Property-Driven Design and Development of Lipids for Efficient Delivery of siRNA

Article abstract of DOI:10.1021/acs.jmedchem.0c01407

Ionizable cationic lipids are critical components involved in nanoparticle formulations, which are utilized in delivery platforms for RNA therapeutics. While general criteria regarding lipophilicity and measured pKa in formulation are understood to have impacts on utility in vivo, greater granularity with respect to the impacts of the structure on calculated and measured physicochemical parameters and the subsequent performance of those ionizable cationic lipids in in vivo studies would be beneficial. Herein, we describe structural alterations made within a lipid class exemplified by 4, which allow us to tune calculated and measured physicochemical parameters for improved performance, resulting in substantial improvements versus the state of the art at the outset of these studies, resulting in good in vivo activity within a range of measured basicity (pKa = 6.0-6.6) and lipophilicity (cLogD = 10-14).

Full text of DOI:10.1021/acs.jmedchem.0c01407

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Property-Driven Design and Development of Lipids for Efficient
Delivery of siRNA
Kumar Rajappan,* Steven P. Tanis,* Rajesh Mukthavaram, Scott Roberts, Michelle Nguyen,
Kiyoshi Tachikawa, Amit Sagi, Marciano Sablad, Pattraranee Limphong, Angel Leu, Hailong Yu,
Padmanabh Chivukula, Joseph E. Payne, and Priya Karmali
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ABSTRACT: Ionizable cationic lipids are critical components involved in
nanoparticle formulations, which are utilized in delivery platforms for RNA
therapeutics. While general criteria regarding lipophilicity and measured pKa in
formulation are understood to have impacts on utility in vivo, greater
granularity with respect to the impacts of the structure on calculated and
measured physicochemical parameters and the subsequent performance of
those ionizable cationic lipids in in vivo studies would be beneficial. Herein, we
describe structural alterations made within a lipid class exemplified by 4, which
allow us to tune calculated and measured physicochemical parameters for
improved performance, resulting in substantial improvements versus the state
of the art at the outset of these studies, resulting in good in vivo activity within a range of measured basicity (pKa = 6.0−6.6) and
lipophilicity (cLogD = 10−14).
propane (DLin-DMA)12−14 1, DLin-MC3-DMA 2 (MC3),5
INTRODUCTION
■
and 3 (L319) (Table 1).15 Lipids 1−3 have been reported to
The recent approval of the first ever lipid nanoparticle-
potently knock down the factor VII (FVII) expression, an
encapsulated siRNA, patisiran (ONPATTRO), against trans-
siRNA paradigm; however, these lipids have not all been
thyretin amyloidosis,1,2 and continuing advances in RNA-based
reported to demonstrate comparable activity in an mRNA
technologies such as CRISPR gene editing, mRNA therapeu-
setting. Lipid 2 (MC3) has been reported to encapsulate and
tics, and vaccines have generated intense interest in safe and
deliver siRNA (patisiran, ONPATTRO) and has also been
effective nonviral vectors for drug delivery. Lipid-promoted
associated with the delivery of mRNA.6 While the above lipids
nanoparticle formulations have shown great promise as a
are considered to be ionizable, meaning that they are charge-
powerful delivery platform for small RNA (siRNA, miRNA,
neutral under plasma pH and they acquire progressively more
anti-miRNA, etc.) therapeutics3−5 in general and mRNA
cationic character in various endosomal compartments after
medicines in particular.6−8 While a small oligonucleotide of
the associated nanoparticles are endocytosed by low-density
<30 nucleotides is amenable for and proven with alternate cell
lipoprotein receptor (LDL-R), further improvements or
selective delivery approaches, such as through ligand-directed
chemical modifications are needed to have lipids that can be
receptor-mediated endocytosis,9−11 a large oligonucleotide
utilized for therapeutic applications of both small RNAs and
with >1000 nucleotides in length such as an mRNA has
mRNAs. Also, a major drawback of many lipids is their
limited delivery options. The stability of RNA outside its
propensity for accumulation in tissues after repeated dosing
cellular environment is a major stumbling block in the
due to insufficient biodegradability. Thus, apart from the safe
development of RNA-based medicines. Hence, a primary
and efficacious delivery of RNAs to the appropriate tissues,
goal of any delivery platform involving RNA is the protection
cells, and ultimately, intracellular compartments where either
of it from plasma nucleases while it is in circulation. An equally
an RNAi may be effected, an mRNA may be introduced for
important consideration is the delivery of the RNA into the
translating into a therapeutic protein, or a gene may be edited,
cytoplasm of a cell where the intended biological outcome
must be realized. Both considerations can be satisfied, to some
extent, if RNA can be encapsulated in a lipid nanoparticle
Received: August 11, 2020
(LNP). Currently, LNP is a clinically validated platform for the
delivery of small RNAs and an emerging one for systemic
delivery of mRNA therapeutics. Several LNPs with various
siRNA cargoes have undergone clinical trials. Most notable
ones are LNPs based on 1,2-dilinoleyloxy-3-dimethylamino-
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Table 1. Alnylam Lipid Acid Chain Length Study
Figure 1. While many of the lipids used for RNA applications are ionizable in nature, further improvements or chemical modifications are
warranted to create lipids that can be utilized for therapeutic applications of RNAs. It was our desire to initiate a systematic study of the impact of
lipid structure on the calculated physicochemical properties (cLogD and c-pKa) and measured (formulated) pKa of the lipids designed for LNP-
facilitated RNA therapeutic applications to design lipids that could function well in both the siRNA and mRNA therapeutic modalities. We planned
to examine the impact of lipophilic chain length, number of chains and their relative orientations, and the nature of ester-forming alcohol to realize
the desired/required LNP performance/biodegradability. Thus, we embarked upon lipid structure−activity relationship (SAR) studies to optimize
performance in a biological system.
