41.4:N 4,0
11;101' kfl 0
-
ra+
11?rN 0 0 0
1(0 ko
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"ilrjok
=
0 k 0
, 0 0
0
0 ,
OH
0
0 0
OH
- 0
OH 0 0 0X
OH OH
0 0 0 OH
is6-0
ximit_cy&0-1-
0 0
OH
0
0
,and
where:
j is an integral number between 1 and 12, and
k is an integral number between 0 and 12.
In some embodiments, j is an integral number independently selected from 1, 2,
3, 4,
5, 6, 7, 8, 9, 10, 11 and 12. In some embodiments, k is an integral number
independently
selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. In some cases, the
present invention
also found that when linkerB contains a six-membered ring fragment, especially
a 4-
Hydroxypiperidinyl group, when it is used as targeted delivery of
pharmaceutical agents,
compared with a five-membered ring, it shows better in vivo stability and
activity, such as the
compound 75 of the present invention.
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Pharmaceutical Agents
In some embodiments a pharmaceutical agent is a diagnostic or therapeutic
drug,
molecule, compound, or combination of drugs, molecules, or compounds that have
a property
of assisting in diagnosis, prevention, treatment, and/or mitigation of a
disease or condition,
.. for example in a cell or subject. In certain embodiments, a pharmaceutical
agent is a drug,
molecule, compound or combination of drugs, molecules, or compounds that have
a property
of assisting in the enhancement of a desirable condition, for example in a
cell or subject.
In some embodiments, a pharmaceutical agent may be an oligonucleotide. In some
embodiments, the oligonucleotide may comprise a siRNA. In some embodiments,
the
oligonucleotide may comprise a double stranded siRNA. In some embodiments, the
double
stranded siRNA may comprise at least one modified ribonucleotide. In some
embodiments of
compositions and methods of the invention, the at least one modified
nucleotide comprises: a
2'-0-methyl nucleotide, 2'-Fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco
nucleotide mimic,
locked nucleotide, unlocked nucleic acid nucleotide (UNA), glycol nucleic acid
nucleotide
(GNA), 2'-F-Arabino nucleotide, 2'-methoyxyethyl nucleotide, abasic
nucleotide, ribitol,
inverted nucleotide, inverted abasic nucleotide, inverted 2'-Ome nucleotide,
inverted 2'-
deoxy nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide,
mopholino
nucleotide, a 3'-Ome nucleotide, a nucleotide comprising a 5'-phosphorothioate
group, or a
5'-(E)-vinyl phosphonate nucleotide (antisense strand only), or a terminal
nucleotide linked
to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2'-amino-
modified
nucleotide, a phosphoramidate, or a non-natural base comprising nucleotide. In
some
embodiments, substantially all (i.e., greater than 85 %) ribonucleotides of
the double stranded
siRNA may be modified.
In some embodiments, all ribonucleotides of the double stranded siRNA may be
modified. In some embodiments, at least one strand of the double stranded
siRNA may
comprise at least one phosphorothioate linkage. In some embodiments, at least
one strand of
the double stranded siRNA may comprise up to 6 phosphorothioate linkages. In
some
embodiments, a double stranded siRNA may comprise at least one locked nucleic
acid (LNA).
As known in the art, a LNA (sometimes referred to as a bridged nucleic acid
(BNA) or an
inaccessible RNA) is a modified RNA molecule where the ribose moiety is
modified with an
extra bridge connecting the 2' oxygen and the 4' carbon, thereby locking the
ribose in the 3'-
endo conformation. LNA is known to increase stability against enzymatic
degradation, and
improve specificity and affinity. In some embodiments, a double stranded siRNA
may
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comprise at least one unlocked nucleic acid (UNA). As known in the art, a UNA
is an acyclic
derivative of RNA lacking a C2'-C3'-bond of the ribose ring of RNA. It is
known to those
skilled in the art that including a UNA in certain positions of an antisense
strand of a siRNA
is tolerated for activity, and may promote reduced off target activity.
In some embodiments, a double stranded siRNA may comprise at least one
glycerol
nucleic acid (GNA). As known in the art, a GNA (sometimes referred to as a
glycol nucleic
acid) is a nucleic acid similar to RNA but differing in the composition of its
sugar-
phosphodiester backbone. It is known to those skilled in the art that
including a GNA in
certain positions of an antisense strand of a siRNA is tolerated for activity,
and may promote
reduced off target activity.
In some embodiments, the oligonucleotide may comprise a siRNA including one or
more modified nucleotides, including but not limited to a 2'-modified
nucleotide (e.g. F and
Me0), an abasic nucleotide, an inverted abasic nucleotide, a locked
nucleotide, UNA an
unlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA).
In some embodiments, the oligonucleotide may comprise a siRNA including one or
more phosphorothioate backbone linkages.
In some embodiments, the oligonucleotide may comprise a single strand siRNA.
In
some embodiments, the oligonucleotide may comprise a small activating RNA. In
some
embodiments, the oligonucleotide may comprise a microRNA (miRNA). In some
embodiments, the oligonucleotide may comprise an antisense oligonucleotide. In
some
embodiments, the oligonucleotide may comprise a short guide RNA (gRNA). In
some
embodiments, the oligonucleotide may comprise a single guide RNA (sgRNA). In
some
embodiments, the oligonucleotide may comprise a messenger RNA (mRNA). In some
embodiments, the oligonucleotide may comprise a ribozyme. In some embodiments,
the
oligonucleotide may comprise a plasmid. In some embodiments, the
oligonucleotide may
comprise an immune stimulating nucleic acid. In some embodiments, the
oligonucleotide
may comprise an antagomir. In some embodiments, the oligonucleotide may
comprise an
aptamer. An aptamer is a short, single-stranded DNA or RNA molecule that can
selectively
bind to a specific target, such as a protein, peptide, carbohydrate, small
molecule, toxin, or
live cell. Aptamers assume a variety of shapes, as they tend to form helices
and single-
stranded loops. The present disclosure is not limited to any particular
aptamer. Aptamers
known and not yet discovered are within the scope of the present disclosure.
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In some embodiments, the oligonucleotide may comprise at least 3 independently
selected nucleotides. In some embodiments, the oligonucleotide may comprise
between 16
and 23 independently selected nucleotides, for example when the
oligonucleotide is a siRNA.
In some embodiments, the oligonucleotide may comprise about 100 independently
selected
nucleotides, for example when the oligonucleotide is a sgRNA. In some
embodiments, the
oligonucleotide may comprise up to fourteen thousand independently selected
nucleotides,
for example when the oligonucleotide is an mRNA.
Functional Groups Capable of Linking to One or More Pharmaceutical Agents
As indicated above, "W" in formula 1 may be a functional group capable of
linking to
one or more pharmaceutical agents.
In some embodiments, the functional group may be a hydroxy group (OH). In some
embodiments, the functional group may be a protected hydroxy group. One
skilled in the art
will appreciate that various protecting groups may be used to protect a
hydroxy group. Each
of the various processing groups are within the scope of the present
disclosure. For example,
and not limitation, the hydroxy group may be protected using at least one of
4,4' -
dimethoxytrityl (DMT), monomethoxytrityl (MMT), 9-(p-methoxyphenyl)xanthen-9-
y1
(Mox), and 9-phenylxanthen-9-y1 (Px).
In some embodiments, the functional group may be a phosphoramidite group
having
the formula:
Ra
"RC Formula 2
where:
Ra is a Cl to C6 alkyl, C3 to C6 cycloalkyl, an isopropyl group, or Ra joins
with Rb
through a nitrogen atom to form a cycle,
Rb is a Cl to C6 alkyl, C3 to C6 cycloalkyl, an isopropyl group, or Rb joins
with Ra
through a nitrogen atom to form a cycle, and
Itc is a phosphite protecting group, phosphate protecting group, or a 2-
cyanoethyl
group.
Itc may be one of various phosphite protecting groups known to those skilled
in the art.
In some embodiments, the phosphite protecting group may comprise at least one
of methyl,
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ally!, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethylethyl, 2-
(trimethylsilyl)ethyl, 2-
(S-acetylthio)ethyl, 2-(S-pivaloylthio)ethyl, 2-(4-nitrophenyl)ethyl, 2,2,2-
trichloroethyl,
2,2,2-trichloro-1, 1-dimethylethyl, 1,1,1,3,3,3-hexafluoro-2-propyl, fluoreny1-
9-methyl, 2-
chlorophenyl, 4-chlorophenyl, and 2,4-dichlorophenyl.
Itc may be one of various phosphate protecting groups known to those skilled
in the
art. In some embodiments, the phosphate protecting group may comprise at least
one of
methyl, ally!, 2-cyanoethyl, 4-cyano-2-butenyl, 2-cyano-1,1-dimethylethyl, 2-
(trimethylsilyl)ethyl, 2-(S-acetylthio)ethyl, 2-(S-pivaloylthio)ethyl, 2-(4-
nitrophenyl)ethyl,
2,2,2-trichloroethyl, 2,2,2-trichloro-1, 1-dimethylethyl, 1,1,1,3,3,3 -
hexafluoro-2-propyl,
fluoreny1-9-methyl, 2-chlorophenyl, 4-chlorophenyl, and 2,4-dichlorophenyl.
In some embodiments, the functional group may be a carboxyl group (CO2H). In
some embodiments, the functional group may be an activated carboxyl group
having the
formula:
I I
f C X
Formula 3
where X is a leaving group.
Various activated carboxyl groups are known to those skilled in the art, all
of which
are within the scope of the present disclosure. In some embodiments, the
leaving group (X)
may be one of carboxylate, sulfonate, chloride, phosphate, imidazole,
hydroxybenzotriazole
(HOBt), N-hydroxysuccinimide (NHS), tetrafluorophenol, pentafluorophenol,
ofpara-
nitrophenol.
In some embodiments, the functional group may be a Michael acceptor. In some
embodiments, the Michael acceptor may have the formula:
Rd Formula 4
where:
E is an electron withdrawing group; and
Rd is hydrogen or a C1-C6 alkyl substitution group on olefin (meaning E and Rd
may
be cis, trans, or iso with respect to the carbon-carbon double bond) .
Various electron withdrawing groups are known to those skilled in the art, all
of
which are within the scope of the present disclosure. In some embodiments, the
electron
withdrawing group (E) may be carboxamide or an ester. In some embodiments, the
electron
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withdrawing group (E) and the carbon-carbon double bond of the Michael
acceptor may be
part of maleimide, a cyclic dicarboximide in which the two carboacyl groups on
nitrogen
together with the nitrogen itself form a 1H-pyrrole-2,5-dione structure.
In some embodiments, the functional group may have the formula:
0 X linkerC
II
Formula 5
where:
linkerC is absent or a spacer attached to a 3' or 5' end of an
oligonucleotide,
X is a methyl group, oxygen, sulfur, or an amino group, and
Y is oxygen, sulfur, or an amino group.
In some embodiments, linkerC of Formula 5 may comprise at least a heterocyclic
compound. In some embodiments, the heterocyclic compound may be an abasic
nucleotide or
an inverted abasic nucleotide.
In some embodiments, the functional group may have the formula:
H linkerC
0 Formula 6
where linkerC is a spacer having one end attached to a nitrogen of a
carboxamide and the
other end attached to a 3' or 5' end of an oligonucleotide.
In some embodiments, linkerB is attached to the carbonyl of the carboxyamide
of
Formula 6.
In some embodiments, linkerC in Formula 6 may comprise at least one PEG. The
PEG may have any number of repeating O-CH2-CH2 units. For example, the PEG may
be
PEG1, PEG2, PEG3, PEG4, PEGS, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12,
PEG13, PEG14, PEG15, PEG16, PEG17, PEG18, PEG19, PEG20, PEG21, PEG22, PEG23,
PEG24, PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31, PEG32, PEG33, PEG34,
PEG35, PEG36, PEG37, PEG38, PEG39, PEG40, PEG41, PEG42, PEG43, PEG44, PEG45,
PEG46, PEG47, PEG48, PEG49, PEG50, PEG51, PEG52, PEG53, PEG54, PEG55, PEG56,
PEG57, PEG58, PEG59, PEG60, PEG61, PEG62, PEG63, PEG64, PEG65, PEG66, PEG67,
PEG68, PEG69, PEG70, PEG71, PEG72, PEG73, PEG74, PEG75, PEG76, PEG77, PEG78,
PEG79, PEG80, PEG81, PEG82, PEG83, PEG84, PEG85, PEG86, PEG87, PEG88, PEG89,
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PEG90, PEG91, PEG92, PEG93, PEG94, PEG95, PEG96, PEG97, PEG98, PEG99, PEG100,
or larger.
In some embodiments, linkerC in Formula 6 may comprise at least one alkyl
group. In
some embodiments, an alkyl group may have 2 carbons, 3 carbons, 4 carbons, 5
carbons, 6
carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons,
13 carbons, 14
carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20
carbons.
In some embodiments, linkerC in Formula 6 may comprise at least one cycloalkyl
group. In some embodiments, a cycloalkyl group may be a C3 cycloalkyl (i.e.,
cyclopropane),
C4 cycloalkyl (i.e., cyclobutene), C5 cycloalkyl (i.e., cyclopentane), C6
cycloalkyl (i.e.,
cyclohexane), C7 cycloalkyl (i.e., cycloheptane), C8 cycloalkyl (i.e.,
cyclooctane), C9
cycloalkyl (i.e., cyclononane), or C10 cycloalkyl (i.e., cyclodecane).
In some embodiments, linkerC in Formula 6 may comprise at least one
heteroatom. In
some embodiments, linkerC may comprise one or more oxygen (0) heteroatoms, one
or more
nitrogen (N) heteroatoms, one or more sulfur (S) heteroatoms, and/or one or
more
phosphorous (P) heteroatoms.
In some embodiments, linkerC in Formula 6 may comprise at least one aliphatic
heterocycle. In some embodiments, linkerC may comprise at least one of
tetrahydrofuran
(THE), tetrahydropyran (THP), morpholine, piperidine, piperazine, pyrrolidine,
and/or
azetidine.
In some embodiments, linkerC in Formula 6 may comprise at least one heteroaryl
group. In some embodiments, linkerC may comprise one or more of imidazole,
pyrazole,
pyridine, pyrimidine, triazole, and. or 1,2,3-triazole.
In some embodiments, linkerC in Formula 6 may comprise at least one
substituted
heteroaryl group. In some embodiments, a substituted heteroaryl group may
include one or
more of the following substitution groups: alkyl, cycloalkyl, hydroxy,
alkoxide, carboxyl,
amine, amide, halide, sulfonyl, and sulfonamide.
In some embodiments, linkerC in Formula 6 may comprise at least one amino
acid.
Various amino acids are known to those skilled in the art. LinkerC is not
limited to including
one or more specific amino acids. For example, linkerC may comprise one or
more arginine
(Arg) amino acids, one or more histidine (His) amino acids, one or more lysine
(Lys) amino
acids, one or more aspartic acid (Asp) amino acids, one or more glutamic acid
(Glu) amino
acids, one or more serine (Ser) amino acids, one or more threonine (Thr) amino
acids, one or
more asparagine (Asn) amino acids, one or more glutamine (Gin) amino acids,
one or more
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cysteine (Cys) amino acids, one or more selenocysteine (Sec) amino acids, one
or more
glycine (Gly) amino acids, one or more proline (Pro) amino acids, one or more
alanine (Ala)
amino acids, one or more valine (Val) amino acids, one or more isoleucine
(Ile) amino acids,
one or more leucine (Leu) amino acids, one or more methionine (Met) amino
acids, one or
more phenylalanine (Phe) amino acids, one or more tyrosine (Tyr) amino acids,
and/or one or
more tryptophan (Trp) amino acids.
In some embodiments, linkerC in Formula 6 may comprise at least one
nucleotide.
Various nucleotides are known to those skilled in the art. LinkerC is not
limited to including
one or more specific nucleotides. For example, linkerC may comprise one or
more
nucleotides comprising a guanine nucleobase, one or more nucleotides
comprising an adenine
nucleobase, one or more nucleotides comprising a cytosine nucleobase, one or
more
nucleotides comprising a thymine nucleobase, and/or one or more nucleotides
comprising a
uracil nucleobase. In some embodiments, linkerC may comprise at least one
abasic nucleotide.
As known in the art, an abasic nucleotide is a nucleotide having an abasic
site, which is a
location that has neither a purine nor a pyrimidine base. For example, linkerC
may comprise
one or more abasic DNAs and/or one or more abasic RNAs. In some embodiments,
linkerC
may comprise at least one inverted abasis nucleotide. For example, linkerC may
comprise
one or more inverted abasic DNAs and/or one or more inverted abasic RNAs.
In some embodiments, linkerC in Formula 6 may comprise at least one
saccharide. In
some embodiments, linkerC may comprise at least one glucose monosaccharide
unit, at least
one fructose monosaccharide unit, at least one mannose monosaccharide unit, at
least one
galactose monosaccharide unit, at least one ribose monosaccharide unit, and/or
at least one
glucosamine monosaccharide unit.
In some embodiments, linkerC in Formula 6 may comprise one or more of:
X X
t
(.? p
ik
I II
y
)1(-,0A X X
11/ 111
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P i
, and/or
where:
j is an integral number between 1 and 12, and
k is an integral number between 0 and 12.
In some embodiments, the functional group may have the formula:
0 IinkerC
S
))-7 N1
0 Formula 7
where linkerC is a spacer with one end attached to one of the succinimide ring
carbons
through a thioether bond and the other end attached to a 3' or 5' end of an
oligonucleotide.
In some embodiments, linkerB is attached to the succinimide nitrogen of
Formula 7.
In some embodiments, linkerC in Formula 7 may comprise at least one PEG. The
PEG may have any number of repeating O-CH2-CH2 units. For example, the PEG may
be
PEG1, PEG2, PEG3, PEG4, PEGS, PEG6, PEG7, PEG8, PEG9, PEG10, PEG11, PEG12,
PEG13, PEG14, PEG15, PEG16, PEG17, PEG18, PEG19, PEG20, PEG21, PEG22, PEG23,
PEG24, PEG25, PEG26, PEG27, PEG28, PEG29, PEG30, PEG31, PEG32, PEG33, PEG34,
PEG35, PEG36, PEG37, PEG38, PEG39, PEG40, PEG41, PEG42, PEG43, PEG44, PEG45,
PEG46, PEG47, PEG48, PEG49, PEG50, PEG51, PEG52, PEG53, PEG54, PEG55, PEG56,
PEG57, PEG58, PEG59, PEG60, PEG61, PEG62, PEG63, PEG64, PEG65, PEG66, PEG67,
PEG68, PEG69, PEG70, PEG71, PEG72, PEG73, PEG74, PEG75, PEG76, PEG77, PEG78,
PEG79, PEG80, PEG81, PEG82, PEG83, PEG84, PEG85, PEG86, PEG87, PEG88, PEG89,
PEG90, PEG91, PEG92, PEG93, PEG94, PEG95, PEG96, PEG97, PEG98, PEG99, PEG100,
or larger.
In some embodiments, linkerC in Formula 7 may comprise at least one alkyl
group. In
some embodiments, an alkyl group may have 2 carbons, 3 carbons, 4 carbons, 5
carbons, 6
carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons,
13 carbons, 14
carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20
carbons.
In some embodiments, linkerC in Formula 7 may comprise at least one cycloalkyl
group. In some embodiments, a cycloalkyl group may be a C3 cycloalkyl (i.e.,
cyclopropane),
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C4 cycloalkyl (i.e., cyclobutene), C5 cycloalkyl (i.e., cyclopentane), C6
cycloalkyl (i.e.,
cyclohexane), C7 cycloalkyl (i.e., cycloheptane), C8 cycloalkyl (i.e.,
cyclooctane), C9
cycloalkyl (i.e., cyclononane), or C10 cycloalkyl (i.e., cyclodecane).
In some embodiments, linkerC in Formula 7 may comprise one or more of:
)i(
0,10,01& ..(=-=-...,,,./.Ø,...õ...-- X
0 1 0 ,-, X ,.,
v., 1,.t..q.
1 I ik --,, 13,- A .1YNII} p
ii
1 y Y Y
4-04 0 0 0 4 )1( 0 X
1
P . .p....-
II II li
Y Y Y
P
ii i I
Y , and/or Y ,
where:
j is an integral number between 1 and 12, and
k is an integral number between 0 and 12.
Multivalent Ligand Clusters Comprising GalNAc Targeting Ligands
In some embodiments, a multivalent ligand cluster of the present disclosure
may have
the general structure of Formula 8:
HO
H0, 51 ...,
' linkerA H I inkerB
HO 0 ¨N
..enN¨w
AHAc 0
H 0 0 ti-1)
0 linkerA )1_4 vN11/
HO 0,¨. rd- rin li
H0 NHAc
HO* fl
0 s"-
HO
-,
HO NHAc
Formula 8
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where:
m is an integral number between 1 and 10,
each n is an independently selected integral number between 1 and 10,
each linkerA is an independently selected spacer, with one end attached to a
TL and
the other end attached to the nitrogen of an alkylcarboxamide,
linkerB is a spacer, with one end attached to a pharmaceutical agent or a
functional
group capable of linking to one or more pharmaceutical agents, and the other
end attached to
a diamine nitrogen, and
W is either one or more pharmaceutical agents, or a functional group capable
of
linking to one or more pharmaceutical agents.
In some embodiments, a multivalent ligand cluster of the present disclosure
may have
the general structure of Formula 9:
linkerA H linkerB linkerC
0-oligonucleotide
NHAc 0
HO 0 )m
0 linkerA AwN
HO N
nfl
HN
HO NHAc
H 0
0
HO
HO NHAc Formula 9
where:
m is an integral number between 1 and 10,
each n is an independently selected integral number between 1 and 10,
each linkerA is an independently selected spacer,
linkerB is a spacer,
linkerC is a spacer or absent,
X is a methyl group, oxygen, sulfur, or an amino group, and
Y is oxygen, sulfur, or an amino group.
In some embodiments, a multivalent ligand cluster of the present disclosure
may have
the general structure of Formula 10:
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HO
linkerA H linkerB linkerC
-N
NHAc 01 0
HO 0 im
0 linkerA
HO
v)n n
HO -NHAc HN 0
HO
HO
0 4\
-NHAc
HO Formula 10
where:
m is an integral number between 1 and 10,
each n is an independently selected integral number between 1 and 10,
each linkerA is an independently selected spacer,
linkerB is a spacer, and
linkerC is a spacer.
In some embodiments, a multivalent ligand cluster of the present disclosure
may have
the general structure of Formula 11:
HO
HO linkerC
4"===)0 linkerA 0
linkerB S-oligonucleotide
HO - O-N
NHAc 0 y
HO 0 rn 0
0 linkerA
HO ON
n fln
HO -NHAc HN 0
HO scssir
HO
HO NHAc Formula 11
where:
m is an integral number between 1 and 10,
each n is an independently selected integral number between 1 and 10,
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each linkerA is an independently selected spacer,
linkerB is a spacer, and
linkerC is a spacer.
The following are example formulas for multivalent ligand clusters comprising
GalNAc or protected GalNAc targeting ligands, various "m" from Formula 1,
various "n"
from Formula 1, and various functional groups capable of linking to one or
more
pharmaceutical agents:
Ac0
AcOo
linkerA
H linkerB
Ac0 - 0 ¨N 1.r
_ N,----õ,,,
NHAc
0 01 õN
Ac0 0 P
0 linkerA )N
, 0
Ac0 )--.0,------ ill
HN 0 CN
Ac0 -N HAc
Ac0
0 1/4
Ac0--):"0 4\
Ac0 -NHAc Formula 12
AcOi
Ac0.,...A0 linkerA linkerB
H
Ac0 . 0¨N.I.r¨N-1 -."---------
NHAc 0, N
0 P-
Ac0 0 O
Ac0
o linkerA N
)--Ø--,---
CN
Ac0 ON
HN 0
Ac0
0 i. rrs <
Ac0
,
t0
Ac0 NHAc Formula 13
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Ac0
Ac0.,..)
0
linkerA H linkerB
Ac0 - -N '-....--^- N-,
_
NHAc 0 I
0,
- N
P
Ac0 0
0 linkerA ).N 0
Ac0 -....Ø---,õ-CN
: Ac0 NHAc HN 0
Ac0 0 A
Ac0 "..R.-
--
, 0
Ac0 NHAc Formula 14
Ac0
Ac0.,
0 linkerB
linkerA H
inkcoo -N,,.......õ.õ,......õA.--, --y--
NHAc ON
0 P
Ac0_ 0 N
o linkerA
Ac0 ---Ø,..--..,N CN
Ac0 -NHAc HN---
0
Ac0 A0 R-
'cµ
Ac0
Ac0 NHAc Formula 15
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Ac0
Ac0.,..)
0
linkerA H F
linkerB
Ac0 : 0-N 1\,,,,_ro F
IlHAc
0 \ 0
Ac0 0 F
0 linkerA 7N F
Ac0 --... 0 ,õ--. hi
HNO
Ac0 -N HAc
Ac0 0 fe,,.-
Ac0 0
Ac0 -N HAc Formula 16
Ac0
F
Ac0
0 0 F
linkerA H linkerB
Aco . 0 -A F..N,--------..
NHAc 0
0 F
Ac0 0
ii
__________ 0 linkerA )__Y_>
Ac0 /)--.0 J,J1M-M-M1W hi
Ac0 -N HAc H N '()
Ac0 0 /Se,.-
--)"...
0
Ac0
Ac0 -NHAc Formula 17
Ac0
Ac0.,..)
0
linkerA FH linkerB
Ac0 - --N N 0 F
_ 1----"õõõ,õi-
NHAc
0 0
F
Ac0_ 0 F
0 linkerA ),N
Ac0
Ac0 -N HAc HN 0
Ac0 0 /c--).....
0
Ac0
Ac0 -N HAc
Formula 18
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Ac0
Ac0..,
0 F
linkerA H linkerB
Ac0 : -N1r\N-1-10 F
NHAc \ 0
0 F
Ac0_ 0 N F
0 linkerA
Ac0 )--.0-----hi
Ac0 -NHAc HI\I"
0
Ac0 Ssssek.
Nt--
Ac00 4\
Ac0 -NHAc Formula 19
AcOi
Ac030 linkerA 0
H Ac0 0 N
linkerB
- -1
N-N 1
IIHAc 0 )7----
Ac0 0 0
0 linkerA zi\i
Ac0 ---.(3,--.,hi
HN 0
Ac0 -NHAc
Ac0 A-
t--
Ac0-*
Ac0 -NHAc Formula 20
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AcOi
Ac0.,..) 0
linkerA H linkerB
Ac0 _ 0¨N 1N .--N 1
NHAc )7----
0 0
Ac0 0
0 linkerA 7,/
Ac0 ---ب..-- N
H
:
Ac0 NHAc HN 0
Ac0 Ss"
Ac0
õ
Ac 0 NHAc Formula 21
AcO
Ac0 i0 linkerA 0
H linkerB
Ac0 - 0 --N ,i..._õ.õ,
N¨N 1
IIHAc 0 )7.----
0
Ac0 0
0 linkerA 7-N7
Ac0 ---.0õ-------N
H
/ Li
Ac0 -NHAc HNr-,
Ac0 A.
k-
Ac00
Ac0 -NHAc Formula 22
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Ac0
AcOoLs 0
linkerA H linkerB
Ac0 - 0¨N .,/N11 ¨NJ
_
NHAc /I
o 0
Ac0_ 0 V
0 linkerA >
Ac0 õ)¨...0,------,11
:
Ac0 NHAc HN ----
0
Ac0-0"... ssszsi.-
0
*
4\
Ac0 0
Ac0 -NHAc Formula 23
I-10
HO.,,,
0
linkerA H linkerB linkerC
HO : 0 ¨NI X
IN-------^'0, 1 _O¨oligonucleotide
NHAc p
0
HO 0 Y
0 linkerA 7,N
HO ---.(:).,----",11
HO -NHAc HN 0
HO-*
0
*
0 4\
HO
HO -NHAc Formula 24
HO
I
HO=-)0 linkerA H linkerB linkerC
X
¨NI NCO--, I ,0¨oligonucleotide
_ P
NHAc
0 11(
HO 0
N
0 linkerA
HO ---Ø,,,----11
HO -NHAc HN 0
H 0 0
--___
HO
HO NHAc Formula 25
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HO
I
HO.,,
0
. linkerA H linkerB linkerC
HO - -N X
NHAc
1.N-0õ_ 1,0-oligonucleotide
P
0
Y
HO 0
0 linkerA m7
HO /)--.0 VWWW,,,, hi
HN 0
HO -NHAc
HO-*0 isi-
,
HO
õ
HO NHAc Formula 26
HO
I
HO.,...)
0
linkerA H linkerB linkerC
X
HO - 0-N r;I'----o- 1 -0-oligonucleotide
_
NHAc P
0 11(
HO 0 N
0 linkerA
HO )--.0
HO -NHAc HN---
0
HO-* 0 S.R.-
(z)
HO
-NHAc
HO Formula 27
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HO
i
H04"..0
linkerA H linkerB linkerC
N-oligonucleotide
IIHAc 0 o 0 H
HO
O linkerA ),N
HO ---Ø----,---11
HN 0
HO -NHAc
HO A
-*O -
HO
HO -NHAc Formula 28
HO
i
HO..
0 linkerB linkerC
linkerA H H
HO - 0 -N N'-----------N-oligonucleotide
_
IIHAc 0
0
HO 0
O linkerA )___/N>
HO ---Ø.,-----,
HO -NHAc HN0
HO--"...0 is
HO "
0 .c\
:
HO NHAc Formula 29
HO
HO"===)0
),.,, ON
H linkerB linkerC
HO - ---N N'------N---oligonucleotide
NHAc H
0 0
HO 0
O linkerA zN
HO ---Ø------wil
HN 0
HO -NHAc
HO -- fil
HO ,
0
HO -NHAc Formula 30
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HOI
HO.,..
0 linkerA linkerB linkerC
H
H0.1)-.. ¨N
N¨N¨oligonucleotide
NHAc - 0 H
0
HO 0
N
/
0 linkerA )Y >
HO />¨Ø,------
HO -NHAc HN ----%
0 W
HO
:
HO NHAc Formula 31
HO
I
HO,,,,
linkerA linkerC
0 0
H linkerB )..,.7S¨oligonucleotide
HO - ¨N
N's--------N
NHAc
>r"
0
HO 0 0
0 linkerA ).N
HO ---Ø,,,,,----, ill
HN 0
HO -NHAc
HO :A
-0)....0-
0
Nt--
HO
HO -NHAc Formula 32
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HO
I
HO.,,..) 0 linkerC
0 linkerA linkerB )\........S----oligonucleotide
H
HO - 0 -N NI N
_
NHAc )7-"----
0
0
HO 0
0 linkerA )__7>
HO ---Ø.,------- il
HO -NHAc HN'0
HOH 0 0 .
0
HO -NHAc Formula 33
I-10
HO30 linkerA linkerC
0
H linkerB ),\S-oligonucleotide
HO - -N N.,,,,_,N
r1HAc 0
)7-----
0
HO 0
0 linkerA ),NV
HO --Ø,,,,,,,,-.N
H
HN 0
HO -NHAc
HO- AR-
0 .Cµ
HO
HO -NHAc Formula 34
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I-10
HO.,..) 0 linkerC
0 linkerB
linkerA H >\......._7S-oligonucleotide
HO - 0 -N--......r\õ..--N''''--N
NHAc )7---
0 0
HO 0 /
0 linkerA 7.YN
/
HO ----,0,---,------N
H
HO -NHAc HN---
0
HO
HO
õ
HO NHAc Formula 35
First Example Method of Preparation
One method for the preparation of examples of compounds with general Formula 1
is
depicted in Scheme 1 below. Starting materials and intermediates may be
purchased from
commercial sources, made from known procedures, or are otherwise illustrated.
The order of
carrying out the steps of the reaction scheme may be varied.
CO2R
CO2R
r )n r( )n
H RO2C*_2.---..,
, , X RO2C-..L.,r,..,1.----)õ, N,pG
¨ n RO2CNH
H2N-h\N'PG
m "nro )n
m
or --%CO2R deprotection
X: a leaving group CO2R
CO2R
or an aldehyde or a ketone
Compound I Compound II Compound III
R: is H, Me, Et, "Pr, IPr,
"Bu, tBu, Bn CO2R CO2H
linkerB
ro )n linkerA
Y¨OH r-(-' )n deprotection TL ¨NH2
¨I.-
" nrc j )n m linkerB \ inro )n m
linkerB
Y: a leaving group,
or an aldehyde, or
a ketone, or a CO2R CO2H
Michael acceptor
or a carboxyl Compound IV Compound V
group or an linkerA
isocyanate H H linkerA
0N¨TL
,,,,,TL 0N¨TL
HN linkerA ) NC OP(NPr2)2
n õ.0IL
HN linkerA i
H
N Pr n z 2
(DN N¨ OH
n Jr.] )n m linkerB tetrazole ON-(1'"---
.N¨C)-1:'\
n jrj )n m linkerB 0
ON¨TL
O N TL ¨
H NC
linkerA H
linkerA
Compound VI Compound VII
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Scheme 1
Scheme 1 starts with a mono-protected diamine (Compound I). The mono-protected
diamine comprises a first nitrogen and a second nitrogen, where the first
nitrogen is a primary
amine, and the second nitrogen is a secondary amine comprising a protecting
group (PG) in
.. Scheme 1).
Various protecting groups are known to those skilled in the art, and may be
used. In
some embodiments, the protecting group may be a benzyl group. In some
embodiments, the
protecting group may be a triphenylmethyl group.
"m" in Scheme 1 may be any integral number. In some embodiments, m in Scheme 1
.. may be an integral number between 1 and 10.
Starting with Compound I, a triester Compound II can be synthesized in one
step. In
some embodiments, Compound II may be synthesized via a SN2 substitution
reaction using
Compound I and one or more appropriate substrates. In some embodiments,
Compound II
may be synthesized via a reductive amination reaction using Compound I and one
or more
appropriate substrates. In some embodiments, Compound II may be synthesized
via a
Michael addition reaction using Compound I and one or more appropriate
substrates.
As illustrated in Scheme 1, Compound II results from various protected
carboxylic
acids being coupled to Compound I. In Compound II, the first nitrogen is a
tertiary amine
comprise a first protected carboxylic acid and a second protected carboxylic
acid, and the
second nitrogen is a tertiary amine comprising the protecting group and a
third protected
carboxylic acid.
Each "n" in Scheme 1 is an independently selected integral number. In some
embodiments, each n in Scheme 1 is an independently selected integral number
between 0
and 10.
Compound III is produced by deprotecting the second nitrogen of Compound II,
resulting in the second nitrogen of Compound III being a secondary amine
comprising the
third protected carboxylic acid. In embodiments where the protecting group is
a benzyl group,
Compound III may be produced by performing a hydrogenation reaction using
Compound II.
In embodiments where the protecting group is a triphenylmethyl group, Compound
III may
be produced by reacting the second compound with at least one acid. Example
acids include,
but are not limited to, hydrochloric acid (HC1) and trifluoroacetic acid
(TFA).
Compound IV is produced by attaching a moiety comprising a hydroxy group to
the
second nitrogen of Compound III, resulting in the second nitrogen of Compound
IV being a
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tertiary amine or an amide comprising the third protected carboxylic acid and
the moiety
comprising the hydroxy group. The moiety comprising the hydroxy group may be
attached to
the second nitrogen using any linkerB described herein above.
In some embodiments, producing triester Compound IV comprises performing a SN2
reaction using Compound III and one or more appropriate substrates. In some
embodiments,
producing Compound IV comprises performing a reductive amination reaction
using
Compound III and one or more appropriate substrates. In some embodiments,
producing
Compound IV comprises performing a Michael addition reaction using Compound
III and
one or more appropriate substrates. In some embodiments, producing Compound IV
comprises performing an amide coupling reaction using Compound III and one or
more
appropriate substrates. In some embodiments, producing Compound IV comprises
performing a nucleophilic addition reaction using Compound III and one or more
appropriate
substrates. Examples substrates include, but are not limited to, isocyaniate
and isothiocyanate.
Triacid Compound V is produced by converting the protected carboxylic acids of
Compound IV into carboxylic acids. In some embodiments, Compound V may be
produced
by reacting Compound IV with one or more acids (e.g., when R in Scheme 1 is an
acid
sensitive group, such as a tert-butyl group). In some embodiments, the one or
more acids may
comprise hydrochloric acid (HC1), hydrobromic acid (HBr), trifluoroacetic acid
(TFA), and
formic acid. In some embodiments, producing Compound V may comprise performing
a
hydrogenation reaction using Compound IV (e.g., when R in Scheme 1 is a benzyl
group). In
some embodiments, producing Compound V may comprise performing a hydrolysis
reaction
using Compound IV.