the terminal fate of the components of the LNP is an
important consideration for successful therapeutic develop-
ment. These considerations have prompted the incorporation
of biodegradable chemical functionalities within the general
chemical motif of traditional lipids of lipid nanoparticle
formulations.15 As shown in Figure 1, the lipids exhibit two
basic structural motifs: DLin-DMA 112−14 has the lipophilic
moiety connectivity via stable ether linkages and DLin-MC3-
DMA 25 exhibits a basic 4-dimethylaminobutyrate head group
annealed to a large lipophilic tail via a secondary ester linkage,
while L319 (Table 1)15 has an identical (see 2) basic, 4-
dimethylaminobutyrate head group attached via a secondary
ester linkage to a lipophilic 9-hydroxyheptadecanedioc acid tail,
which also presents cleavable primary-ester linkages in the
form of (Z)-2-nonenol esters. The ether linkages of 1 are not
readily cleaved hydrolytically; thus, oxidative/hepatic metab-
olism is likely needed to degrade 1. By comparison, the
secondary ester moiety of 2 could undergo hydrolysis through
various esterases, leaving a large lipophilic core to be degraded
oxidatively. L319 (Table 1) presents two sets of ester functions
as esterase targets with the largest residual entity to be cleared
in the form of 9-hydroxyheptadecanedioc acid.
single entity bearing the basic nitrogen of the ionizable lipid.
Ester linkages were selected as the potential biodegradable
entities that were to be resident on the lipophilic moieties and
the basic nitrogen was to be introduced onto the lynchpin-N
through a readily assembled, robust, thiocarbamate unit, as
shown in 4. The assembly of a structure such as 4 would allow
diversity in acid chain length (m = n or m ≠ n), ready
alteration of the ester-forming alcohol (R1 = R2 or R1 ≠ R2),
the introduction of linear, primary alcohols and branched,
secondary alcohols during ester formation, and alteration of
the basicity of the nitrogen head group by changing the
distance between S and N as well as changing nitrogen
substitution (R3/R4). Further, we anticipated that the use of a
lynchpin-N would facilitate the syntheses of lipids 4.
FVII siRNA knockdown was selected as an in vivo primary
screen to determine the appropriateness of lipid design and to
serve as a proxy for mRNA delivery to hepatocytes. Lipid
design notions were initiated using a central nitrogen as a
lynchpin for the attachment of two lipophilic moieties and a
An early study of the impact of acid chain length on
measured pKa and FVII knockdown has been reported by
Alnylam.15a) Table 1 presents the lipids in this study and the
associated measured pKa and FVII knockdown information.
There is a progression in the decrease of measured pKa from
B
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Figure 2. Synthesis of symmetrically esterified lipids 10. Conditions: (a) PhCH2NH2, K2CO3, CH3CN, 80 °C; (b) H2 (70 psi), BOC2O, 10% Pd/
C, EtOH; (c) NaOH, THF, MeOH, H2O; (d) R1OH, EDC-HCl, DMAP, CH2Cl2; (e) CF3CO2H, CH2Cl2, 0 °C to RT; (f) i. triphosgene, CH2Cl2,
pyridine; ii. amino-thiol, pyridine.
the 7.0 reported for entry 1 where the acid chain length is 4 to
pKa = 6.26 for entry 4 with a C-10 acid chain length. The
overall lipid tail length is kept roughly the same by decreasing
the size of the Z-2-alkenol used to form the ester. The optimal
pKa with respect to the FVII ED50 (mg/kg) is found in the
RESULTS AND DISCUSSION
■
The data of Table 1, for L319, prompted us to initiate our
study utilizing the symmetrical octanoate chain length of L319
(Table 1, m = n = 6) and a symmetrical linear diester (R1 = R2)
where the alcohol fragment was the same (Z)-2-nonenol
employed in 3. The first construct plan was completed by
selecting an S-to-N chain length of two carbons (o = 1) and a
symmetrical R3 = R4 = CH3. This will be followed by altering
R3 = R4 to CH2CH3, and then S-to-N chain length could be
altered to three carbons (o = 2) with the symmetrical R3 = R4
= CH3 and R3 = R4 = CH2CH3. All subsequent lipid syntheses
could be conducted as outlined in Figure 2.
form of L319 (Table 1) with a pKa = 6.38 and a FVII ED50
<
0.01 mg/kg. These data have been reported15b) to correspond
to a 90% knockdown at 0.1 mg/kg and a 97% knockdown at
0.3 mg/kg. L319 was found to be eliminated from plasma,
falling to below the level of quantification (0.8 pmol/mL), at 8
h post dose.