Compound VI may be produced by perform an amide coupling reaction using the
Compound V. In Compound VI the first nitrogen is a tertiary amine comprising a
first amide
and a second amide, and the second nitrogen is a tertiary amine comprising the
moiety
comprising the hydroxy group and a third amide. The first amide, the second
amide, and the
third amide may each be coupled to an independently selected targeting ligand.
linkerA in
Scheme 1 may be any linkerA described herein. TL in Scheme 1 may be any TL
described
herein.
Compound VII is produced by converting the hydroxy group (attached to linkerB)
of
Compound VI to a phosphoramidite group using a phosphitylation reaction. As
illustrated in
Scheme 1, in some embodiments converting the hydroxy group to the
phosphoramidite group
may be performed after performing the amide coupling reaction to produce
Compound VI.
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Second Example Method of Preparation
Another method for the preparation of examples of compounds with general
Formula
1 is depicted in Scheme 2 below. Scheme 2 allows for a compound with general
Formula 1 to
have one or more different targeting ligands. Scheme 2 allows for stepwise
introduction of
targeting ligands. Starting materials and intermediates may be purchased from
commercial
sources, made from known procedures, or are otherwise illustrated. The order
of carrying out
the steps of the reaction scheme may be varied.
Rlo c
1 H 2 'H-Fix X H H
PG,NN,pG2 _____________________________________________________________
RaO2CN4iN,pG2 _,...Ra02CiNN, 2
PG
H m CO2Ra \ inx i m n H
m
or
PG', deprotection
Compound I X: a leaving group Compound ll Compound III
or an aldehyde or a ketone
Rb0 C H R'02CNH2
2 Ra0 C
2N-idN'PG2 deprotection
or CO2Rb nro ) n
ny m
________________________________________________ .-
)nY
CO2Rb CO2Rb
X: a leaving group
or an aldehyde or Compound V
Compound VI
a ketone
OH
linkerB
Rc02 C , µ,, R8O2C N ,(iNH X CO2Rc RaO2C ,(yn, N 4/1 N
n "n2 linkerB CO2RG
Y¨OH
r(c)nli r )nY
or -7---'¨0O2Rc
CO2Rb Y: a leaving group CO2Rb
X: a leaving group or an aldehyde or
or an aldehyde or Compound VI a ketone or a
a ketone Michael reaction Compound VII
acceptor or a
carboxyl group or
an isocyanate
OH OH
linkerB linkerB
linkerA Fe(D2C
0
H -(1
Ra02C,H,F i), N41N1-_,(,,,
k i CO2H IL ¨NH2 N
N
m ¨ nz nio ) ny m ri2
deprotection
r0 )nY
amide coupling NH
CO2Rb CO2Rb TL',
Compound VIII Compound IX
OH OH
linkerB 1 linkerB
Ra02C,i _y \ _(/, N 0 linkerA Ra02C ri2 N 0
M x N
nro m IL ¨N H2 x N-(iii
deprotection N ri2
) HnY r())nY CO2H TO
amide coupling NH
CONH¨TL2 TL1
Compound X Compound XI
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OH OH
linkerB 0 linkerB
HO2Ci,>ri Nj(,)N 0 linkerA 0
nz ---2 nx 1\1-N
deprotection rO) TL NH HN
nY NH nz
NH
TL3 )1111
TL1 amide coupling
CO2H CONH-TL2 TL'
Compound XII Compound XIII
CN
0 linkerB
phosphitylation
reaction HN)-L(")ix N-h) 0
TL3 r(j) NHnY CONH-TL2 TL', Compound XIV
Scheme 2
Scheme 2 starts with a double-protected diamine (Compound I). The double-
protected
diamine comprises a first nitrogen and a second nitrogen, where the first
nitrogen is a
secondary amine comprising a first protecting group (PG), and the second
nitrogen is a
primary or a secondary amine comprising a second protecting group (PG2).
The first protecting group and the second protecting group may be different,
thereby
allowing different linkerAs and targeting ligands to be attached to the
diamine scaffold.
Various protecting groups are known to those skilled in the art, and may be
used. In some
embodiments, the first protecting group may be a benzyl group, and the second
protecting
group may be a tert-butyloxycarbonyl (Boc) group.
"m" in Scheme 2 may be any integral number. In some embodiments, m in Scheme 2
may be an integral number between 1 and 10.
Compound II may be produced by coupling a first protected carboxylic acid to
the
first nitrogen of Compound I, resulting in the first nitrogen of Compound II
being a tertiary
amine. One skilled in the art will appreciate that, by differentiating the
protecting groups in
Compound I, the protecting groups may be strategically removed and replaced.
For example,
in embodiments where the first protecting group is a benzyl group and the
second protecting
group is a Boc group, the amine with the benzyl group (and not the amine with
the Boc group)
of Compound I may undergo a SN2 substitution reaction, a reductive amination
reaction, or a
Michael addition reaction with one or more appropriate reagents to form
Compound II.
Compound III may be produced by removing the first protecting group from
Compound II. In Compound III, the first nitrogen is a secondary amine having
the first
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protected carboxylic acid, and the second nitrogen is a primary or a secondary
amine having
the second protecting group. In some embodiments, producing Compound III may
comprise
performing a hydrogenation reaction (e.g., when the first protecting group is
a benzyl group).
Compound IV may be produced by coupling a second protected carboxylic acid to
the
first nitrogen of Compound III, resulting in the first nitrogen of Compound IV
being a tertiary
amine. In some embodiments, producing Compound IV may comprise performing a
SN2
substitution reaction using Compound III and one or more other appropriate
reagents. In
some embodiments, producing Compound IV may comprise performing a reductive
amination reaction using Compound III and one or more other appropriate
reagents. In some
embodiments, producing Compound IV may comprise performing a Michael addition
reaction using Compound III and one or more other appropriate reagents. In
some
embodiments, producing Compound IV may comprise performing an amide coupling
reaction using Compound III and one or more other appropriate reagents. In
some
embodiments, producing Compound IV may comprise performing a nucleophilic
addition
reaction using Compound III and one or more other appropriate reagents.
Compound V may be produced by removing the second protecting group from
Compound IV, resulting in the first nitrogen of Compound V being a tertiary
amine
comprising the first protected carboxylic acid and the second protected
carboxylic acid, and
the second nitrogen of Compound V being a primary amine. In some embodiments
(e.g.,
when the second protecting group is a Boc group), Compound V may be produced
by
reacting Compound IV with at least one acid. Example acids include, but are
not limited to,
hydrochloric acid (HC1) and trifluoroacetic acid (TFA). Compound VI may be
produced by
coupling a third protected carboxylic acid to the second nitrogen of Compound
V, resulting in
the second nitrogen of Compound VI being a secondary amine. In some
embodiments,
Compound VI may be produced by performing a SN2 substitution reaction using
Compound
V and one or more other appropriate reagents. In some embodiments, Compound VI
may be
produced by performing a reductive amination reaction using Compound V and one
or more
other appropriate reagents. In some embodiments, Compound VI may be produced
by
performing a Michael addition reaction using Compound V and one or more other
appropriate reagents.
Compound VII may be produced by attaching a moiety comprising a hydroxy group
to the second nitrogen of Compound VI, resulting in the second nitrogen of
Compound VII
being a tertiary amine or an amide or a urea. The moiety comprising the
hydroxy group may
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be attached to the second nitrogen using any linkerB described herein above.
In some
embodiments, Compound VII may be produced by performing a SN2 substitution
reaction
using Compound VI and one or more other appropriate reagents. In some
embodiments,
Compound VII may be produced by performing a reductive amination reaction
using
Compound VI and one or more other appropriate reagents. In some embodiments,
Compound
VII may be produced by performing a Michael addition reaction using Compound
VI and one
or more other appropriate reagents. In some embodiments, Compound VII may be
produced
by performing an amide coupling reaction using Compound VI and one or more
other
appropriate reagents. In some embodiments, Compound VII may be produced by
performing
a nucleophilic addition reaction using Compound VI and one or more other
appropriate
reagents.
In the example of Scheme 2, Ra, Rb, and Itc may be sufficiently different such
that
targeting ligands may be selectively attached, for example in one scenario Ra,
Rb, and Itc may
be methyl, benzyl and tert-butyl groups, respectively. The following describes
such selective
attachment of targeting ligands.
Compound VIII is produced by converting the third protected carboxylic acid of
Compound VII into a first carboxylic acid. In some embodiments, Compound VIII
may be
produced by reacting Compound VII with one or more acids (e.g., when Itc in
Scheme 2 is an
acid sensitive group, for example a tert-butyl group).
Compound IX may be produced by performing an amide coupling reaction using
Compound VIII. In Compound IX, the first nitrogen comprises the first
protected carboxylic
acid and the second protected carboxylic acid, and the second nitrogen of
Compound IX
comprises a first amide having a first targeting ligand coupled thereto and
the moiety
comprising the hydroxy group.
Compound X is produced by converting the second protected carboxylic acid of
Compound IX into a second carboxylic acid. In some embodiments, producing
Compound X
may comprise performing a hydrogenation reaction using Compound IX (e.g., when
Rb in
Scheme 2 is a benzyl group).
Compound XI may be produced by performing an amide coupling reaction using
Compound X. In Compound XI, the first nitrogen comprises the first protected
carboxylic
acid and a second amide having a second targeting ligand coupled thereto, and
the second
nitrogen of Compound XI comprises the first amide having the first targeting
ligand coupled
thereto and the moiety comprising the hydroxy group.
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Compound XII is produced by converting the first protected carboxylic acid of
Compound XI into a third carboxylic acid. In some embodiments, producing
Compound XII
may comprise performing a hydrolysis reaction using Compound XI (e.g., when Ra
in
Scheme 2 is a methyl group).
Compound XIII may be produced by performing an amide coupling reaction using
Compound XII. In Compound XIII, the first nitrogen comprises the second amide
having the
second targeting ligand coupled thereto and a third amide having a third
targeting ligand
coupled thereto, and the second nitrogen of Compound XI comprises a first
amide having a
first targeting ligand coupled thereto and the moiety comprising the hydroxy
group.
The first amide may be coupled to the first targeting ligand using any
independently
selected linkerA described herein. The second amide may be coupled to the
second targeting
ligand using any independently selected linkerA described herein. The third
amide may be
coupled to the third targeting ligand using any independently selected linkerA
described
herein.
One or more of the first targeting ligand, the second targeting ligand, and
the third
targeting ligand may be independently selected to be one or more of the
targeting ligands
described herein.
The hydroxy group may be coupled to the second nitrogen using any linkerB
described herein.
In some embodiments, the hydroxy group may be converted to a phosphoramidite
group using a phosphitylation reaction. In some embodiments, the hydroxy group
may be
converted to the phosphoramidite group producing Compound XIV.
Each "nx", "nY" or "d" in Scheme 2 is an independently selected integral
number. In
some embodiments, each"nx", "nY" or "d" in Scheme 2 is an independently
selected
integral number between 0 and 10.
Certain Elements of Preparation and Use
Embodiments of multivalent ligand clusters of the invention can be prepared
and used
to deliver oligonucleotide agents to cells, tissues, and organs. Non-limiting
examples of
agents that can be delivered include therapeutic agents such as siRNA.
Delivery methods
using multivalent ligand clusters of the invention can be used to deliver
siRNAs and other
agents conjugated to a target ligand cluster of the invention to in vitro and
in vivo cells.
Multivalent ligand clusters of the invention can be used as a delivery vehicle
with which to
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deliver agents, such as but not limited to agents comprising nucleic acids, to
a cell. As used
herein, the term "multivalent ligand cluster/pharmaceutical agent complex"
means a
multivalent ligand cluster as described herein that is linked to a
pharmaceutical agent as
described herein. In some embodiments of the invention, the pharmaceutical
agent is an
siRNA.
Another aspect of the present disclosure, the dsRNA agent comprises 2'-fluoro
modified nucleotides at position 2, 7, 12, 14 and 16 of the antisense strand
(counting from the
first paired nucleotide from the 5' end of the antisense strand), and/or 2'-
fluorine-modified
nucleotides at position 9, 11 and 13 of the sense strand (counting from the
first paired
nucleotide from the 3' end of the sense strand). In some embodiments, no other
positions of
the dsRNA agent contain 2' fluorine-modified nucleotides. In some embodiments,
all
nucleotides of the antisense strand and /or sense strand of the dsRNA agent
are modified
nucleotides. In some embodiments, the dsRNA agent has 2'-fluoro modified
nucleotides at
position 2, 7, 12, 14 and 16 of the antisense strand and/or 2' fluorine-
modified nucleotide at
positions 9, 11 and 13 of the sense strand, with other positions containing
modified
nucleotides selected from: 2'-0-methyl nucleotide, 2'-deoxy nucleotide, 2'3'-
seco nucleotide
mimic, locked nucleotide, unlocked nucleic acid nucleotide (UNA), glycol
nucleic acid
nucleotide (GNA), 2'-F-Arabino nucleotide, 2'-methoyxyethyl nucleotide, abasic
nucleotide,
ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2'-Ome
nucleotide, inverted
2'-deoxy nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified
nucleotide, mopholino
nucleotide, and 3'-0Me nucleotide, a nucleotide including a 5'-
phosphorothioate group, or a
terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid
bisdecylamide group,
a 2'-amino-modified nucleotide, a phosphoramidate, or a non-natural base
containing
nucleotide. In some embodiments, the dsRNA agent includes an E-
vinylphosphonate
nucleotide at the 5' end of the guide strand. In certain embodiments, the
dsRNA agent
includes at least one phosphorothioate internucleoside linkage. In certain
embodiments, the
sense strand includes at least one phosphorothioate internucleoside linkage.
In some
embodiments, the antisense strand includes at least one phosphorothioate
internucleoside
linkage. In some embodiments, the sense strand includes 1, 2, 3, 4, 5, or 6,
phosphorothioate
internucleoside linkages. In some embodiments, the antisense strand includes
1, 2, 3, 4, 5, or
6, phosphorothioate internucleoside linkages. In certain embodiments, the
sense strand is
complementary or substantially complementary to the antisense strand, and the
region of
complementarity is between 16 and 23 nucleotides in length. In some
embodiments, the
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region of complementarity is 19-21 nucleotides in length. In certain
embodiments, the region
of complementarity is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30
nucleotides in length. In some embodiments, each strand is no more than 30
nucleotides in
length. In some embodiments, each strand is no more than 25 nucleotides in
length. In some
embodiments, each strand is no more than 23 nucleotides in length. In certain
embodiments,
the dsRNA agent includes at least one modified nucleotide and further includes
one or more
targeting groups or linking groups. In some embodiments, the one or more
targeting groups
or linking groups are conjugated to the sense strand. In some embodiments, the
targeting
group comprises N-acetyl-galactosamine (GalNAc). In some embodiments, the
targeting
group contains a structure of GalNAc described above.
In some aspects of the invention, a multivalent ligand cluster may be used to
deliver a
pharmaceutical agent to a cell in a subject. Means of administering a
multivalent ligand
cluster/pharmaceutical agent complex to a subject may include art-known
methods. As a non-
limiting example, a multivalent ligand cluster/pharmaceutical agent complex
may be locally
delivered in vivo by direct injection or by use of an infusion pump. In some
aspects of the
invention, a multivalent ligand cluster/pharmaceutical agent complex is in a
pharmaceutical
composition and may be referred to as a pharmaceutical agent. In some
embodiments, a
pharmaceutical agent of the invention is administered to a subject in an
amount effective to
prevent, modulate the occurrence, treat, or alleviate a symptom of a disease
state in the
subject.
Cells and Subjects
As used herein, a subject shall mean a human or vertebrate mammal including
but not
limited to a dog, cat, horse, goat, cow, sheep, rodent, and primate, e.g.,
monkey. Thus, the
invention can be used to treat diseases or conditions in human and non-human
subjects. For
instance, methods and compositions of the invention can be used in veterinary
applications as
well as in human prevention and treatment regimens. In some embodiments of the
invention,
a vertebrate subject is a mammal.
In certain embodiments of the invention, a multivalent ligand
cluster/pharmaceutical
agent complex of the invention is delivered to and contacted with a cell. In
some
embodiments of the invention, a contacted cell is in culture, and in other
embodiments a
contacted cell is in a subject. Types of cells that may be contacted with a
multivalent ligand
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cluster/pharmaceutical agent complex of the invention include, but are not
limited to, liver
cells, muscle cells, cardiac cells, circulatory cells, neuronal cells, glial
cells, fat cells, skin
cells, hematopoietic cells, epithelial cells, sperm, oocytes, muscle cells,
adipocytes, kidney
cells, hepatocytes, or pancreas cells. In some embodiments, the cell contacted
with a
multivalent ligand cluster/pharmaceutical agent complex of the invention is a
liver cell.
Dosage
Dosage levels for the medicament and pharmaceutical compositions that may be
delivered using a multivalent ligand cluster/pharmaceutical agent complex of
the present
disclosure can be determined by those skilled in the art by routine
experimentation. In at least
some embodiments, a unit dose may contain between about 0.01 mg/kg and about
100 mg/kg
body weight of siRNA. Alternatively, the dose can be from 10 mg/kg to 25 mg/kg
body
weight, or 1 mg/kg to 10 mg/kg body weight, or 0.05 mg/kg to 5 mg/kg body
weight, or 0.1
mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 1 mg/kg body weight, or 0.1
mg/kg to 0.5
mg/kg body weight, or 0.5 mg/kg to 1 mg/kg body weight, or 1 mg/kg to 3 mg/kg
body
weight.
The pharmaceutical composition may be a sterile injectable aqueous suspension
or
solution, or in a lyophilized form. The pharmaceutical compositions and
medicaments of the
present disclosure may be administered to a subject in a pharmaceutically
effective dose.
Administration Methods
A variety of administration routes for a multivalent ligand
cluster/pharmaceutical
agent complex of the invention are available. The particular delivery mode
selected will
depend upon the particular condition being treated and the dosage required for
therapeutic
efficacy. Methods of this invention, generally speaking, may be practiced
using any mode of
administration that is medically acceptable, meaning any mode that produces
effective levels
of treatment without causing clinically unacceptable adverse effects. In some
embodiments of
the invention, a multivalent ligand cluster/pharmaceutical agent complex of
the invention
may be administered via an oral, enteral, mucosal, percutaneous, and/or
parenteral route. The
term "parenteral" includes subcutaneous, intravenous, intramuscular,
intraperitoneal, and
intracisternal injection, or infusion techniques. Other routes include but are
not limited to
nasal (e.g., via a gastro-nasal tube), dermal, vaginal, rectal, and
sublingual. Delivery routes of
the invention may include intrathecal, intraventricular, or intracranial. In
some embodiments
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of the invention, a multivalent ligand cluster/pharmaceutical agent complex of
the invention
may be placed within a slow release matrix and administered by placement of
the matrix in
the subject.
A multivalent ligand cluster/pharmaceutical agent complex of the invention may
be
administered in formulations, which may be administered in pharmaceutically
acceptable
solutions, which may routinely contain pharmaceutically acceptable
concentrations of salt,
buffering agents, preservatives, compatible carriers, adjuvants, and
optionally other
therapeutic ingredients. According to methods of the invention, the
multivalent ligand
cluster/pharmaceutical agent complex may be administered in a pharmaceutical
composition.
In general, a pharmaceutical composition comprises the multivalent ligand
cluster/pharmaceutical agent complex of the invention and a pharmaceutically-
acceptable
carrier. Pharmaceutically acceptable carriers are well known to the skilled
artisan and may be
selected and utilized using routine methods. As used herein, a
pharmaceutically-acceptable
carrier means a non-toxic material that does not interfere with the
effectiveness of the
biological activity of the active ingredients (e.g., the ability of the
delivered nucleic acid, for
example the siRNA to prevent and/or treat a disease or condition to which it
is directed).
Pharmaceutically acceptable carriers may include diluents, fillers, salts,
buffers,
stabilizers, solubilizers, and other materials that are well-known in the art.
Illustrative
pharmaceutically acceptable carriers are described in U.S. Pat. No. 5,211,657
and others are
known by those skilled in the art. Such preparations may routinely contain
salt, buffering
agents, preservatives, compatible carriers, and optionally other therapeutic
agents. When used
in medicine, the salts should be pharmaceutically acceptable, but non-
pharmaceutically
acceptable salts may conveniently be used to prepare pharmaceutically-
acceptable salts
thereof and are not excluded from the scope of the invention. Such
pharmacologically and
pharmaceutically-acceptable salts include, but are not limited to, those
prepared from the
following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric,
maleic, acetic,
salicylic, citric, formic, malonic, succinic, and the like. Also,
pharmaceutically-acceptable
salts can be prepared as alkaline metal or alkaline earth salts, such as
sodium, potassium or
calcium salts.
In some embodiments of the invention, a multivalent ligand
cluster/pharmaceutical
agent complex of the invention maybe administered directly to a tissue. Direct
tissue
administration may be achieved by direct injection, or other art-known means.
A multivalent
ligand cluster/pharmaceutical agent complex of the invention may be
administered once, or
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alternatively may be administered in a plurality of administrations. If
administered multiple
times, a multivalent ligand cluster/pharmaceutical agent complex of the
invention may be
administered via different routes. For example, the first (or the first few)
administrations may
be made directly into an affected tissue or organ while later administrations
may be systemic.
A multivalent ligand cluster/pharmaceutical agent complex of the invention,
when it
is desirable to have it administered systemically, may be formulated for
parenteral
administration by injection (e.g., by bolus injection or continuous infusion).
Formulations for
injection may be presented in unit dosage form (e.g., in ampoules or in multi-
dose containers),
with or without an added preservative. The pharmaceutical compositions may
take such
forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and
may contain
formulatory agents such as suspending, stabilizing, and/or dispersing agents.
Preparations for parenteral administration include sterile aqueous or non-
aqueous
solutions, suspensions, and emulsions. Examples of non-aqueous solvents are
propylene
glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable
organic esters such
as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions,
emulsions, or
suspensions, including saline and buffered media. Parenteral vehicles include
sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated
Ringer's, or fixed
oils. Intravenous vehicles include fluid and nutrient replenishers,
electrolyte replenishers
(such as those based on Ringer's dextrose), and the like. Preservatives and
other additives
may also be present such as, for example, antimicrobials, anti-oxidants,
chelating agents, and
inert gases and the like. Lower doses will result from other forms of
administration, such as
intravenous administration. In the event that a response in a subject is
insufficient at the
initial doses applied, higher doses (or effectively higher doses by a
different, more localized
delivery route) may be employed to the extent that patient tolerance permits.
Multiple doses
per day may be used as needed to achieve appropriate systemic or local levels
of one or more
multivalent ligand cluster/pharmaceutical agent complexes of the invention, to
result in a
desired level of the pharmaceutical agent, for example a desired level of the
siRNA.
Both non-biodegradable and biodegradable polymeric matrices can be used to
deliver
one or more multivalent ligand cluster/pharmaceutical agent complexes of the
invention to a
cell and/or subject. In some embodiments, a matrix may be biodegradable.
Matrix polymers
may be natural or synthetic polymers. A polymer can be selected based on the
period of time
over which release is desired, generally in the order of a few hours to a year
or longer.
Typically, release over a period ranging from between a few hours and three to
twelve
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months can be used. The polymer optionally is in the form of a hydrogel that
can absorb up to
about 90% of its weight in water and further, optionally is cross-linked with
multivalent ions
or other polymers.
In certain embodiments of the invention, a multivalent ligand
cluster/pharmaceutical
agent complex of the invention may be delivered using the bioerodible implant
by way of
diffusion, or by degradation of the polymeric matrix. Illustrative synthetic
polymers for such
use are well known in the art. Biodegradable polymers and non-biodegradable
polymers can
be used for delivery of one or more of a multivalent ligand
cluster/pharmaceutical agent
complex of the invention using art-known methods. Such methods may also be
used to
deliver one or more multivalent ligand cluster/pharmaceutical agent complexes
of the
invention for treatment. Additional suitable delivery systems can include time-
release,
delayed release or sustained-release delivery systems. Such systems can avoid
repeated
administrations of a multivalent ligand cluster/pharmaceutical agent complex
of the invention,
increasing convenience to the subject and the health-care provider. Many types
of release
delivery systems are available and known to those of ordinary skill in the
art. [See for
example: U.S. Pat. Nos. 5,075,109; 4,452,775; 4,675,189; 5,736,152; 3,854,480;
5,133,974;
and 5,407,686 (the teachings of each of which are incorporated herein by
reference)]. In
addition, pump-based hardware delivery systems can be used, some of which are
adapted for
implantation.
Use of a long-term sustained release implant may be particularly suitable for
prophylactic treatment of subjects and for subjects at risk of developing a
recurrent disease or
condition to be prevented and/or treated with an siRNA delivered using a
multivalent ligand
cluster of the invention. Long-term release, as used herein, means that the
implant is
constructed and arranged to delivery therapeutic levels of the active
ingredient for at least 30
days, 60 days, 90 days, or longer. Long-term sustained release implants are
well-known to
those of ordinary skill in the art and include some of the release systems
described above.
Therapeutic formulations of one or more multivalent ligand
cluster/pharmaceutical
agent complexes of the invention may be prepared for storage by mixing the
multivalent
ligand cluster/pharmaceutical agent complex having the desired degree of
purity with
optional pharmaceutically acceptable carriers, excipients or stabilizers
[Remington's
Pharmaceutical Sciences 21st edition, (2006)], in the form of lyophilized
formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at
the dosages and concentrations employed, and include, but are not limited to:
buffers such as
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phosphate, citrate, and other organic acids; antioxidants including ascorbic
acid and
methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol;
resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about
10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic
polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine,
histidine, arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates
including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars
such as
sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal
complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN ,
PLURONICS or polyethylene glycol (PEG).
The multivalent ligand cluster/pharmaceutical agent complexes of the present
disclosure may be formulated as pharmaceutical compositions. The
pharmaceutical
compositions may be used as medicaments, alone or in combination with other
agents. The
multivalent ligand cluster/pharmaceutical agent complex of the present
disclosure can also be
administered in combination with other therapeutic compounds, either
administrated
separately or simultaneously (e.g., as a combined unit dose). In at least some
embodiments,
the present disclosure includes a pharmaceutical composition comprising one or
more
multivalent ligand cluster/pharmaceutical agent complex according to the
present disclosure
in a physiologically/pharmaceutically acceptable excipient, such as a
stabilizer, preservative,
diluent, buffer, and the like.
A pharmaceutical composition of the invention may be administered alone, in
combination with each other, and/or in combination with other drug therapies,
or other
treatment regimens that are administered to subjects with a disease or
condition.
Pharmaceutical compositions used in the embodiments of the invention
preferably are sterile
and contain an effective amount of a multivalent ligand cluster/pharmaceutical
agent complex
to prevent or treat a disease or condition, to which the pharmaceutical agent,
for example a
siRNA, is directed.
The dose or doses of a pharmaceutical composition of the invention that are
sufficient
to treat a disease or condition when administered to a subject can be chosen
in accordance
with different parameters, in particular in accordance with the mode of
administration used
and the state of the subject. Other factors may include the desired period of
treatment. In the
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event that a response in a subject is insufficient at the initial doses
applied, higher doses (or
effectively higher doses by a different, more localized delivery route) may be
employed to
the extent that patient tolerance permits. In some embodiments of the
invention, dosing is
used that has been determined using routine means such as in clinical trials.
Examples
Example 1. Multivalent Ligand Clusters Comprising GalNAc Targeting Ligands
In some embodiments of the invention, a multivalent ligand cluster may
comprise
GalNAc targeting ligands. The following are example compounds of multivalent
ligand
clusters comprising acetyl group protected GalNAc targeting ligands, core C2
and C3
diamines, branching acetyl and propanoyl amides, PEG2 and PEG3 linkerAs,
various
linkerBs as described herein, and various functional groups capable of linking
to one or more
pharmaceutical agents, as described herein. The acetyl protecting group on the
below
GalNAc ligands can be readily removed after conjugation is completed to
generate GalNAc
targeting ligands.
OAc
Ac0
0
Ac0
NHAc C)-N1-1
OAc LO I
Ac0
0 0
Ac0
N
NHAc 0\/N),-N ON
Ac0 OAc
/
0 HN
Ac0
NHAc Compound 1
OAc
AcO
0 õ
Ac0
NHAc
,N
OAc
AcO\ 0 C)
0 f, N CN
Ac0
NHAc
/0
Ac0 OAc
HN
NHAc Compound 2
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OAc
AcO__\...,..
0
Ac00_,õ_,,---,0-------.,..0---_,--------HN ,0
NHAc y
N C)P-N
(S
OAc
Ac0 0
0
Ac0 0 0 N ) N CN
NHAc H
/ -0
Ac0 OAc
HN
NHAc Compound 3
OAc
Ac0
0
Ac0 0õ,_,--..0,-----õ_--0----HNO 0 \/
NHAc
P
0
OAc
Ac0 0
A
CN
Ac0 0 0 ON
NHAc H
/C:1
Ac0 OAc
HN
0 rl
NHAc Compound 4
OAc
Ac0
Ac0..\..?__\ 0NH 0
NHAc
0
Y
N -
N
P
OAc 0
Ac0
0
Ac00 / CN
(:)
/\NN
NHAc N
Ac0 OAc H / -0
HN
Ac000,.)
NHAc Compound 5
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OAc
Ac0\____\_.\.
"----õ--"
N -õ,..._õ----. N
NHAc P
H
0
Ac0
Ac0
Ac000N N( CN
NHAc H
0
AGO OAc 0
0
Ac0 0 o N H
NHAc Compound 6
OAc
Ac0
Ac0\00
NHAc NI-1 0
0 0
N 0
OAc F F
Ac0
0
Ac0\--- 0 /
-"\--\ 0NNx F F
NHAc
H
Ac0 OAc "21
0 HN
Ac0 0,)
NHAc Compound 7
OAc
Ac0
Ac000
NHAc N H0
0 0
N )N
OAc /
AcO
0
\..... 0
0
Ac0 0 0 AN
NHAc \/N
Ac0 OAc H
/0
HN
NHAc Compound 8
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OAc
AcO\
AcOA___ 0
\ , _ ..õ.,....._õ---...0
\----'
NHAc NH(:)
Y
-,, ,---, N ,0P
,N.,,,_õ--
OAc
6
Ac0
AcO 00,,----,0
Z\NN
NHAc N CN
H
Ac0 OAc / 0
Ac0\0 HN0)
NHAc Compound 9
OAc
Ac0
0
Ac0 __________________________ HN 0
YNHAc
N P
OAc 6
Ac0\\ 0
NI CN
NHAc H
'
Ac0 OAc / 0
HN
Ac0000)
NHAc Compound 10
OAc
AcO\
0 r,
Ac0 =._, 0
NI-1 0
NHAc
\/
N
0P
N
OAc 0
Ac0
0
/ Ac000 )NN CN
NHAc N
H ,.
AcR OAc / '0
Ac0\---o 0 HN
\ ..,--...0
NHAc
\----
----._.---
Compound
11
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OAc
Ac0
0 Ac0 0
O
NHAc NFI 0 \/
N P-
OAc
O Ac0
0 r, 0
CN
\/N
NHAc
Ac0 OAc H
"21
0 Ac0 O HNcl.)
NHAc Compound 12
OAc
Ac0 I
\---\ 0
Ac000
NI-1 0
NHAc \/
0-...õ N
N P-
OAc C)
Ac0
Ac0 \___\.......
0 0
)N CN
00
\/N
NHAc
Ac0 H
0Ac "21
0 Ac0 0 HN
n
`) \ /
NHAc Compound 13
OAc
Ac0
0
Ac0 00C)/-=--HN 0
NHAc Y
N C)--P-N
OAc C)
AcO______\.. 0
0 Ac0 CN 0c)ON/ N
NHAc H
Ac0 OAc "21
HN
NHAc Compound 14
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OAc
Ac0
0
Ac0 0 (:)C)/---- H N
NHAc
6.
OAc
Ac0 0
0
/ CN
Ac0 Nõ,,
NHAc H
Ac0 OAc
HN
0 Ac0 ,r., ,0..õ.õõ)
NHAc Compound 15
OAc
Ac0
Ac0\0 _
NHAc NH 0
\------
N 0-, N
P-
OAc
(1:1
Ac0
Ac00
0
_ ,.---,o
CN
NHAc
Ac0
OAc H
"D
0 Ac0 ________________________ HN 00)
\
NHAc Compound 16
OAc
AcO\
AcOA____ 0 n
\ %-) ..õ..,,õ/". '---,
NHAc NH 0
\/
P-
OAc 0
AcO
Ac0 ____\....__
0 0 CN
0 o
NHAc
Ac0 OAc H / 0
Ac0_,..\ HN_.(0.,.----,,,
0------NHAcCompound 17
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OAc
Ac0 K
HN
Y
NHAc
0, N
N P-
OAc 0
Ac0 0
CNAc0 LI ,õ,,_,,,.., ^.õ 0 ==="'-''',. ',.. .--'......NN N
NHAc H
/ -
OAc 0
Ac0\____\.._. HN
0
NHAc Compound 18
OAc
AcO___\.(p.
Ac0 00 ----------HN 0 \/
NHAc
0 N
0
OAc
Ac0 0 CN
0 N
NHAc H
Ac0 OAc "D
HN
0 r,
NHAc Compound 19
OAc
AcO\ ."----,/
0
Ac0 0
Nõ--1\__...---.N--------õ- ----p-N
NHAc
1
0
Ac0 H
Ac0
0 (:)0z\ N----,./N CN
Ac0
NHAc H
0 0
AcOOAc
Ac0 0 (:) NH
0
NHAc
Compound 20
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OAc
Ac0,___\._.__\
0 Y 0
Ac0 ,...,-, ; ...õ,õ../^,_
NHAc
H
0
Ac0
Ac0
CN
0 ,,,,,--- 0 ,,.z.õ,,,,
Ac0 0 N
NHAc H
Ac0 cOAc 0 C)
AcOOoNH
NHAc Compound 21
OAc
Ac0 0 \/
0
Ac0 0c)ON.N(D-,ID-NI
NHAc H
0
Ac0
Ac00.0NN CN
Ac0 H
NHAc 0 0
Ac0
0Ac NH
0 r,
NHAc Compound 22
OAc
Ac0 0 \/
0
Ac0 0(DON)-0,õID,N1
NHAc H
0
Ac0
Ac0 CN
0
HN -,/\N
NHAc
0 0
Ac0 OAc HN
0 Ac0 ,-% ,_, 0 -.,....õ--
NHAc Compound 23
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OAc
Ac0..õ\...._
\/
0 0
Ac0 n ._,,..õ._,---,
0..,....,..--õN,..__._.--,N 0, N
NHAc P-
1
Ac0 H
0
Ac0
0 00,..,zõ,,
Ac0 NN CN
NHAc H
Ac0 OAc 0 (D
0
Ac0 OoNH
NHAc Compound 24
OAc
Ac0
\----
0 0
Ac0 0
ON,-J-1,_____,-.--õ,,___....0,10,1\
NHAc
O
Ac0 H
Ac0
0 CN
0,/---0 ,,/
Ac0 N-____ IN
NHAc H
Ac0 OAc 0 C)
NH
NHAc Compound 25
OAc
AcO____7_....\ 0
Y
Ac0 N--11,,..õ.---..N 0, N
P-
NHAc H
6
Ac0
Ac0_,\____, CN
,--- N
0 _,,,----0, ¨
Ac0 0 H
NHAc 0 0
Ac0 OAc NH
AcOn 5 NHAc Compound 26
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OAc
Ac0 0
Y
0
0, N
Ac0 0 0C)/\N.)-\ N P-
NHAc H
0
Ac0
AcO____\:._\7, /
u 0 7\0C)7-'1-1N,,/\" CN
\
Ac0
NHAc
0 0
AGO OAc HN
NHAc Compound 27
OAc
Ac0\____\.....____
0 r,
Ac0 k_,õ___-----..0
NH0
NHAc \/
0
0õ, N
N P-
OAc
0
AcOL
\__ 0 0
Ac0 sr, ,õ...,õ---,....0 N '" m CN
NHAc
Ac0 OAc H / -0
HN
Ac000)
NHAc Compound 28
OAc
Ac0\___.\...,_
0
Ac0 0.õ-----.Ø----..õ-0-,..-------HN O 0
yNHAc
N
0P, N
-
OAc 0
Ac0 0
zN CN
NHAc H
/o
Ac0 OAc HN
0 Ac0 ,-, ,-=õ.,----,0,-----õ_õ0õ)
NHAc Compound 29
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OAc
Ac0
\/
0 0 0
Ac0 Ot,
t_)....---,N ,--1-1-..õ---,, 0, ,N
NHAc N P
Ac0 H
0
Ac0
0 0,/----0
Ac0 N-___ N CN
NHAc H
Ac0 OAc 0 C)
NH
0
NHAc Compound 30
OAc
Ac0 0 0
0
Ac0 0(:)0N-J-N 0, N
P-
NHAc H
O
Ac0
Ac0
CN
N \
0,,,,7,---.,0,---------
Ac0 H
0
NHAc 0
Ac0 OAc NH
NHAc Compound 31
OAc
Ac0 0 0
Y
0
Ac0 0c)ON)-N 0, N
P-
NHAc H
O
Ac0
Ac0.4_\7 0
N m
CN
/
Ac0 (Dov '-1-1 ..õ/\"
NHAc
0 (:)/
Ac0 OAc HN
0 r,
NHAc Compound 32
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OAc
AcO____\52..,
Ac0 00
N '1\1H
HAc 0
H \/
-,N .----,_õ-N
OAc P
Ac0 0
0 0 0
Ac0 Oc:1 7N
NHAc CN
H
Ac0 OAc / '0
0 HN
Ac0 00)
NHAc Compound 33
OAc
AcO___..\___\
0
H N 0
NHAc H
Y
Th\l-rN N
OAc 0 P-
AcOl 0 O
cN
NHAc H
/0
Ac0
0Ac
HN
0
AGO 0 c:10
NHAc Compound 34
OAc
AcO\..._\.....____
0
Ac0 00
NI-1 0
NHAc
0 0
N OH
OAc
AcO\.....__
0 0
Ac0 00 K.