For our planned study, we intended to examine the impact
of acid chain length, starting from that exhibited by L319:
octanoate (m = n = 6) decreasing systematically to butanoate
(m = n = 2) and then to acetate (m = n = 0). The data of Table
1 (viz L354 and L356) suggest some issues associated with
chain shortening; however, the small data set argues for a more
thorough examination. Toward that end, diversity in
esterification was envisioned to utilize linear alcohols ((Z)-2-
nonenol) as well as symmetrically branched alcohols (5-
nonanol, 6-undecanol, 7-tridecanol, 8-pentadecanol, and 9-
heptadecanol) for ester formation with R1 = R2 or R1 ≠ R2.
Nitrogen would be altered with respect to calculated basicity
by restricting o = 1, 2 and R3 = R4 = CH3, CH2CH3. Syntheses
of the targeted entities were expected to be facilitated by the
symmetrical chain lengths of the acid moieties with R1 = R2 for
the esters being introduced via esterification of a diacid post-
nitrogen alkylation or through nitrogen alkylation with an
omega-bromo-ester. Differentially esterified entities (R1 ≠ R2)
could be pursued either through sequential esterification of a
diacid with the selected alcohols or through nitrogen alkylation
with differentially esterified omega-bromo-esters. We em-
barked upon this study with a poorly developed understanding
of the interplay between lipid lipophilicity as measured by c-
pKa and desired measured pKa; however, the measured pKa
range of 6.20−6.50 had been described as optimal.5 Herein, we
report the development of an SAR understanding that led to
the evolution of more optimized lipid designs and a platform
approach, which was called LUNAR, for encapsulation and
delivery of small RNAs, with plans for future utilization with
large RNAs such as mRNAs.
In the event, commercially available methyl-8-bromo-
octanoate and benzyl amine were combined to afford benzylic
amine 5a, which was utilized crude. Hydrogenolysis (10% Pd/
C, 70 psi H2) in the presence of BOC-anhydride led to
protected diester 6a in 81% yield after purification. Bis-ester
hydrolysis (NaOH, THF, and aq. MeOH) provided crude di-
acid 7a, which underwent bis-esterification with (Z)-2-nonenol
(EDC-HCl and DMAP) to give BOC-blocked diester 8a in
72% yield. The synthesis was completed by BOC-removal
(TFA and CH2Cl2) yielding crude amino-diester 9a which was
converted to an intermediate carbamyl chloride with
triphosgene (CH2Cl2, pyridine), and reaction of the carbamyl
chloride with commercially available 2-(dimethylamino)-
ethanethiol-HCl in pyridine to yield target lipid 10a in 79%
yield over the last 2 steps.
Additional symmetrical octanoate lipids of Table 2 were
prepared from 9a by replacing 2-(dimethylamino)-ethanethiol-
Table 2. Data for Symmetrically Esterified Lipids 10 and
Intermediates 5−9
compound
m
R1OH = R2OH
(CH2)o
R3
yield (%)
5a
6a
7a
8a
6
6
6
6
6
6
6
6
6
crude
81
crude
72
crude
79
71
H
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
9a
10a
10b
10c
10d
1
1
2
2
CH3
CH2CH3
CH3
74
78
CH2CH3
C
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Figure 3. Synthesis of nonsymmetrically esterified octanoate-derived lipids 15.
HCl with 2-(diethylamino)-ethanethiol-HCl to provide 10b
(71%), 3-(dimethylamino)-propanethiol-HCl16 to yield 10c
(74%), and 3-(diethylamino)-propanethiol-HCl17 to give 10d.
With the octanoate-derived bis-esters, utilizing (Z)-2-
nonenol as the alcohol, we turned our attention to the
preparation of octanoate-derived lipids containing one linear
alcohol-derived ester ((Z)-2-nonenol) and one branched chain
alcohol-derived ester. As outlined above (vide supra), we
planned to incorporate the symmetrically branched chain
alcohols, 5-nonanol, 6-undecanol, 7-tridecanol, 8-pentadeca-
nol, and 9-heptadecanol, for this effort. Two pathways of
synthesis were considered: The first was the construction of
differentially esterified versions of 8-bromo-octanoic acid and
8-BOC-amnio-octanoic acid and the alkylative addition of the
bromoester to an 8-amino-octanoate ester. The second
approach called for a selective mono-esterification of a
symmetrical diacid18 such as 8 (m = n = 6; R1 = R2 = H)
followed by esterification with alcohol #2. The former of these
approaches was investigated first as shown in Figure 3.
Commercially available BOC-blocked amino acid 11 was
esterified with 5-nonanol to provide ester 12a (p = 3, Table 3),
which was utilized without purification. BOC removal (TFA
and CH2Cl2) afforded amino ester 13a (p = 3, 48% over two
steps), which was then coupled with 8-bromo-octanoic acid Z-
2-nonenylester,19 leading to amine 14a (p = 3) in a modest
28% yield. The target lipid was realized after 14a was
converted to an intermediate carbamyl chloride with
triphosgene (CH2Cl2 and pyridine) and carbamyl chloride
was reacted with commercially available 2-(dimethylamino)-
ethanethiol-HCl in pyridine to yield target lipid 15a in 46%
yield. Replacing 5-nonanol with 6-undecanol in the ester-
ification of 11 leads to 12b, deprotection yields 13b (56%),
alkylation with 8-bromo-octanoic acid Z-2-nonenylester affords
14b (22%), and conversion to the target thiocarbamate gives
target lipid 15b (70%, 8.6% overall). Similarly, the use of 7-
tridecanol, 8-pentadecanol, and 9-heptadecanol leads to lipids
15c (6.7% overall), 15d (12% overall), and 15e (6.7% overall).