NHAc -...õ-----õN "N
Ac0 OAc H"D
0 HN
Ac0 ,)
NHAc Compound 35
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OAc
Ac0
0 (-1
Ac0 _________
NHAc
NO 0
OH
OAc
Ac0
0
AcOO N )N
NHAc
AcR eOAc /
AcOO 0 HN
NHAc Compound 36
OAc
AcO\
1-IN
\ 0 0 0
NHAc
/W0H
OAc
Ac0 0
AcO 0()ON N
NHAc
/0
Ac0 OAc
HN
Ac00 00j
NHAc Compound 37
OAc
Ac0
0
Ac0 0 (:)(:) H N 0 0
NHAc
OH
OAc
AcO
0
0
Ac0
NHAc
Ac0 OAc
HN
NHAc Compound 38
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OAc
Ac0\_____\..
0
Ac0 0
0.,...,__------õNH 0
NHAc
0 0
N 0
OAc
0
F F
Ac0%_.
Ac0
0 ,-, ....-----..
0 )NN F F
NHAc N
AcR e0Ac
H
/(:)
0 Ac0 HN00)
NHAc Compound 39
OAc
AcO_____7.__
0
Ac0 00C)/----HN 0 0 0
NHAc
N 0
F
OAc F
Ac0 L 0
N )N F F
NHAc H
/ '0
OAc
Ac0 HN
Ac0%._),.......õ..---..,0õ----..,,.....õØ........õ---
NHAc Compound 40
OAc
AcO______\..
0
Ac0 HN }21 0
0
NHAc
N 0
OAc F F
Ac0\_....\_____ 0
N N F F
NHAc H
Ac0 OAc "21
HN
0 ,-,
Ac0 4_,00,,)
NHAc Compound 41
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OAc
Ac0
0 0 0 0
Ac0 r,
jNI--1N-LOH
NHAc
Ac0 H
Ac0
0 Ac0 00NN
NHAc H
0 CD
OAc
Ac0\____7._
0
Ac0 (:) NH
0
NHAc Compound 42
OAc
Ac0
0 0 0 0
Ac0 Lr, ,......,,,----,,,
N ...----1.----,N OH
NHAc
H
Ac0
Ac0
K. /
0 Z'
Ac0 0 O N--...__/\"
NHAc H
Ac0 OAc 0 O-
AcOO NH
\ 0
NHAc Compound 43
OAc
Ac0 0 0 0
0 n
Ac0 Li -,..------0.-----..õ- --õ,---"-- N ---1-N OH
NHAc H
Ac0
Ac0o c:1N
H Ac0
0
NHAc 0
Ac0 OAc NH
0
Ac0 0c)(D/
NHAc Compound 44
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OAc
Ac0 0 0 0
0
Ac0 00(D/\N )N OH
NHAc H
Ac0
Ac0
0
HN ,./
Ac0 ,_",
NHAc
0 (i
Ac0
0Ac HN
0
Ac0 00 /
NHAc Compound 45
OAc
Ac0
0 Ac0 %.,
,......,-----.Ø.....,.....Nr_11.,õ _.........õ..õ..._)õ,
NHAc N 0
Ac0 H F F
Ac0
0 o__õ../---oõ_/,,,,,
Ac0 N N F F
NHAc H
0 0
Ac0OAc
0
Ac0 0 NH
0
NHAc Compound 46
OAc
Ac0
0 0 0 0
Ac0 0.õ..----,_
u---------N-k------N 0 NHAc
F F
Ac0 H
Ac0
/ F F
0 10,õ_z--0,,,õy,,,
Ac0 N--_,N
NHAc H
0 0
Ac0 zOAc
\ 0
Ac0 c;po NH
NHAc Compound 47
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OAc
AcOL, 0 0 0
N õõ-.N 0
NHAc H
F F
Ac0
Ac0x,õ\______\7
Ac0 N F F
0 0 ,,,7--07----..,
H
0
NHAc 0
AcO\ OAc NH
\---"
NHAc Compound 48
OAc
Ac0 0 0 0
0 Ac0 n 4-,.,.._..----..oõ-----,,._-
0,,.....õ-----,N)I¨.,
N 0
NHAc H
F F
Ac0
Ac0\_____\,_\7 k, / F F
Ac0 () 077 -1-INõ/"
NHAc
0 0/
Ac0
\_..........\,0Ac HN
0
Ac0 0 0
0
NHAc Compound 49
OAc
Ac0
0 Ac0 0
ONI-1 0
NHAc
0 0
)CN
OAc N /
Ac0
Ac00
0 0
_ ,..,..õ----...0
NHAc
Ac0 OAc H
/ '0
0 Ac0 n ,,...õ...-----õ0õ) HN
NHAc Compound 50
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OAc
AcO______\______
0
Ac0 0(j(D./---HNO 0
0
NHAc
\ N )N
OAc /
0
Ac0\______\______ 0
0
Ac0 000N /N
NHAc H
Ac Ac0 0 HN
0
Ac0 0(j0)
NHAc Compound 51
OAc
Ac0\____\_.._.
0
0
NHAc
M\I)CN
/
OAc 0
Ac0 0
0 r, Ac0
N N =-=-..õ_.õ-----,0...---O-,___..--N,
NHAc H ,.
Ac0 OAc
HN
0 r,
NHAc Compound 52
OAc
Ac0\____\______
0 0 0 0
Ac0 0(DN 1.).NN
NHAc
/ Ac0 H
Ac0 0
0 0ozõ,,,,,
Ac0 N-,,N \
NHAc H
0 CD
OAc
Ac0
0
Ac0 Oo NH
NHAc Compound 53
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OAc
AcO Ac0 n 0 0 0
N HAc
Ac0 0
Ac0
0 Ac 00 N N
N HAc
Ac0 cOAc 0 0
AcO\Z4_00 N H
NHAc Compound 54
OAc
AcO\L__ 0 0 0
0
Ac0 Oc)zONN
NHAc
Ac0 0
AcO
0
Ac0 0 N
0
NHAc 0
AcO\ OAc NH
0
NHAc Compound 55
OAc
Ac0 0 0 0
0
Ac0 0(j(DN
NHAc
Ac0 0
AcO
Ac0 \Z'ovvC)HN
NHAc
0
Ac0 OAc HN
0
Ac0 0
NHAc Compound 56
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OAc
AcO\
AcOy 0(.1
\`'
NHAc NH 0
0 0
N 1\2-10H
OAc
Ac0\________\
0 0
Ac0 00 7N
ODMT
-N,N N
NHAc
,.
Ac0 OAc H /0
0 Ac0 0 HN
0.,)
NHAc Compound 57
OAc
AcO\
AcOA___ 0,
\ v..õ.____----,..õ
NHAc NI-1,0 0 0
N NQ,10H
OAc
Ac0\___\___
ODMT
0 0
/ Ac0 0
`-'NV\NN
NHAc
,.
Ac0 OAc H / '0
AcO.\01
HN
_ ..-----,.0
\/
NHAc Compound 58
OAc
Ac0\_..7____\
0
Ac0 0(:)0/"----HN 0 0 0
NHAc
N NQ = IOH
OAc
AcO___\..._\., 0 ODMT
0
NHAc H
-
Ac0 OAc / 0
HN
NHAc Compound 59
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OAc
Ac0\____\____
0 ,
Ac0 1-1N, 0
" 0 0
NHAc
N NQ-10H
OAc
Ac0\____.\._... 0 ODMT
0
m/ n
7,,,õ IN ,..,
0 N
NHAc H
Ac0 OAc " 21
HN
Ac00 0
NHAc Compound 60
OAc
Ac0
0 Ac0 n .,...,,..,......---.,0
NHAc NH 0
0 0
N
rp-i0H
OAc
Ac0.___v_s
0 n 0
Ac0,,,,...õ----......0
),NN ODMT
NHAc N
AcR e0Ac
H
"21
Ac0\--o 0 HN
\----\ ,_,,__õ----,...0,........._.)
''
NHAc Compound 61
OAc
Ac0\..._.
0
Ac0 0()
NHAc NI-1 o
0 0
N
\O,
OAc
Ac0\____\_.......
0 0 ODMT
Ac0 00 /
vcr\IN
NHAc N
Ac0 OAc H
/ -0
AcO0 HN0)
NHAc Compound 62
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OAc
Ac0.___.\______
0
0 0
NHAc
N)LKI
1 4 . OH
OAc
Ac0.___.\______ 0
0 Ac0 000N7' N 00 MT
NHAc H
Ac0 OAc
HN
0 Ac0 ,-) ,_,0
NHAc Compound 63
OAc
AcO\
HN 0 0
\ 0
NHAc
N
1p..10H
OAc
Ac0 0
N7-NV 00 MT
0
Ac0 000
NHAc H i.
Ac0
OAc
HN
0
Ac0 000)
NHAc Compound 64
OAc
AcO___7_____\
0 0 0 0
Ac0 0
N
0..,,_õ,...,----õ
NHAc N NQ 'OH
Ac0 H
Ac0
0 O
0 0z
Ac0 NN DMT
NHAc H
0
Ac0 OAc 0
0
Ac0 OoNH
NHAc Compound 65
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OAc
AcO____\....._
0 0 0 0
Ac0 r, ._,..õ..._,..--,,
0.õ,......,-----õN __----,õ,_,---,õN
NHAc NQ ,I0H
H
Ac0
Ac0 ODMT
0 0_,..7-----0/
Ac0 '--/-'-'N" m
NHAc H
0 0
Ac0 zOAc
\ 0
Ac0 OoNH
NHAc Compound 66
OAc
Ac0 0 0 0
0
Ac0 0c)ON m
NL'Q ,I0H
NHAc H
Ac0 ODMT
AcO_____\______\õ 07'N_V\/N \
0 0........"--,07---õz
AGO H
0
NHAc 0
Ac0 OAc NH
NHAc Compound 67
OAc
Ac0 0 0 o
0
Ac0 N.)-N NQ IOH
NHAc H
Ac0
ODMT
Ac0,___;_.\7, /
Ac0 u (D0(3-'FIN,/\"
NHAc
0 0
Ac0 OAc HN
0
Ac0 0(30
NHAc Compound 68
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OAc
AcO_____.\?.. 0 0 0
Ac0 Or.,
L)N,-1---..,...õ,---..N
NHAc
p 1(::IH
Ac0 H
Ac0
0 0_,7---.0,.,,,,,
Ac0
NN ODMT \
NHAc H
Ac0 OAc 0 C)
0
Ac0 Oo NH
NHAc Compound 69
OAc
Ac0
0 0 0 0
Ac0 0,,
N..õ----...N
0H
NHAc
=1
H
Ac0
Ac0
ODMT
0 Ac0 0,, N m,...z----0,,z, /
--,.._'1
NHAc H
Ac0 OAc 0 (D
0
Ac0 0 NH
0
NHAc Compound 70
OAc
Ac0 0 0 0
Ac0 0c)ON.)-N
Np-i0H
NHAc H
Ac0
Ac0(:)0N ODMT
Ac0 H
NHAc 0 0
Ac0 OAc NH
0 Ac0 r% ,-,-..0,-----...õ.0,õ_,..--
NHAc Compound 71
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OAc
Ac0\____\_..., 0 0 0
0 r,
Ac0 N r)-_,,,..--,
N N = .10H
NHAc H
Ac0
Ac0 N m
OD MT
____=-iNõ,..
NHAc
0 0,-.
Ac0 OAc HN
0 r,
NHAc Compound 72
OAc
Ac0j
µ...-0
AcOA___ () \ µJE.,
\ ..,.,
NHAc NI-1,0 0 0
N 11)
OAc Q. iOH
AcO\.......
0
ODMT
0
Ac0 0 n /
µ-'N NZ"\ N
NHAc
Ac0 OAc H
")
Ac0\_. _.0 HN
0
NHAc Compound 73
OAc
Ac0
Ac00
_ .õ.,...õ.õ...-..,,
1/4-1 NH 0
NHAc
0 0
N [\il ----... 0 H
OAc
Ac0
0 ODMT
AcO\00 /
)N x
NHAc N
Ac0 OAc H
"31
Ac00
HN
_ õ.õõ----..,o
NHAc Compound 74
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OAc
AGO
0
Ac0
NHAc NH 0
0 0
OAc
Ac0 0 \
0 0
Ac0 00 LCN
NHAc \/N7NN
Ac0 OAc
/
0 HN
Ac0
NHAc Compound 75
Example 2. Preparation of Intermediate Compounds
In Scheme 3 below, Intermediate-A was synthesized by treating commercially
available galactosamine pentaacetate (Compound I) with trimethylsilyl
trifluoromethanesulfonate (TMSOTf) in dichloromethane (DCM). This was followed
by
glycosylation with Cbz protected 2-(2-aminoethoxy)ethan-1-ol to give Compound
II. The
Cbz protecting group was removed by hydrogenation to afford Intermediate-A as
a
trifluoroacetate (TFA) or HC1 salt. Intermediate B was synthesized based on
the same scheme
except Cbz protected 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol was used as the
starting
material. Scheme 3 allows access to variation of linkerA, as well as variation
of targeting
ligands.
CF3C00
Ac0
AcOseThr/NHAc
OAc Intermediate A
H3+
AcO/fY.'iNHAc CF3C00
OAc Intermediate B
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1. TMSOTf
Ac0.44..õ0.õ.00NHCbz
AcOvThr'''NHAc 0 AcOsfY.'/NHAc
2. HC-J(D'N)0 OAc
OAc
Ac0,07NH3+ CF3C00
H2, Pd/C, TFA
THF AcOf.'iNHAc
OAc
Intermediate-A
Scheme 3
To a solution of Compound 1(20.0 g, 51.4 mmol) in DCE (100 mL) was added
TMSOTf (17.1 g, 77.2 mmol). The resulting reaction solution was stirred at 60
C for 2 hrs,
and then at 25 C for 1 hr. Cbz protected 2-(2-aminoethoxy)ethan-1-ol (13.5 g,
56.5 mmol) in
DCE (100 mL) dried over 4 A powder molecular sieves (10 g) was added dropwise
to the
abovementioned reaction solution at 0 C under N2 atmosphere. The resulting
reaction
mixture was stirred at 25 C for 16 hrs under N2 atmosphere. The reaction
mixture was
filtered and washed with sat. NaHCO3 (200 mL), water (200 mL) and sat. brine
(200 mL).
The organic layer was dried over anhydrous Na2SO4, filtered and concentrated
under reduced
pressure to give a crude product, which was triturated with 2-Me-THF/heptane
(5/3, v/v, 1.80
L) for 2 hrs, filtered and dried to give Compound 11 (15.0 g, 50.3% yield) as
a white solid.
To a dried and argon purged hydrogenation bottle was carefully added 10% Pd/C
(1.50 g), followed by THE (10 mL) and then a solution of Compound 11 (15.0 g,
26.4 mmol)
in THF (300 mL) and TFA (3.00 g, 26.4 mmol). The resulting mixture was
degassed and
purged with H2 three times and stirred at 25 C for 3 hrs under H2 (45 psi)
atmosphere. TLC
(DCM: Me0H = 10:1) indicated Compound II was consumed completely. The reaction
mixture was filtered and concentrated under reduced pressure. Residue was
dissolved in
anhydrous DCM (500 mL) and concentrated. This process was repeated 3 times to
give
Intermediate-A (14.0 g, 96.5% yield) as a foamy white solid. 1H NMR (400 MHz
DM50-d6):
6 ppm 7.90 (d, J= 9.29 Hz, 1 H), 7.78 (br s, 3 H), 5.23 (d, J 3.26 Hz, 1 H),
4.98 (dd, J=
11.29, 3.26 Hz, 1 H), 4.56 (d, J= 8.53 Hz, 1 H), 3.98 - 4.07 (m, 3 H), 3.79 -
3.93 (m, 2 H),
3.55 -3.66 (m, 5 H), 2.98 (br d, J= 4.77 Hz, 2 H), 2.11 (s, 3 H), 2.00 (s, 3
H), 1.90 (s, 3 H),
1.76 (s, 3 H).
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Intermediate-B was synthesized using similar procedures for synthesis of
Intermediate-A. 11-INMR (400 MHz DMSO-d6): 6 ppm 7.90 (br d, J = 9.03 Hz, 4
H), 5.21 (d,
J= 3.51 Hz, 1 H), 4.97 (dd, J= 11.1 Hz, 1 H), 4.54 (d, J= 8.53 Hz, 1 H), 3.98 -
4.06 (m, 3 H),
3.88 (dt, J = 10.9 Hz, 1 H), 3.76- 3.83 (m, 1 H), 3.49 - 3.61 (m, 9 H), 2.97
(br s, 2 H), 2.10 (s,
.. 3 H), 1.99 (s, 3 H), 1.88 (s, 3 H), 1.78 (s, 3 H). Mass calc. for
C20H34N2011: 478.22; found:
479.3 (M+H ).
Example 3. Preparation of Compound /
Scheme 4 below was used to prepare Compound 1 identified in Example 1 above.
.. Commercially available 2,2',2",2"-(propane-1,3-
diylbis(azanetriy1))tetraacetic acid
(Compound I in Scheme 4) was converted to dianhydride Compound II. Upon
treatment of 6-
aminohexan-1-ol followed by hydrolysis, Compound II was converted to triacid
Compound
III. Amide coupling between Compound III and Intermediate-A afforded Compound
IV.
Treatment of Compound IV with 2-Cyanoethyl N,N-
diisopropylchlorophosphoramidite and a
.. catalytic amount of 1H-tetrazole afforded the phosphoramidite Compound 1.
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H(DC:'
Oy---,
N N Ac20, pyridine
ON N0
H 2 N .0H
OH y yOH Oy y 0
H20 (0.5 eq)/DMF
OH 0 0 0
II
I
OAc
AcOv....\.,
0
Ac0 0.õ._,-----,õ
H,i-J....._.------
0,,
NHAc NH 0N.õ,_,..w.OH
H
(7)y N N amide coupling N N
OH
OH y LOH ____________________________ v- OAc
AcO\ 0 0
0
OH 0 Ac0-...y01.0,..---0----....i. 73+ _ Ac0Z--\ 0õ......-
-Nio N
III AceY'NHAc 2 NHAc N N
OAc H
Ac0 OAc
"D
HN
Ac00.õ.----..0,,,,j
NHAc
OAc Iv
Ac0.,
0
Ac0 0,---.0
Nip.1\1, NHAc 'N1-1,0
H
LCN OAc Nfr\l'O¨p N,
o _,.. AcOl 6
A ....\____\.,
0 0 LC N
c0 0,o tetrazole )-N1,
N
AcOs OAc H
HN
AcOn _0,,)
NHAc
Compound1
Scheme 4
To a stirring solution of Ac20 (8.83 g, 86.5 mmol) and pyridine (193 mg, 2.45
mmol)
was added tetraacid Compound 1(5.0 g, 16.3 mmol). After purging with N2 for 3
times, the
reaction mixture was stirred at 65 C for 12 hrs under N2 atmosphere. After
cooling, the
reaction mixture was filtered to remove insoluble solid. Filtrate was
concentrated in vacuum.
Toluene was added to the residue and volatiles were evaporated. This process
was repeated 3
times to give compound 11 (2.20 g, 49.8% yield) as a yellow oil. 1H NMR (400
MHz DMS0-
d6): 6 ppm 3.65 (s, 8 H), 2.46 (br t, J= 7.19 Hz, 4 H), 1.55 - 1.65 (m, 2 H).
To a mixture of Compound 11 (1.80 g, 6.66 mmol) and imidazole (3.63 g, 53.2
mmol)
in DMF (18 mL) was added 6-aminohexan-1-ol (624 mg, 5.33 mmol) and pyridine
(263 mg,
3.33 mmol) sequentially. The mixture was left stirring at 50 C for 5 hrs
under N2 atmosphere.
The reaction mixture was concentrated under reduced pressure. Residue was
purified by
reversed phase prep-HPLC. Compound III was obtained (1.90 g, contains 8.4% DMF
and
63.2% imidazole by weight). 1H NMR (400 MHz DMSO-d6): 6 ppm 4.00 (br t, J =
6.50 Hz, 1
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H), 3.44- 3.56 (m, 6 H), 3.34- 3.39 (m, 3 H), 3.20- 3.27 (m, 2 H), 3.02 - 3.12
(m, 2 H), 2.80
-2.86 (m, 1 H), 2.79 - 2.87 (m, 1 H), 2.64 - 2.70 (m, 2 H), 1.49- 1.69 (m, 3
H), 1.33 - 1.73
(m, 4 H), 1.18- 1.46 (m, 3 H).
To a solution of Compound III (950 mg, purity 28.3% 0.66 mmol) and
Intermediate-A
(1.01 g, 2.32 mmol) in DMF (10 mL) was added DIEA (385 mg, 2.99 mmol), HOBt
(358 mg,
2.65 mmol) and EDC (508 mg, 2.65 mmol) sequentially. The resulting reaction
mixture was
stirred at 25 C for 3 hrs under N2 atmosphere. LC-MS indicated desired
product. The
reaction mixture was purified by reverse phase prep-HPLC. Fractions containing
desired
product were combined and concentrated to afford Compound IV (200 mg, 18.2%
yield) as a
white solid. lEINMR (400 MHz DMSO-d6): 6 ppm 8.03 -8.11 (m, 3 H), 7.84 (d, J=
9.26 Hz,
3 H), 5.21 (d, J= 3.38 Hz, 3 H), 4.97 (dd, J= 11.19, 3.31 Hz, 3 H), 4.54 (d,
J= 8.50 Hz, 3 H),
4.34 (br t, J= 4.88 Hz, 1 H), 4.03 (s, 9 H), 3.82 - 3.92 (m, 3 H), 3.73 - 3.81
(m, 3 H), 3.44 -
3.60 (m, 10 H), 3.38 - 3.43 (m, 8 H), 3.19- 3.27 (m, 6 H), 3.01 -3.07 (m, 8
H), 2.39 - 2.48 (m,
6 H), 2.10 (s, 9 H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.78 (s, 9 H), 1.55 (br s,
2 H), 1.33 - 1.44 (m,
4 H), 1.23 (br s, 4 H). LCMS: [M+2H-]/2, 828Ø
To a solution of compound IV (200 mg, 120 umol) in anhydrous DCM (2.0 mL) was
added diisopropylammonium tetrazolide (22.9 mg, 132 umol), followed by
dropwise addition
of 3-bis(diisopropylamino)phosphanyloxypropanenitrile (145 mg, 483 umol) at 25
C under
N2. The reaction mixture was stirred at 25 C for 2 hrs. LC-MS indicated
compound IV was
consumed completely. The reaction was quenched by addition of a mixture of
brine and
saturated NaHCO3 solution (1:1, 5 mL) at -20 C and the resulting mixture was
stirred at 0 C
for 1 min. Layers were separated. The aqueous phase was extracted with
additional DCM (5
mL). The combined organics were washed with brine / saturated aq. NaHCO3
solution (1:1, 5
mL), dried over Na2SO4, filtered and concentrated to - 1 mL of volume. This
solution was
added dropwise to MTBE (20 mL) while stirring. This resulted in formation of
white solid,
which was isolated by centrifuge. This process was repeated one more time.
Solid was then
dissolved in anhydrous CH3CN and volatiles were removed. This process was
repeated 3
times to give Compound 1 (103 mg, 45.9% yield) as a colorless oil. lEINMR
(CDC13): 6 ppm
7.74 -7.88 (m, 3 H), 6.70 -7.02 (m, 3 H), 5.37 (br s, 3 H), 5.14- 5.27 (m, 3
H), 4.77 (br d, J
= 7.78 Hz, 3 H), 4.13 -4.27 (m, 6 H), 3.95 (br s, 10 H), 3.71 -3.82 (m, 4 H),
3.47 - 3.70 (m,
20 H), 3.42 (br s, 3 H), 3.13 - 3.29 (m, 10 H), 2.63 -2.68 (m, 6 H), 2.15 -
2.23 (m, 9 H), 2.07
(s, 9 H), 2.02 (s, 9 H), 1.89 (s, 9 H), 1.53 - 1.77 (m, 6 H), 1.37 -1.18 (m,
16 H). 3113NMR
(CDC13): 6 ppm 147.14.
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Example 4. Preparation of Compound 2
Compound 2 (in Example 1) was synthesized using the same procedure based on
Scheme 4 above except Intermediate B was used instead of Intermediate A. 1E1
NMR
(CD03): 6 ppm 8.01 - 8.09 (m, 1 H), 7.59 - 7.61 (m, 2 H), 7.21 - 7.23 (m, 1
H), 6.66 - 6.85
(m, 3 H), 5.35 (br s, 3 H), 5.06 - 5.25 (m, 3 H), 4.72 - 4.84 (m, 3 H), 4.05 -
4.25 (m, 10 H),
3.76 -4.00 (m, 12 H), 3.46 -3.62 (m, 32 H), 3.20 (br s, 10 H), 2.61 -2.68 (m,
6 H), 2.16 -
2.18 (m, 9 H), 2.05 (s, 9 H), 1.96 -2.02 (m, 18 H), 1.61 - 1.66 (m, 4 H), 1.52
(br s, 2 H), 1.36
(br s, 4 H), 1.17- 1.19 (m, 12 H). 3113 NMR (CDC13): 6 ppm 147.07.
Example 5. Preparation of Compound 3
Scheme 5 below can be used to prepare Compound 3 identified in Example 1
above.
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Br--õ........--,õ
H OBn H H TFA/DCM H
Boc,N NI-12 ,.._ Boc,NN
OBn
'OBn
K2CO3/DMF
I II
OtBu OtBu OH OH
o
tBu,0õk,Br
H ro HCOOH CD r0
______________ )). N _,... NOBn r\J 1\1 oBn
DIEA/DMF
tBu,
0 0 HOO
III
IV
OAc
Ac0._\.,
0
Ac0 0õ,,-----Ø-----
õ,,0,---HN O
Ac0 NHAc
Ac?NOBn
'y)'NHAc CF3CO2-
OAc Pd/C, H2
OAc
Intermediate B
____________________________ ).- AcO 0
0
THE
amide coupling NHAc N
H
AcO, OAc HN
Ac00.õõ-----.0,---,,O..õ,)
NHAc
V
OAc
Ac0\&7___\,,
0 OAc
Ac0 0õ,--.0O,HNO ---.T,N.p,Ny, Ac0\&...\_._.\õ
0 ,
Ac0
NHAc ,,,--.0----õ,0,..õ----HN
L.N---,,,OH 0 I
ION NHAc
-.NO-p N,,,,
OAc o I
AcON&41, 0 i _, OAc
Ac0 0,---.0,---.....õ0.,,N)1...õN., tetrazole AcON&...\
0 0 i L
Ac0 0,--Ø-
.õ0,---õNN CN
,
NHAc H
NHAc H
Ac0 OAc
HN
"D "D
Ac0 OAc
HN
Ac0\,.....,1õ0 n ,---...---._,O,õ)
NHAc ¨ Ac0,0 0-----...- -
0,..)
-....-----
NHAc
VI
Compound 3
Scheme 5
Starting from tert-butyl (3-aminopropyl)carbamate, it can be alkylated with
benzyl
protected 2-bromoethanol (SN2 substitution) to give Compound I. The Boc group
can then be
removed under acidic condition to afford Compound II, which can be alkylated
with tert-
butyl 2-bromoacetate to afford triester Compound III. tert-Butyl protecting
groups can then
be removed upon treatment of formic acid to generate triacid Compound IV.
Amide coupling
with Intermediate-A affords Compound V. The benzyl protecting group may then
be
removed by hydrogenation to afford compound VI. Phosphoramidite Compound 3 can
be
synthesized by treating Compound VI with 2-Cyanoethyl N,N-
diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole.
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Example 6. Preparation of Compound 4
Scheme 6 below was used to prepare Compound 4 identified in Example 1 above.
o
o
0) ,Br 0 ?(D 0 Pd/C H2
H
H 2 N 40 N _õ..
0 -).-
DIEA
I,
0
OH
0 ?Ci<
H 0 0 OH
"_,,N.õ..---.õ,N.j=L.o..--<
0
HCI
-).-
NNA(:)< ________________________________ ..-
0 e\/\/\OH dioxane
amide coupling
II III
OAc
+ AcC
NH3\_K
Ac0,....(2,0,00,-HN,00
0 AcO'Thr 'NHAc CF3CO2" NHAc
OAc
LNK._,......õ--...õOH
0 'LCDH 0 Intermediate B
OAc
Ho)C-N--NOH ______________________________ ) __ AcO _ 0
clOH amide coupling Ac0.4:).. 0' ....,---.,NN.,
NHAc H
VI Acp OAc HN/0
Ac0...P.,,0,-00,
NHAc
Ac0OAc V
Ac0\_...\::.,\,,,
u - (:)(D/.-.HN 0 0
,N.12,,N
NHAc
1 0, 1 i\li'.(3---p N 1.--
L C N K 0
OAc
_____________ ).- AcO, 0 LCN
tetrazole Ac0....\!.Ø.õ----Ø-----,0,NN:
NHAc H
AGO OAc
HN/N:)
Ac00,00,
NHAc
Compound 4
Scheme 6
Starting from benzyl protected propane-1,3-diamine, it was alkylated with tert-
butyl
2-bromoacetate to afford triester Compound I. The benzyl protecting group was
removed by
hydrogenation to afford secondary amine Compound II. Amide coupling with 6-
hydroxyhexanoic acid afforded Compound III. tert-Butyl protecting groups were
then
removed upon treatment of HC1 in dioxane to generate triacid Compound IV.
Amide
coupling between triacid compound IV and Intermediate-A was performed to
afford
Compound V. Phosphoramidite Compound 4 was synthesized by phosphitylation of
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Compound V with 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite and a
catalytic
amount of 1H-tetrazole.
To a solution of N1-benzylpropane-1,3-diamine (5.00 g, 30.4 mmol) in DMF (100
mL)
was added tert-butyl 2-bromoacetate (23.7 g, 121 mmol), followed by addition
of DIEA
(23.61 g, 182 mmol) dropwise. The resulting reaction mixture was stirred at 25-
30 C for 16
hrs. LCMS showed Ni-benzylpropane-1,3-diamine was consumed completely.
Reaction
mixture was diluted with H20 (500 mL) and extracted with Et0Ac (500 mL x 2).
The
combined organics were washed with sat. brine (1 L), dried over anhydrous
Na2SO4, filtered,
and concentrated under reduced pressure to give crude product, which was
purified by silica
gel column chromatography (gradient: petroleum ether:ethyl acetate from 20:1
to 5:1).
Compound 1(12.1 g, 78.4% yield) was obtained as a colorless oil. 1H NMR (400
MHz,
CDC13): 6 ppm 7.26 - 7.40 (m, 5 H), 3.79 (s, 2 H), 3.43 (s, 4 H), 3.21 (s, 2
H), 2.72 (dt, J=
16.9, 7.34 Hz, 4 H), 1.70 (quin, J= 7.2 Hz, 2 H), 1.44 - 1.50 (m, 27 H).
A dried hydrogenation bottle was purged with Argon three times. Pd/C (200 mg,
10%)
.. was added, followed by Me0H (5 mL) and then a solution of Compound 1(1.00
g, 1.97
mmol) in Me0H (5 mL). The reaction mixture was degassed under vacuum and
refilled with
H2. This process was repeated three times. The mixture was stirred at 25 C for
12 hrs under
H2 (15 psi) atmosphere. LCMS showed Compound I was consumed completely. The
reaction
mixture was filtered under reduced pressure under N2 atmosphere. Filtrate was
concentrated
under reduced pressure to give Compound 11 (655 mg, 79.7% yield) as yellow
oil, which was
used for the next step without further purification. 1H NMR (400 MHz, CDC13):
6 ppm 3.44
(s, 4 H), 3.31 (s, 2 H), 2.78 (t, J= 7.1 Hz, 2 H), 2.68 (t, J= 6.9 Hz, 2 H),
1.88 (br s, 1 H), 1.69
(quin, J= 7.03 Hz, 2 H), 1.44 - 1.50 (s, 27 H).
A mixture of Compound 11 (655 mg, 1.57 mmol), 6-hydroxyhexanoic acid (249 mg,
1.89 mmol), DIEA (1.02 g, 7.86 mmol) , EDCI (904 mg, 4.72 mmol), and HOBt (637
mg,
4.72 mmol) in DMF (6 mL) was degassed and purged with N2 three times, and then
was
stirred at 25 C for 3 hrs under N2 atmosphere. LCMS indicated desired product.
The
reaction mixture was diluted with H20 (10 mL) and extracted with Et0Ac 20 mL
(10 mL x
2). Organics were combined and washed with sat. brine (20 mL), dried over
anhydrous
Na2SO4, filtered, and concentrated to give crude product, which was purified
by silica gel
column chromatography (gradient: petroleum ether:ethyl acetate from 5:1 to
1:1) to afford
Compound III (650 mg, 77.8% yield) as a yellow oil. 1H NMR (400 MHz, CDC13): 6
ppm
3.90 - 3.95 (s, 2 H), 3.63 (t, J = 6.40 Hz, 2 H), 3.38 - 3.45 (m, 6 H), 2.72
(t, J= 6.65 Hz, 2 H),
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2.40 (t, J= 7.28 Hz, 2 H), 1.55 - 1.75 (m, 8 H), 1.44 (s, 27 H). Mass calc.
for C27H50N208:
530.36; found: 531.3 (M+H ).
A mixture of Compound III (5.5 g, 10.3 mmol) in HC1/dioxane (2M, 55 mL) was
stirred at 25 C for 3 hrs. LCMS showed complete consumption of Compound III.
Reaction
mixture was filtered, washed with EtOAc (50 mL), and dried reduced pressure to
give crude
product. It was dissolved in CH3CN (50 mL), volatiles were removed under
vacuum. This
process was repeated three times to give Compound IV (2.05 g, 54.5% yield) as
a white solid.
1E1 NMR (400 MHz, D20): 6 ppm 4.21 (s, 1 H), 4.07 (d, J= 4.5 Hz, 4 H), 3.99
(s, 1 H), 3.45 -
3.52 (m, 3 H), 3.42 (t, J= 6.5 Hz, 1 H), 3.32 - 3.38 (m, 1 H), 3.24 -3.31 (m,
1 H), 2.37 (t, J=
7.4 Hz, 1 H), 2.24 (t, J= 7.4 Hz, 1 H), 1.99 (dt, J= 15.5, 7.53 Hz, 1 H), 1.85
- 1.94 (m, 1 H),
1.85 - 1.94 (m, 1 H), 1.39 - 1.56 (m, 4 H), 1.19- 1.31 (m, 2 H).