A consistent issue associated with the chemistry of Table 3 is
the poor yield of the alkylative step, leading to 14a−14e.
Future endeavors will examine the selective mono-esterifica-
Table 3. Synthesis of Nonsymmetrically Esterified
Octanoate-Derived Lipids 15
compound
(CH2)p
(CH2)o
R3
yield (%)
12a
13a
14a
15a
12b
13b
14b
15b
12c
13c
14c
15c
12d
13d
14d
15d
12e
13e
14e
15e
3
3
3
3
4
4
4
4
5
5
5
5
6
6
6
6
7
7
7
7
crude
49
28
69
crude
56
22
70
crude
51
18
73
crude
49
34
72
crude
51
17
1
1
1
1
1
CH3
CH3
CH3
CH3
CH3
77
tion of a diacid as a paradigm to avoid the poor alkylation
yields.
With the lipids of Table 2 (10a−10d) and Table 3 (15a−
15e) available, we wished to examine the impact of the
structural changes represented in the lipids on the calculated
and measured physicochemical properties, the ability to
formulate into a nanoparticle-encapsulating RNA, nanoparticle
size, and the in vivo activity in the FVII knockdown assay.
The data of Table 4 include cLogD20 and calculated pKa,20
as well as measured pKa (in formulation), a Δ pKa value that
indicates the difference between calculated and measured pKa
values, measured nanoparticle size, and in vivo activity in the
FVII knockdown assay. Calculated LogD values (cLogD) range
from a low value of 11.01 for 10c (calculated pKa = 9.35) to a
high value of 16.08, associated with 15e, which exhibits the
D
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Table 4. Impact of Structure on Physicochemical Properties and In Vivo Activity in the FVII Knockdown Assay
large branched heptadecanol moiety and a modestly basic (c-
pKa = 8.68) amine. All of the lipids of Table 4 formulated well-
affording nanoparticle sizes ranging from 73 nm (15b) to 84
nm (10d), enabling us to measure basicity (pKa) in
formulation using the TNS assay and to test them in vivo in
the FVII knockdown assay. The aforementioned paradigms to
alter basicity (vide supra), increasing the distance between the
heteroatoms of the head group and altering the nature of the
nitrogen substituents, are apparent in the lipids of Table 4.
Lipid 10a, with a 2-C spacer between S and N, has a calculated
pKa (c-pKa) of 8.68, while the 3-C spacer of lipid 10c has a c-
pKa = 9.35. The measured pKa values for lipids 10a and 10c,
6.03 and 6.77, respectively, then lead to a Δ pKa value, the
difference between the calculated and measured pKa values of a
consistent 2.65/2.58. These Δ values suggest that lipids afford
an expected increase in basicity with the structural change, with
the basic nitrogen in a similar environment. The anticipated
change in basicity wrought by changing the substitution on
nitrogen from methyl to ethyl results in a very different
outcome/analysis. Lipid 10b (2-C spacer, N-ethyl) has a c-pKa
= 9.62, a measured pKa = 5.50 and a Δ pKa = 4.12. Lipid 10d
(3-C spacer, N-ethyl) exhibits a c-pKa = 10.20, a measured pKa
= 6.60, and a Δ pKa = 3.60. These two lipids exhibit more than
a 1 pKa unit greater deviation between calculated and
measured pKa values when compared to lipids 10a and 10c,
suggesting that the change from N-methyl to N-ethyl
substitution, which increases lipophilicity at the basic terminus
as well as impacting basicity with this substitution, has resulted
in an environmental alteration, leading to a lower measured
pKa than might have been anticipated. The ability to
manipulate the measured pKa in this fashion allows us some
latitude in design to alter the ultimate measured basicity in
formulation to achieve a desired target value. The remaining
lipids of Table 4 (15a−15e) restrict the S-to-N spacer to two
atoms and the substitution of nitrogen to N-methyl while
introducing a single branched chain alcohol to form an ester to
E
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Figure 4. Syntheses of butanoate- and acetate-based lipids 10. Conditions: (a) PhCH2NH2, K2CO3, CH3CN, 80 °C; (b) H2 (70 psi), BOC2O, 10%
Pd/C, EtOH; (c) NaOH, THF, MeOH, H2O; (d) i. R1OH, EDC-HCl, i-Pr2NEt, DMAP, CH2Cl2; ii. R2OH, EDC-HCl, i-Pr2NEt, DMAP, CH2Cl2;
(e) CF3CO2H, CH2Cl2; (f) i. triphosgene, CH2Cl2, pyridine; ii. amino-thiol, pyridine.
determine if there is an impact of increasing branched alcohol
size on measured parameters. Given the general target
measured pKa in formulation of ca. 6.0 and the observed
impact of measured pKa on FVII knockdown, the data of Table
4 provide little granularity with respect to the impact of
structural modification upon pKa and FVII knockdown until
one adds N-ethyl substituents to the 2-C S-to-N spacer of 10b
(measured pKa = 5.50; FVII knockdown, 14%) and a large
branched substituent (9-heptadecanol) to the C-8 acid in the
form of 15e (measured pKa = 5.59; FVII knockdown, 40%).