A mixture of Compound IV (150 mg, 0.413 mmol), Intermediate-B (693 mg, 1.45
mmol), DIEA (267 mg, 2.07 mmol), EDCI (277 mg, 1.45 mmol), and HOBt (195 mg,
1.45
mmol) in DMF (2.6 mL) was stirred at 25 C for 3 hrs under N2 atmosphere. LCMS
indicated
desired product. The reaction mixture was purified by reversed phase prep -
HPLC to afford
Compound V as a white solid after lyophilizing (186 mg, 0.106 mmol, 25.7%
yield). 1E1
NMR (400 MHz, CDC13): ppm 6 7.91 - 8.13 (m, 1 H), 7.70 (br s, 1 H), 7.02 (br
s, 1 H), 6.54 -
6.84 (m, 3 H), 5.27 (br d, J= 3.0 Hz, 2 H), 5.26- 5.30 (m, 1 H), 4.99 - 5.15
(m, 3 H), 4.66 -
4.76 (m, 3 H), 3.98 -4.17 (m, 10 H), 3.83 - 3.95 (m, 8 H), 3.63 -3.76 (m, 4
H), 3.46 - 3.60 (m,
30 H), 3.40 (br s, 6 H), 3.12 - 3.18 (m, 4 H), 2.56 (br d, J= 7.2 Hz, 2 H),
2.22 - 2.39 (m, 2 H),
2.09 (s, 9 H), 1.98 (s, 9 H), 1.87 - 1.95 (m, 18 H), 1.69 (br d, J= 6.25 Hz, 2
H), 1.50 (br s, 2
H), 1.37 (br d, J= 7.0 Hz, 2 H).
To a solution of Compound V (180 mg, 0.103 mmol) in anhydrous DCM (3.6 mL)
was added diisopropylammonium tetrazolide (19.44 mg, 0.114 mmol), followed by
dropwise
addition of 3-bis(diisopropylamino)phosphanyloxypropanenitrile (124 mg, 0.412
mmol) at
ambient temperature under N2. The reaction mixture was stirred at 20 - 25 C
for 2 hrs.
LCMS indicated Compound V was consumed completely. After cooling to -20 C,
the
reaction mixture was added to a stirred solution of brine/saturated aq. NaHCO3
(1:1, 5 mL) at
0 C. After stirring for 1 min, DCM (5 mL) was added. Layers were separated.
Organics were
washed with brine/saturated aq. NaHCO3 solution (1:1.5 mL), dried over Na2SO4,
filtered,
and concentrated to - 1 mL of volume. The residue solution was added dropwise
to 20 mL
MTBE with stirring. This resulted in precipitation of white solid. The mixture
was
centrifuged, and solid was collected. This process was repeated one more time.
The solid
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collected was dissolved in anhydrous CH3CN. Volatiles were removed. This
process was
repeated two more times to afford Compound 4 (106 mg, 52.8% yield) as a white
solid. 11-1
NMR (400 MHz, CDC13): ppm 6 7.94- 8.18 (m, 1 H), 7.69 (br s, 1 H), 6.66 -7.10
(m, 3 H),
5.35 (d, J = 3.5 Hz, 3 H), 5.07- 5.25 (m, 3 H), 4.76 -4.86 (m, 3 H), 4.01 -
4.31 (m, 10 H),
3.91 -4.01 (m, 8 H), 3.74 -3.86 (m, 4 H), 3.52- 3.71 (m, 30 H), 3.42 -3.50 (m,
6 H), 3.15 -
3.25 (m, 4 H), 2.52 - 2.70 (m, 4 H), 2.22 - 2.45 (m, 2 H), 2.15 -2.22 (s, 9
H), 2.06 (s, 9 H),
1.95 - 2.03 (m, 18 H), 1.77 (br s, 2 H), 1.58 - 1.66 (m, 4 H), 1.40 (m, 2 H),
1.08 - 1.24 (m, 12
H). 31PNMR (CDC13): ppm 6 147.12.
Example 7. Preparation of Compound 5
Compound 5 was synthesized using the same procedure based on Scheme 6 except
Intermediate A was used instead of Intermediate B. lEINMR (400 MHz, CDC13):
ppm 6 7.71
- 8.06 (m, 2 H), 7.36 - 7.49 (m, 0.5 H), 6.59 - 7.14 (m, 3 H), 6.34 - 6.43 (m,
0.5 H), 5.36 (br d,
J=3.01 Hz, 3 H), 5.10- 5.31 (m, 3 H), 4.57 - 4.85 (m, 3 H), 3.85 -4.22 (m, 18
H), 3.29 -3.81
(m, 30 H), 3.13 - 3.26 (m, 4 H), 2.61 -2.68 (m, 4 H), 2.26 -2.42 (m, 2 H),
2.13 -2.19 (m, 9
H), 2.05 (s, 9 H), 1.97 - 2.01 (m, 9 H), 1.94 - 1.96 (m, 9 H), 1.63 (br s, 4
H), 1.35 - 1.46 (m, 2
H), 1.16- 1.19 (m, 12 H). 31PNMR (CDC13): ppm 6 147.15.
Example 8. Preparation of Compound 6
Scheme 7 below can be used to prepare Compound 6 identified in Example 1
above.
ootBu
1401
U
0 0
404
OAc
Acc0
A01, 0
NHAcO,
- NH
0
OAc N p N
AcO\
C)
Ac0_) , 0
NHAcN N H CN
AGO OAc
0 NHO
Ac0
NHAc
Compound 6
Scheme 7
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Starting from the benzyl protected propane-1,3-diamine (Compound I), a Michael
addition reaction with tert-butylacrylate may be performed to afford triester
Compound II.
Once Compound II is synthesized, the same procedure for synthesis of Compound
4 in
Scheme 6 may be followed for remaining steps to synthesize Compound 6.
Example 9. Preparation of Compound 7
Scheme 8 below can be used to prepare Compound 7 identified in Example 1
above.
o
o
0 H..0< H 0 cBzCI 0 H...0< 0 NCI
)- N N
___________________________________________________________________ ).--
N Nj-(j<
0
0 TEA
0J'OBn
I II
OAc
Ac0.,
0
0 Ac0 0,,..---..
0_ --_
NH3' NHAc ¨ NH
,(:) 0
0 1)-OH 0 Ac0-*"y 1' 0
AcO'NHAc CF3CO2-
'NJ(0Bn Pd/C H2
1 OH OAc -
OAc
I.-
______________________________________ ).-- Ac0\,L__o 0 LD
00Bn Ac0 __ 0õ,..---.n
amide coupling
NHAc --..õ....---.
III N
Ac0 OAc H
"D
Ac0n HN
_,..---.,0.)
NHAc
IV
OAc
Ac0\,.......\
AGO 0,---,..o_ OAc
0 Ac0...v.s,\.,
NHAc ¨ NH 0 0
'CNN 0)- Ac0 0,,..---,
0,
NHAc ¨ NI-V) 0 0
OAc
AcOa 0
0 i 0
_,.. OAc -.N.-
11.õ.......--.õ ....õ-kOH
Ac0O,.--..o,
NHAc " N)IN' AcO__...\(. 0 LD
H Ac0 0,,..---.
0
Ac0 (:)Ac ":1 NHAc -.õ....---.
N
Ac0\t4I,0..--,, HN H
Aco OAc
to
NHAc `) HN
Ac0n _õ----.0,,,_,)
V NHAc
VI
OAc
AcO__\.___\.,
0
Ac0 0,---..0
NHAc 'NFIto 0 0
EDC OAc N)L0
AcO__\_..._\.,
Ac0 0,----..
)- ,
F
2,3,5,6-tetrafluorophenol NHAc 0N N
Ac0 OAc H /c) F F
0 , HN
Ac0...\,,,µ,.õ----.0,..,.._.)
NHAc
Compound-7
Scheme 8
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Starting from secondary amine Compound I (Compound II in Scheme 6), Cbz
protection may be used to afford Compound II. The tert-Butyl groups of
Compound II may
be removed by treatment with acid to give triacid Compound III. Amide coupling
of
Compound III with Intermediate-A may afford Compound IV. The Cbz protecting
group of
Compound IV may be removed by hydrogenation to afford secondary amine Compound
V,
which may react with glutaric anhydride to afford carboxylic Compound VI. The
carboxylic
acid of Compound VI may be converted to tetrafluorophenyl ester by standard
procedure to
afford Compound 7.
Example 10. Preparation of Compound 8
Scheme 9 below can be used to prepare Compound 8 identified in Example 1
above.
OAc
AcO
0 0 0 OAc
Ac0
NHAc 0
0 r1 N) Ac0 __ 0
NHAc
NI-1
0
,0 0
OAc NH 0/
0 LD OAc N
Ac0 u N
0 L-j
0 0/
NHAc
Ac0 C)0 N
Ac0 OAc NHAc
HN AcO OAc
NHAc HN
NHAc
Compound-8
Scheme 9
Compound I (Compound V in Scheme 8) may be reacted with NHS conjugated
maleimide compound 2,5-dioxopyrrolidin-1-y1 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-
1-
yl)propanoate to afford Compound 8.
Example 11. Preparation of Compound 74
Scheme 10 below can be used to prepare Compound 74 identified in Example 1
above.
0 j<
)010-110.0 CnzCI 7E" õtcr.k. mom/
A1OH041198n Ho NAH
Ori''Ven
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-1/4
AcC Ac
.1AA ,ALAA
A ,r1 TFA
NI'
- k &TWA
Ne"
KM
IINAc
IV V
OAc
ACO
OAc OH
NHAc 0 Ac0 0
<-0DMTr
Fi 0 0
OH
H,N NHAc
0Ac
Nj.)11-0-0H
Int-D
Ac0 N AAccOoa 0 N 0 N
NHAc
H NHAc
Ac0 0Ac
AwAc0 0 0_/-0__7"-NH
AGO NHAc NHAc
Compound 74
VI
Scheme 10
Starting from compound I (as same from example 6 Compound II in Scheme 6).
To a solution of Compound 1(275 g, 660 mmol, 1.00 eq.) in DCM (2.75 L) was
added TEA (133 g, 1.32 mol, 2.00 eq.), then Cbz-Cl (169 g, 990 mmol, 1.50 eq.)
was added
dropwise into the reaction mixture. The mixture was stirred at 25 C for 2
hrs. LCMS showed
Compound I was consumed completely and one main peak with desired mass was
detected.
The reaction mixture was diluted with NaHCO3 (800 mL) and extracted. The
combined
organic layers were washed with brine 500 mL (500 mL * 1), dried over Na2SO4,
filtered and
concentrated under reduced pressure to give crude product, which was purified
by column
chromatography (5i02, PE/EA=100/1 to 5/1) to give Compound 11 (290 g, 527
mmol, 75.7%
yield) as a colorless oil.
1H NMR: 400 MHz, DMSO-d6 6 ppm 7.23 -7.40 (m, 5 H), 5.00 - 5.12 (m, 2 H), 3.86
- 3.95 (m, 2 H), 3.23 - 3.39 (m, 6 H), 2.55 -2.67 (m, 2 H), 1.56 - 1.64 (m, 2
H), 1.31 - 1.46
(m, 27 H).
To a solution of Compound II (145 g, 263 mmol, 1.00 eq) in HCOOH (2.9 L). The
mixture was stirred at 60 C for 12 hrs under air atmosphere. LCMS showed
Compound III
was consumed completely and one main peak with desired mass was detected. The
reaction
was diluted with toluene and acetonitrile (ACN, 1500 mL each), and the mixture
was
concentrated in vacuum to remove the formic acid azeotropically. The residue
was diluted
with 1:1 ACN : toluene (-750 mL) and concentrated. The residue was diluted
with ACN
(1000 mL), and concentrated. This process was repeated one more time to give
crude product
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as a solid. The crude product was triturated with ACN (700 mL) at 60 C for 2
hrs, filtered
and dried to give compound III (210 g, quantitative yield) as a white solid.
11I NMR: 400 MHz, DMSO-d6 6 ppm 7.26 -7.40 (m, 5 H), 5.02 - 5.10 (m, 2 H),
3.89 -
4.00 (m, 2 H), 3.36 - 3.45 (m, 4 H), 3.24 - 3.34 (m, 2 H), 2.59 - 2.72 (m, 2
H), 1.40 (s, 2 H).
To a solution of Compound III (100 g, 261 mmol, 1.00 eq.), intermediate A (502
g, 915.
mmol, 3.50 eq., TFA) in DMF (1.00 L) was added TBTU (327 g, 1.02 mol, 3.90
eq.), TEA
(212 g, 2.09 mol, 291 mL, 8.00 eq.). The mixture was stirred at 25 C for 1
hr. LCMS
showed Compound III was consumed completely and one main peak with desired
mass was
detected. The reaction mixture was added into H20 (4000 mL). The resulting
mixture was
extracted with MTBE (2000 mL *2) to remove impurities. The remaining aqueous
portion
was extracted with DCM (3000 mL * 2). The combined DCM extracts were washed
with 10%
citric acid (2000 mL * 2), saturated NaHCO3 (2000 mL * 2), brine 2000 mL,
dried over
Na2SO4, filtered and concentrated under reduced pressure to give Compound IV
(260 g, 159
mmol, 60.9% yield) as a white solid.
1H NMR: 400 MHz, DMSO-d6 6 ppm 7.99 - 8.08 (m, 2 H), 7.93 (br d, J=5.50 Hz, 1
H), 7.79
- 7.86 (m, 3 H), 7.26 - 7.39 (m, 5 H), 5.22 (d, J=3.13 Hz, 3 H), 4.95 - 5.08
(m, 5 H), 4.54 (br
d, J=8.38 Hz, 3 H), 4.03 (s, 9 H), 3.81 - 3.93 (m, 5 H), 3.76 (br d, J=4.88
Hz, 3 H), 3.44 -
3.62 (m, 10 H), 3.34- 3.43 (m, 6 H), 3.24 (br d, J=6.13 Hz, 7 H), 3.02 - 3.09
(m, 4 H), 2.40 -
2.47 (m, 2 H), 2.10 (s, 9 H), 1.99 (s, 9 H), 1.89 (s, 9 H), 1.77 (s, 9 H),
1.57 - 1.68 (m, 2 H).
The 2.00 L hydrogenation bottle was purged with Ar for 3 times and added dry
Pd/C (9 g)
carefully. Then Me0H (50 mL) was added to wet the Pd/C completely, followed by
the
solution of Compound IV (90 g, 55.1 mmol, 1.00 eq.) and TFA (6.29 g, 55.1
mmol, 1.00 eq.)
in Me0H (850 mL) slowly under Ar atmosphere. The resulting mixture was
degassed and
purged with H2 for 3 times, and then the mixture was stirred at 25 C for 10
hrs under H2
atmosphere. LCMS showed Compound IV was consumed completely and one main peak
with desired mass. The reaction mixture was filtered under reduced pressure
carefully under
N2 atmosphere. The filtrate was concentrated under reduced pressure to give
compound V
(160 g, 90.2% yield).
11I NMR: 400 MHz, DMSO-d6 6 ppm 9.12 (br s, 2 H), 8.50 (br t, J=5.19 Hz, 1 H),
8.10 (br t,
J=5.50 Hz, 2 H), 7.85 - 7.91 (m, 3 H), 5.22 (d, J=3.25 Hz, 3 H), 4.95 - 5.01
(m, 3 H), 4.52 -
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4.58 (m, 3 H), 4.03 (s, 9 H), 3.84 - 3.93 (m, 3 H), 3.75 - 3.83 (m, 3 H), 3.39
- 3.61 (m, 17H),
3.23 -3.32 (m, 7H), 3.15 -3.18 (m, 3 H), 2.97 - 3.05 (m, 2 H), 2.54 - 2.61 (m,
2 H), 2.10 (s, 9
H), 2.00 (s, 9 H), 1.89 (s, 9 H), 1.77 - 1.80 (m, 9 H), 1.70 - 1.76 (m, 2 H).
To a solution of compound V (5.0 g, 3.10 mmol, 1.0 eq, TFA salt) in DCM (50
mL)
was added glutaric anhydride compound 5A, 531 mg, 4.65 mmol, 1.5 eq) at 25 C,
then TEA
(1.26 g, 12.4 mmol, 1.73 mL, 4.0 eq.) was added to the mixture dropwise. The
mixture was
stirred at 25 C for 1.0 hr. LC-MS showed compound V was consumed completely
and a
main peak with desired product mass. The resulting reaction mixture was
triturated with
isopropyl ether for two times (50 mL * 2) vacuum dried to afford compound VI
(crude, 5.5 g)
as a brown solid.
To a solution of compound VI (2.6 g, 1.61 mmol, 1.0 eq) in DMF (26 mL) was
added
int-D (protected (R)-3-aminopropane-1,2-diol) (952 mg, 2.42 mmol, 1.5 eq),
TBTU (1.04 g,
3.23 mmol, 2.0 eq) and DIEA (625.53 mg, 4.84 mmol, 843.03 uL, 3.0 eq). The
mixture was
stirred at 25 C for 1.0 hr. LC-MS showed compound int-D (protected (R)-3-
aminopropane-
1,2-diol) was consumed completely. The resulting reaction mixture was
triturated with
isopropyl ether (260 mL) to afford crude product. It was purified by column
chromatography
(SiO2, DCM/Me0H = 100/1 to 10/1, 0.1% Et3N) to give compound 74 (900 mg, 28.0%
yield)
as a white solid.
Compound 74 1E1 NMR: (400 MHz, DMSO-d6) 6 ppm 8.06 (br d, J=6.00 Hz, 2
H), 7.83 (br d, J=8.50 Hz, 3 H), 7.66 (dt, J=10.22, 5.21 Hz, 1 H), 7.39 (br d,
J=7.75 Hz, 2 H),
7.20 - 7.30 (m, 8 H), 6.87 (br d, J=8.63 Hz, 4 H), 5.21 (br d, J=3.00 Hz, 3
H), 4.98 (br dd,
J=11.07, 2.81 Hz, 4 H), 4.49 - 4.59 (m, 3 H), 4.02 (br s, 9 H), 3.83 - 3.91
(m, 5 H), 3.75 -
3.80 (m, 3 H), 3.73 (s, 6 H), 3.54 - 3.59 (m, 5 H), 3.48 (br d, J=7.25 Hz, 8
H), 3.24 (br d,
J=5.63 Hz, 9 H), 3.07 (br d, J=13.13 Hz, 4 H), 2.87 - 2.97 (m, 7 H), 2.43 (br
d, J=7.38 Hz, 2
H), 2.30 (br d, J=6.38 Hz, 1 H), 2.16 (br d, J=7.50 Hz, 1 H), 2.09 (s, 9 H),
1.99 (s, 9 H), 1.89
(s, 9 H), 1.77 (s, 9 H), 1.65 (br dd, J=12.76, 6.25 Hz, 3 H), 1.50 - 1.57 (m,
1 H).
Compound 73 could be prepared per the procedure described in compound 74,
except
protected (S)-3-aminopropane-1,2-diol was used instead of the protected (R)-3-
aminopropane-1,2-diol.