The remaining lipids of Table 4 exhibit measured pKa values
ranging from 6.77 (10c) to 5.79 (15c) with FVII knockdown
observed in the 60−80% range.
amine (K2CO3 and CH3CN, 80 °C) to give 5b (73%, Table
5). Hydrogenolysis (H2 and Pd/C) in the presence of BOC-
anhydride led to di-ester 6b (95%), which then afforded the
target butanoate precursor diacid 7b (aq. NaOH, MeOH, and
THF, 89%). The precursor to all butanoate-derived, non-
symmetric diesters 8b is then prepared by mono-esterification
with (Z)-2-nonenol (EDC-HCl, i-Pr2NEt, and DMAP, 53%).
A second esterification with 7-tridecanol (EDC-HCl, i-Pr2NEt,
and DMAP) then converts 8b to 8c (71%), which yields amine
9b (78%) after BOC removal (TFA and CH2Cl2). The initial
nonsymmetrically esterified butanoate-based lipid 10e is then
prepared by formation of the carbamyl chloride with
triphosgene followed by its reaction with 2-(dimethylamino)-
ethanethiol-HCl in pyridine to provide 10e in 69% yield. The
remaining nonsymmetrically esterified, butanoate-based lipids
of Table 5 (10f−10i) are prepared in a similar fashion. The
synthesis of the first symmetrically esterified, bis-butanoate
lipid 10j proceeds in a similar fashion with the reaction of 7b
with 6-undecanol (EDC-HCl, i-Pr2NEt, and DMAP), giving 8f
(48%). BOC removal (TFA and CH2Cl2) provides 9e (67%),
and the reaction of 9e first with triphosgene and then with 2-
(dimethylamino)-ethanethiol-HCl in pyridine yields 10j
(71%). The remaining bis-butanoate-based symmetrically
esterified lipids (10k, 10m, 10n, 10o, 10p, and 10q) are
synthesized in a similar fashion. The syntheses of the
nonsymmetrically esterified, bis-acetate-based lipids (10r−
10v), and the symmetrically esterified bi-acetate-derived lipids
(10w−10z and 10aa) parallels the bis-butanoate methods
discussed above, and these lipids were derived from
commercially available 2-[tert-butoxycarbonyl-
(carboxymethyl)amino]acetic acid.
Within the bis-butanoic acid manifold of lipids, our initial
study would again (vide supra) examine the impact of the size
of a branched chain alcohol utilized for esterification in a
disymmetrically esterified molecule where (Z)-2-nonenol was
introduced as the linear component (Table 6). The five
disymmetric lipids of Table 6, 10e−10i, have had 7-tridecanol,
8-pentadecnol, 8-pentadecanol, 9-heptadecanol, and 9-hepta-
decanol added as their branched components, respectively.
The result is that 10e−10i span a range of lipophilicities from a
level far below that seen in Table 4 (10e cLogD = 9.89 through
10h and 10i cLogD = 11.93). The impact of lower lipophilicity
is first seen upon formulation where 10e (cLogD = 9.89)
affords a larger nanoparticle size (93 nM) when compared to
As we move forward to the symmetrical butanoate versions
of 10 (m = n = 2; R1 = R2) and acetate versions of 10 (m = n =
0; R1 = R2), as well as the nonsymmetrical butanoate 10 (m =
n = 2; R1 ≠ R2) and acetate versions of 10 (m = n = 0; R1 ≠
R2), we will restrict our designs to entities wherein the ester
moieties include R1 = linear ((Z)-2-nonenol) and R2
branched (increasing in size from 7-tridecanol, 8-pentadecanol
to 9-heptadecanol) and the situation wherein R1 = R2
=
=
branched (increasing in size from 6-undecanol, 7-tridecanol, 8-
pentadecanol to 9-heptadecanol). These choices were derived
from the data of Table 1, wherein little physicochemical impact
was observed for bis-linear esters regardless of acid chain
length, and Table 4 where, in the bis-octanoate chain length,
physicochemical impact was observed in the guise of 15e, with
a single large branched ester moiety held at the octanoate
chain length. Based on the data of Table 4, it is likely that we
will observe a large environmental impact on the measured pKa
at m = n = 2 as the size of the branch increases and an even
greater impact in the shorter acetate series (m = n = 0).
While the syntheses of the symmetrically esterified
butanoate and acetate congeners could be carried out as
shown in Figure 2, the poor yields obtained for the syntheses
of the differentially esterified lipids exemplified in Figure 2 and
Table 3 caused us to adopt a modified synthetic protocol.
Utilizing the same di-acid that would afford access to a
symmetrically esterified target lipid, we envisioned a selective
mono-esterification18 followed by the addition of the second,
different alcohol as a better route to the desired target
nonsymmetrically esterified lipids. The syntheses of the
aforementioned butanoate and acetate congeners are outlined
in Figure 4. Methyl-4-bromo-butyrate was reacted with benzyl
F
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Table 5. Synthesis of Butanoate- and Acetate-Based Lipids
compound
m
R1OH
R2OH
(CH2)o
R3
yield (%)
5b
6b
7b
8b
8c
9b
10e
8d
9c
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
73
95
89
53
71
78
69
68
79
74
71
67
79
72
67
48
67
71
70
69
68
47
66
60
76
75
77
73
29
57
82
58
55
74
63
56
84
55
61
59
54
75
66
57
58
66
68
60
60
76
64
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
6-undecanol
6-undecanol
6-undecanol
6-undecanol
6-undecanol
7-tridecanol
7-tridecanol
7-tridecanol
8-pentadecanol
8-pentadecanol
8-pentadecanol
8-pentadecanol
8-pentadecanol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
(Z)-2-nonenol
6-undecanol
6-undecanol
6-undecanol
6-undecanol
8-pentadecanol
8-pentadecanol
8-pentadecanol
8-pentadecanol
9-heptadecanol
9-heptadecanol
9-heptadecanol
H
7-tridecanol
7-tridecanol
7-tridecanol
1
CH3
8-pentadecanol
8-pentadecanol
8-pentadecanol
8-pentadecanol
9-heptadecanol
9-heptadecanol
9-heptadecanol
9-heptadecanol
6-undecanol
6-undecanol
6-undecanol
6-undecanol
6-undecanol
7-tridecanol
7-tridecanol
7-tridecanol
8-pentadecanol
8-pentadecanol
8-pentadecanol
8-pentadecanol
8-pentadecanol
H
10f
10g
8e
1
2
CH3
CH2CH3
9d
10h
10i
8f
1
2
CH3
CH2CH3
9e
10j
10k
10m
8g
9f
10n
8h
1
2
2
CH3
CH3
CH2CH3
2
CH3
9g
10o
10p
10q
8i
8j
9h
10r
8k
9i
10s
8m
9j
10t
10u
10v
8n
1
2
2
CH3
CH3
CH2CH3
6-undecanol
6-undecanol
6-undecanol
1
2
CH3
CH3
8-pentadecanol
8-pentadecanol
8-pentadecanol
9-heptadecanol
9-heptadecanol
9-heptadecanol
9-heptadecanol
9-heptadecanol
6-undecanol
6-undecanol
6-undecanol
6-undecanol
8-pentadecanol
8-pentadecanol
8-pentadecanol
8-pentadecanol
9-heptadecanol
9-heptadecanol
9-heptadecanol
1
2
2
CH3
CH3
CH2CH3
9k
10w
10x
8o
9m
10y
10z
8p
2
2
CH3
CH2CH3
2
2
CH3
CH2CH3
9n
10aa
1
CH3
those associated with 10f−10i (70−78 nM). Lipids 10e, 10f,
and 10h have the 2-C spacing between S and N as well as N-
methyl substitution. As was the case with the lipids of Table 4,
this combination, with c-pKa = 8.68, led to measured pKa
values in the TNS assay of 10e pKa = 6.05, 10f pKa = 6.04, and
10h pKa = 5.96, with Δ pKa values for these three lipids of
2.63, 2.64, and 2.72, respectively. The introduction of the 3-C
spacing between S and N along with N-ethyl substituents in
the guise of lipids 10g and 10i results in a similar increase in
calculated pKa to 10.20, as seen in Table 4, and measured pKa
values of 6.66 for 10g and 6.58 for 10i. The environmental
impact of this added lipophilicity at the basic terminus of the
lipid in the shorter butanoate-derived lipids mirrors that
reported in Table 4, resulting in Δ pKa values of 3.54 and 3.62
for 10g and 10i, respectively. In this small series of
nonsymmetrically esterified lipids, an increase in the size of
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Table 6. Further Study of the Impact of Structure on Physicochemical Properties and In Vivo Activity in the FVII Knockdown
Assay
the single branched component did not result in a significant
impact on the measured pKa. All lipids in the 10e−10i
subseries are sufficiently basic so as to expect a sizeable in vivo
effect in the FVII knockdown assay. Lipids 10f−10i afford
FVII knockdown values of 82, 96, 80, and 75%, respectively;
however, the less lipophilic lipid 10e (cLogD = 9.89, pKa =
6.05) performs poorly in the FVII knockdown assay (13%),
suggesting the need for cLogD >10.00 for good in vivo activity
in addition to the pKa requirement (ca. 6.00 or greater).
The symmetrically bis-branched alcohol-based butanoate
lipids 10j through 10q utilize 6-undecanol (10j, 10k, and
10m), 7-tridecanol (10n), and 8-pentadecanol (10o, 10p, and
10q) in the bis-esterification step. The impact of the placement
of 2-branched chain ester in the butanoate lipid framework can
be seen by a comparison of the 2-C spacing between S and N,
with N-methyl mono-branch exhibiting lipids 10e (pKa = 6.05,
Δ pKa = 2.63), 10f (pKa = 6.04, Δ pKa = 2.64), and 10h (pKa
= 5.96, Δ pKa = 2.72) with the related bis-branched lipids 10j
(pKa = 5.71, Δ pKa = 2.97) and 10o (pKa = 5.61, Δ pKa =
3.07). While there was little impact on the measured pKa and
Δ pKa with increasing branched moiety size in the 10e/10f/
10h series, moving to a bis-branched lipid paradigm results in a
lower measured pKa for 10j by ca. 0.3 pKa units and 0.4 pKa
units for 10o. Within a series of bis-branched lipids, one can
alter the measured pKa by increasing the 2-C S-to-N spacing to
three carbons and, as has been seen previously, lipophilicity
can be increased and some of the increased basicity can be
attenuated by altering from N-methyl to N-ethyl in the 3-C
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spacing moiety. Consider the series 10j (6-undecanol, cLogD =
9.94, pKa = 5.71), 10k (6-undecanol, cLogD = 9.63, pKa =
6.80), 10m (6-undecanol, cLogD = 9.95, pKa = 6.56) and the
series 10o (8-pentadecanol, cLogD = 14.01, pKa = 5.61), 10p
(8-pentadecanol, cLogD = 13.70, pKa = 6.38), and 10q (8-
pentadecanol, cLogD = 14.03, pKa = 5.31). Lipophilicity, as
measured by cLogD, can be altered toward desired ranges and
pKa can be altered from the <6.00 values of 10j and 10o to
>6.00. These trends afford us latitude in design to achieve
desired levels of lipophilicity and basicity for the applications of
interest. The lipophilicity impact on formulatability is again
seen in 10k, where a cLogD value of 9.63 is associated with
larger nanoparticle size of 94 nM. The lipids 10j, 10m, 10n,
10o, 10p, and 10q with cLogD values ranging from 9.94 to
14.03 formulate well and afford nanoparticles of 64−89 nM in
size. The combination of lipophilicity and measured pKa has an
obvious effect on FVII knockdown in this short series of lipids.