Example 12. Preparation of Compound 75
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Scheme 11 below can be used to prepare Compound 75 identified in Example 1
above.
OAc
OAc
Ac0
NHAc 0
0 Ac0
NHA EINI- 0
AcO I
OAc ) 0 OH HND-OH
OAc
Ac0 0ON -
NHAc AGO
OH
IL NV
Ac0 0/N11 o
A GAO. OA -
Actoz.-43N11
Ac NFIAc
II
OAAcO
Ac0
N N
NITAc
0
1CN CN
OAc
N
AGO 0 0
NHAc /1,0
Ac0
AcO
/,) 0-7 -
Ac0 NHAL
compound 75
Scheme 11
Compound II was synthesized based on Scheme 11. Starting from compound I
(compound VI in Scheme 10), coupling with piperidine-4-ol afforded compound
II.
Phosphoramidite Compound 75 was synthesized by treating Compound II with 2-
Cyanoethyl
N,N diisopropylchlorophosphoramidite and a catalytic amount of 1H-tetrazole.
Compound 75 1E1 NMR (400 MHz in DMSO-d6): 6 ppm 8.05 (br d, J = 6.50 Hz, 2 H),
7.81 (br d, J=9.01 Hz, 3 H), 5.22 (d, J=3.25 Hz, 3 H), 4.98 (dd, J=11.26, 3.25
Hz, 3 H), 4.55
(br d, J=8.50 Hz, 3 H), 4.03 (s, 9 H), 3.64 - 3.97 (m, 12 H), 3.55 - 3.63 (m,
6 H), 3.50 (br s, 5
H), 3.40 (br d, J=6.13 Hz, 6 H), 3.17 - 3.30 (m, 9 H), 3.07 (br d, J=14.26 Hz,
4 H), 2.76 (t,
J=5.82 Hz, 2 H), 2.18 -2.47 (m, 6 H), 2.10 (s, 9 H), 1.99 (s, 9 H), 1.89 (s, 9
H), 1.78 (s, 9 H),
1.52- 1.74(m, 6H), 1.12- 1.19(m, 12H). 31P NMR (DMSO-d6): ppm 6 145.25.
Example 13. LinkerBs Attached to Functional Groups Capable of Linking to One
or More
Pharmaceutical Agents
It will be appreciated that various linkerB's of the present disclosure may be
attached
to various functional groups (W) capable of linking to one or more
pharmaceutical agents. In
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particular, linkerB may comprise a diol moiety in which one of the alcohols is
protected as a
DMT ether, while the other alcohol may be linked directly or indirectly to a
solid phase
synthesis solid support material. Upon removal of the DMT group, the free
alcohol is
generated, which can be phosphitylated by reacting with a phosphoramidite to
initiate
oligonucleotide chain growth. Thus, target ligand clusters of the present
disclosure can be
attached at the 3'-end of an oligonucleotide. A non-limiting list of linkerB's
of the present
disclosure that may be attached to the 3'-end of an oligonucleotide includes
the structures
represented below and their stereoisomers:
o rODMI
H 0 0 0
Q.LHir N i OH QrH
N,,,, ,0)..H. ,, .o.
0 k 11
c_ 'XA(--)I-Nr ''''k01-1
L''ODMT o ..0Divn.
o
, ,
,
r,ODIVIT 0
OH
0
0
i 11
0 cli,,N 0
0
k
0 0 ODMT ODMT
, ,
,
0
OH 0¨t OH
0
a 0 0
\11rj--50EXAT Qt1,,,,ry NI75-0DMT N -i-,
ODMT
0 0 0
, ,
,
0
OH
0 OH 0 03H''''''
Q
OOMT
t1...cliN `1),c11L k,ODMT Xill
*-----L
0 'i ODMT
, , ,
0
F
OH 0
irkii;" 'OH
0
0 0
`1).jf--y= .,. 0 ....0DMT ~L,,.:11..p -.0 11111 ODMT
,or ,
where:
j is an integral number between 0 and 12, and
k is an integral number between 0 and 12.
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All target ligand clusters of the present disclosure can also be attached at
the 5'- end
of the oligonucleotide, including but not limited to the 5'- end of the sense
strand in dsRNA,
or the 5' -end of the antisense strand in dsRNA:
Example 14. Preparation of Ligand Cluster Conjugated siRNA
Sense and antisense strand sequences of siRNA were synthesized on
oligonucleotide
synthesizers using a well-established solid phase synthesis method based on
phosphoramidite
chemistry. Oligonucleotide chain propagation is achieved through 4-step
cycles: a
condensation, a capping, an oxidation, and a deprotection step for addition of
each nucleotide.
Syntheses were performed on a solid support made of controlled pore glass
(CPG, 1000 A).
Monomer phosphoramidites were purchased from commercial sources. Ligand
cluster
attached phosphoramidites were synthesized according to the procedures of
Examples 3-12
herein. 5-Ethylthio-1H-tetrazole was used as an activator. 12 in THF/Py/H20
and phenylacetyl
disulfide (PADS) in pyridine/MeCN was used for oxidation and sulfurization
reactions,
respectively. After the final solid phase synthesis step, solid support bound
oligomer was
cleaved and protecting groups were removed by treating with a 1:1 volume
solution of 40 wt. %
methylamine in water and 28% ammonium hydroxide solution. Crude single strand
product
was isolated by lyophilization and purified by ion pairing reversed phase HPLC
(IP-RP-
HPLC). Purified single strand oligonucleotide product from IP-RP-HPLC was
converted to
sodium salt by dissolving in 1.0 M Na0Ac and precipitation by addition of ice
cold Et0H.
Annealing of equimolar complementary sense stand and antisense strand
oligonucleotide in
water was performed to form the double strand siRNA product, which was
lyophilized to
afford a fluffy white solid.
Example 15. In Vivo Evaluation of GalNAc Ligand Cluster Conjugated siRNAs
To evaluate delivery efficacy, GalNAc ligand clusters were conjugated to the
5' end
or 3' end of a sense strand of a known active FXII siRNA from literature.
See Liu et al. "An
investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the
treatment of
hereditary angioedema". RNA. 2019 Feb;25(2):255-263. doi:
10.1261/rna.068916.118.
Sequence and modification information of this FXII siRNA is summarized in
Table 1. Shown
in Table 1 are also six examples of GalNAc ligand clusters conjugated to FXII
siRNA.
Conjugation of GalNAc cluster to 5'-end or 3' end sense strand of FXII siRNA
was
carried out as part of solid phase synthesis outlined in Example 14. Their
structure drawings
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are shown below Table 1. Mass of these six compounds and the positive control
compound is
summarized in Table 2.
These compounds were tested for efficacy of knocking down mouse FXII. FXII is
a
secreted protein mainly produced in hepatocyte. Reduction of FXII expression
in plasma after
.. siRNA treatment correlates strongly with reduction of FXII mRNA in
hepatocyte. Since these
GalNAc ligand clusters are conjugated to the same FXII siRNA with known
activity, delivery
efficacy can be assessed and compared by measuring degree of reduction of FXII
expression
in plasma for each conjugate.
Mice were given a single subcutaneous injection of siRNA compounds (see Table
1
below) at 0.5 or 1 mg/kg or PBS. The literature compound (GalNAc ligand
cluster conjugated
to 3'-end of sense strand) was included in this study as the positive control.
Plasma samples
were collected pre-dosing, and at day 7, 14 and /or 28 post-dosing.
Concentration of mouse
FXII protein was measured by ELISA assay following literature procedure. See
Liu et at. "An
investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the
treatment of
.. hereditary angioedema". RNA. 2019 Feb;25(2):255-263. doi:
10.1261/rna.068916.118).
Knockdown activity was calculated for percent reduction of FXII protein in
mouse plasma
normalized to PBS treated group and is summarized in Table 3. FXII siRNA
conjugated with
GalNAc ligand GLS-1 and GLS-2 showed significant activity knocking down mouse
FXII
protein expression in mouse plasma at both dosages at day 7, 14 and/or 28 post-
dosing. This
activity compares favorably to the positive control. The data confirms GalNAc
ligand clusters
based on diamine scaffold attached to 5'-end of sense strand are highly
efficacious in
delivering siRNA into hepatocyte in vivo.
Table 1. FXII siRNA compounds. Upper case letters: 2'-deoxy-2'-fluoro (2'-F)
ribonucleotide;
lower case letters: 2'-0-methyl (2'-0Me) ribonucleotide; (*) indicates PS
linkage. L96 is
the trivalent GalNAc ligand cluster from literature. (GalNAc3 as in
Jayaprakash, et al.,
(2014) J. Am. Chem. Soc., 136, 16958-16961)
Compound Sense Sequence 5'->3' SEQ ID Antisense Sequence 5'->3'
SEQ ID
NO NO
Positive a*a*cucaauAaAgUgcuuuga*a-L96
control 1 u*U*caaaGcacuUuAuUga 8
AD00127 g*u*u
AD00130 GLS-1-*a*a*cucaauAaAgUgcuuuga*a 2
AD00131 GLS-2-*a*a*cucaauAaAgUgcuuuga*a 3
AD00197 GLS-5-*a*a*cucaauAaAgUgcuuuga*a 4
AD00448 GLS-15-*(Invab)* 5
aacucaauAaAgUgcuuugaa*(Invab)
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AD00449 GLS-15-*a*acucaauAaAgUgcuuuga*a 6
AD00831 a*a*cucaauAaAgUgcuuuga*a-GLS-14 7
Table 2. Mass of FXII siRNA compounds
Calculated Mass Observed Mass
Compound
Sense Strand Antisense Strand Sense Stand Antisense Stand
Positive control 8784.68 8785.31
AD00130 8482.28 6918.66 8482.74 6918.95
AD00131 8350.12 8350.60
AD00197 8307.05 6918.66 8307.84 6918.89
AD00448 8734.265 6918.66 8734.45 6918.76
AD00449 8374.075 6918.66 8374.49 6918.76
AD00831 8364.04 6918.66 8364.70 6918.44
O
HRH :
,0 0 -
NHAc H ii,S
N N ,P
0 \co_
OH 0
HON&...\...9.._\, 0
HO 0,.--.0---..õ0õ--NN N---,
NHAc H /ND
HO\ OH HN
_________________ ¨ 0 ¨
NHAc GLS-1
OH
0
\
NHAc NI-1 0
0
H
----,,,r
0 0
0
HOOH
0
i-ion
_o Nz N /
NHAc x
H
HO\ eC/F1
/0
HO----\O, HN
\ 0)
NHAc GLS-2
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OH
HO L
NHAca,,,,.--,
0
OH L i
S-
HO\&\!..,.., 0
HO 00 7
NHAc
HO /OH 14 /c
Hr
NHAc 0
GLS-5
OH
H0\43,\...õ
HO 0,0,.,.."õõ..õ
NHAc NH 00 0
TN)t .õ-----}-,,,,-----1 0
OH VS-4
H0
.,;(4,
0
NHAc
rn
HO OH )
HN
H 0 H
'-'"0-,,,,,)
NHAc GLS-15
5
OH
HO._:).\
HO 0,,.....õ,----,
0-...,_,------,
NHAc NH 0
0 0
OH
N
OH FN1 :i-
HO.__\...__. P 0
O's1 -
0 0
HO C)0 )-NN
NHAc N
HO /:)F1 H "D
HO\t_470 HN
0
NHAc
GLS-14
Table 3. Percent reduction of FXII protein in mouse plasma normalized to PBS
treated group.
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Compound Dosage Day 7 Day 14 Day 28
ID
(mg/kg) Knockdown STD Knockdown STD Knockdown STD
Positive
control 1 74% 0.011 81% 0.011 67%
0.011
AD00127
0.5 67% 0.044 62% 0.041 45% 0.041
AD00130 1 82% 0.030 82% 0.016 71%
0.016
0.5 61% 0.075 72% 0.034 51% 0.067
AD00131 1 84% 0.048 80% 0.045 66%
0.020
Example 16. In Vivo Evaluation of GalNAc Ligand Cluster Conjugated siRNAs
Mice were given a single subcutaneous injection of siRNA compounds at 1 mg/kg
or
PBS. Plasma samples were collected at day 14 post-dosing. Concentration of
mouse FXII
protein was measured by ELISA assay following literature procedure. See Liu et
al. "An
investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the
treatment of
hereditary angioedema". RNA. 2019 Feb;25(2):255-263. doi:
10.1261/rna.068916.118).
Knockdown activity was calculated for percent reduction of FXII protein in
mouse plasma
normalized to PBS treated group and is summarized in Table 4. FXII siRNA
conjugated with
GalNAc ligand GLS-5 and GLS-15 showed significant activity knocking down mouse
FXII
protein expression in mouse plasma. The data confirms GalNAc ligand clusters
based on
diamine scaffold attached to 5'-end of sense strand are highly efficacious in
delivering
siRNA into hepatocyte in vivo.
there also found that when linkerB contains a six-membered ring fragment, when
it is
used as targeted delivery of pharmaceutical agents, especially a 4-
Hydroxypiperidinyl group,
such as the compoundAD00448. AD00449,
it shows better in vivo stability and activity.
Table 4. Percent reduction of FXII protein in mouse plasma normalized to PBS
treated group.
Compound Dosage Day 14
ID (mg/kg) Knockdown
STD
AD00197 1 83.1% 0.015
AD00448 1 86.0% 0.013
AD00449 1 80.0% 0.031
Example 17. In Vivo Evaluation of GalNAc Ligand Cluster Conjugated siRNAs
AD00831
Mice were given a single subcutaneous injection of siRNA compounds at 2 mg/kg
or
PBS. Plasma samples were collected at day 7 and 14 post-dosing. Concentration
of mouse
FXII protein was measured by ELISA assay following literature procedure. See
Liu et al. "An
investigational RNAi therapeutic targeting Factor XII (ALN-F12) for the
treatment of
hereditary angioedema". RNA. 2019 Feb;25(2):255-263. doi:
10.1261/rna.068916.118).
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Knockdown activity was calculated for percent reduction of FXII protein in
mouse plasma
normalized to PBS treated group. The percent of knockdown at days 7 and 14
post dosing
was 87% and 88%. The data confirms GalNAc ligand clusters based on diamine
scaffold
attached to 3'-end of sense strand are highly efficacious in delivering siRNA
into hepatocyte
in vivo.
Example 18
In Vivo testing of ANGPTL3 siRNA Duplexes
At 14 days before dosing of siRNAs, female C57BL/6J mice were infected by
intravenous administration of a solution of adeno-associated virus 8 (AAV8)
vector encoding
human ANGPTL3 and luciferase gene. At day 0, mice were subcutaneously
administered a
single dose of AD00112-2 (Table 5. ) at 1, 3 or 10 mg/kg or PBS. Blood samples
were
collected at day 0, before dosing of siRNA and at day 7, at termination. Serum
samples were
isolated and luciferase activity of serum samples was measured per
manufacturer' s
recommended protocol. Since expression of human ANGPTL3 level correlates with
expression level of luciferase, measurement of luciferase activity is the
surrogate for
measuring ANGTPL3 expression. Percent remaining of luciferase activity was
calculated by
comparing luciferase activity in samples from pre- (day 0) and post (day 7)
treatment of
siRNA for each mouse and normalized by the change of luciferase activity in
the samples
from the control treated mice during the same period of time. Result is
summarized in Table
6. AD00112-2 demonstrated dose dependent activity suppressing expression of
ANGPTL3,
which confirms again GalNAc ligand clusters based on diamine scaffold are
highly
efficacious in delivering siRNA into hepatocyte in vivo.
Table 5. ANGPTL3 siRNA compounds. Upper case letters: 2 -deoxy-2 -fluoro
(2 -F) ribonucleotide; lower case letters: 2 -0-methyl (2 -0Me)
ribonucleotide; (*)
indicates PS linkage.
Compound Sense Sequence 5'->3' SEQ Antisense Sequence 5'->3'
SEQ
ID NO ID NO
AD00112 (GLS-15)*(Invab)* 9 10
-2 gaauggaaGgUuAuacucuaa*(In u*U*agagUauaaCcUuCcau
vab) * *
u c
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Table 6 provides experimental results of in vivo studies (percent reduction of
luciferase
activity). The duplex sequence and modification of AD00112-2 is shown in Table
5.
Dose Day 7, relative to PBS
Duplex AD#
(mg/kg) (Mean SD)
AD00112-2 1 0.33 0.06
AD00112-2 3 0.20 0.09
AD00112-2 10 0.11 0.03
Equivalents
Although several embodiments of the present invention have been described and
illustrated herein, those of ordinary skill in the art will readily envision a
variety of other
means and/or structures for performing the functions and/or obtaining the
results and/or one
or more of the advantages described herein, and each of such variations and/or
modifications
is deemed to be within the scope of the present invention. More generally,
those skilled in the
art will readily appreciate that all parameters, dimensions, materials, and
configurations
described herein are meant to be illustrative and that the actual parameters,
dimensions,
materials, and/or configurations will depend upon the specific application or
applications for
which the teachings of the present invention is/are used. Those skilled in the
art will
recognize, or be able to ascertain using no more than routine experimentation,
many
equivalents to the specific embodiments of the invention described herein. It
is, therefore, to
be understood that the foregoing embodiments are presented by way of example
only and that,
within the scope of the appended claims and equivalents thereto, the invention
may be
practiced otherwise than as specifically described and claimed. The present
invention is
directed to each individual feature, system, article, material, and/or method
described herein.
In addition, any combination of two or more such features, systems, articles,
materials, and/or
methods, if such features, systems, articles, materials, and/or methods are
not mutually
inconsistent, is included within the scope of the present invention.
All definitions, as defined and used herein, should be understood to control
over
dictionary definitions, definitions in documents incorporated by reference,
and/or ordinary
meanings of the defined terms.
The indefinite articles "a" and "an," as used herein in the specification and
in the
claims, unless clearly indicated to the contrary, should be understood to mean
"at least one."
The phrase "and/or," as used herein in the specification and in the claims,
should be
understood to mean "either or both" of the elements so conjoined, i.e.,
elements that are
conjunctively present in some cases and disjunctively present in other cases.
Other elements
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may optionally be present other than the elements specifically identified by
the "and/or"
clause, whether related or unrelated to those elements specifically
identified, unless clearly
indicated to the contrary.
All references, patents and patent applications, and publications that are
cited or
referred to in this application are incorporated by reference herein in their
entirety.
156