Lipid 10j (cLogD = 9.94, pKa = 5.71) has a poor FVII
knockdown of 5%, while lipid 10k (cLogD = 9.63, pKa = 6.80),
which has a lower c-pKa but higher pKa, is associated with an
improved FVII knockdown of 51%. Moving toward greater
lipophilicity and pKa > 6.00 (10n cLogD = 11.67, pKa = 6.56;
10p cLogD = 13.70, pKa = 6.38; 10q cLogD = 14.03, pKa =
6.31) leads to improved in vivo performance with FVII
knockdown values of 98, 99, and 97% for these three lipids.
The bis-acetate-based lipids 10r−10aa (Table 6) present an
interesting arena to examine the impact of lipophilicity/
basicity/branched alcohol size upon physicochemical calcu-
lated and measured parameters and in vivo performance. The
mono-branched ester containing lipid 10r (6-undecanol) has a
low cLogD (8.29) and a low pKa in formulation (5.39) likely
due to the environmental impact of the branch in such close
proximity to the basic-N (Δ pKa = 3.17). The combination of
low lipophilicity and low pKa is also associated with
formulatability issues, which are seen in an increased
nanoparticle size (108 nM). The 9-heptadecanol containing
mono-branched lipid 10t is in better lipophilicity space
(cLogD = 11.34); however, the larger branched chain ester
results in further erosion of measured pKa (5.13, Δ pKa = 3.43)
but a more normal nanoparticle size (72 nM) due to the
increase in lipophilicity compared to 10r. Neither of these
lipids performs well in vivo (0% FVII knockdown) as 10r is
insufficiently lipophilic as well as too weakly basic, while 10t
exhibits low basicity in formulation. As shown above in Table 6
and also in Table 4, basicity in formulation can be altered by
increasing the S-to-N spacing to three carbons, leading to 10s
(8-heptadecanol, cLogD = 9.98, pKa = 6.44, Δ pKa = 2.86),
10u (9-heptadecanol, cLogD = 11.00, pKa = 6.38, Δ pKa =
2.92), and 10v (cLogD = 11.30, pKa = 6.20, Δ pKa = 3.96).
Lipids 10s, 10u, and 10v formulate well (nanoparticle size,
66−72 nM) are sufficiently basic, and their use in vivo results in
moderate (10s, 62%) to potent (10u, 97%; 10v, 82%) FVII
knockdown. The lower lipophilicity of 10s is likely the cause of
the modest in vivo activity.
cLogD = 9.26) but are sufficiently basic (10w pKa = 6.17, Δ
pKa = 3.13; 10x pKa = 6.21, Δ pKa = 3.95). The low
lipophilicity associated with 10w and 10x leads to a 0% FVII
knockdown despite good measured basicity. As increasing the
size of the branched chain moiety has been shown to be
associated with an environmental impact and lessened
measured basicity (15e and 10o), achieving appropriate levels
of lipophilicity in this series by increasing the size of the
branched moieties should push us toward decreasing basicity.
This is exactly what is observed in lipids 10y, 10z, and 10aa.
The introduction of two 8-heptadecanol fragments into 10v
(3-C S-to-N spacer, N-methyl) leads to a cLogD = 13.04 but
poor measured basicity (pKa = 5.68, Δ pKa = 3.92). Lipid 10z
bears identical branched chain fragments and S-to-N spacing
but N-ethyl substitution, resulting in a cLogD = 13.34 and an
even lower measured basicity (pKa = 5.62, Δ pKa = 4.54). As
expected, lipids 10y and 10z are poorly active in the FVII
knockdown at 31% each. Increasing the size of the branched
alcohol moieties to 9-heptadecanol further increases the
lipophilicity of lipid 10aa to cLogD = 15.42 and the association
of these fragments with a 2-C S-to-N linker (N-methyl)
provides a very low measured pKa = 4.65 (Δ pKa = 3.91).
Given the space 10aa occupies, it was not examined in vivo.
To more clearly show the connection between two variables,
pKa and lipophilicity, upon in vivo activity, the lipids of the
current study are presented in Table 7, sorted from higher to
lower measured pKa with associated cLogD and FVII
Table 7. Interrelationship between cLogD and Measured
pKaImpact on In Vivo FVII Knockdown
lipid
cLogD
pKa
FVII knockdown (%) (0.03 mg/kg)
10k
10k
10c
10g
10d
10i
10n
10m
10s
10p
10u
10q
10x
10v
10w
10e
10f
10a
10h
15d
15a
15b
15c
10j
10y
10z
10o
15e
10r
10t
9.63
9.63
6.80
6.80
6.77
6.66
6.60
6.58
6.56
6.56
6.44
6.38
6.38
6.31
6.21
6.20
6.17
6.05
6.04
6.03
5.96
5.89
5.81
5.81
5.79
5.71
5.68
5.62
5.61
5.59
5.39
5.13
51
51
70
96
57
75
98
61
62
99
97
97
0
11.01
10.92
11.34
11.93
11.67
9.95
9.98
13.70
11.00
14.03
9.26
11.30
8.96
82
0
9.89
13
82
63
80
71
71
68
70
5
31
31
70
40
0
10.91
11.32
11.93
15.02
11.37
12.38
13.95
9.94
13.04
13.34
14.01
16.08
8.29
The introduction of the second branched chain ester onto
the bis-acetate core was expected to place us in poor calculated
and measured physicochemical space given the data derived
from 10r−10v, and these expectations were met as shown
toward the end of Table 6. Lipids 10w−10z were constructed
with the 3-C S-to-N spacing to increase c-pKa, alternating
between the N-methyl and N-ethyl motifs. Lipids 10w and 10x
have bis-6-undecanoate esters on the bis-acetate core; they are
less lipophilic than might be desired (10w cLogD = 8.96, 10x
11.34
0
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knockdown data presented. As mentioned previously, the pKa
range of 6.20−6.50 had been reported in the literature to be
optimal for good FVII response.5 These data clearly show that
the in vivo response is a multivariable equation with strong ties
to lipophilicity. At what should be the range of optimum pKa
impact, pKa ca. 6.20−6.50 where maximal in vivo activity would
be expected based on the assumptions mentioned above, two
lipids 10w and 10x were found to be associated (Table 7) with
FVII knockdown of 0%, and two lipids 10s and 10m exhibit
FVII knockdown of 61−62%. These data track nicely with
variable 2. The calculated cLogD of lipids 10w, 10x, 10s, and
10m is <10.00, while the remaining lipids of the optimal pKa
ca. 6.20−6.50 range afford greater FVII knockdown (75−99%)
with greater lipophilicity (cLogD = 11.00−14.03).
more hindered esters more slowly with 73% of 10f and 100%
of 10p remaining at the 2 h time point. More in-depth
evaluations of the biodegradability of the lipids of this study are
underway and the results will be presented in due course. All
three lipids were tolerated at a dose of 3.0 mg/kg. The 3.0 mg/
kg dose was selected in the tolerability study window to
examine how a 100× dose increase (from a 0.03 mg/kg) was
tolerated. No abnormal observations were noted for the tested
lipids at either dose level.
CONCLUSIONS
■
The study of lipids with bis-octanoate chain lengths (10a−10d
and 15a−15e) provided an initial surprise when the
modification of basic nitrogen substituents from methyl to
ethyl at the same S-to-N chain length produced a decrease in
measured basicity in formulation despite a ca. 1 pKa unit
increase in predicted basicity (10a to 10b; 10c to 10d). An
increase in the S-to-N chain length from two carbons to three
carbons at the same nitrogen substitution (methyl or ethyl;
10a to 10c and 10b to 10d) does result in an expected increase
in measured basicity. These variations in measured basicity are
readily seen in the Δ pKa parameter (the difference between
predicted and measured basicities), and they suggest that there
is an environmental impact on measured basicity by increasing
lipophilicity at the basic terminus. In this bis-octanoate lipid
series (10a−10d and 15a−15e), another environmental
impact on the measured pKa in formulation is observed
when the large branched chain alcohol, 9-heptadecanol, is
introduced in the synthesis of 15e (c-pKa = 8.68, pKa = 5.59, Δ
pKa = 3.09). As the bis-acid chains are shortened to butyrate
(10e−10q) an identical N-methyl to N-ethyl impact on
measured pKa is seen, while as might be expected, the effect of
the size of a single branched chain alcohol in ester formation is
modest (10e/10h). Within this bis-butyrate series, a large
change in measured vs calculated basicity is more readily
apparent when 2-branched chain alcohols are introduced (10o
and 10q). In the bis-acetate derived lipids (10r−10aa), the
environmental impact of a single branched chain alcohol is
observed in 10t and is more profoundly seen in the bis-
branched chain lipids 10y−10aa. Throughout the lipids of
Tables 4 and 6, good in vivo activity in the FVII knockdown
assay is observed when cLogD is 10.0 or greater and the
measured pKa is ca. 5.80 or greater. A higher measured pKa at a
similar cLogD generally leads to a greater FVII knockdown.
Low cLogD values such as those reported for 10w and 10x
lead to no FVII knockdown despite good measured basicity
(6.17 and 6.21, respectively); these trends are shown for the
entire series of lipids in Table 7.
We have reported22 the utility of lipid 10a in mRNA delivery
via lipid nanoparticles to treat a factor IX (FIX)-deficient
mouse model of hemophilia B. Delivery of human FIX (hFIX)
mRNA encapsulated in lipid 10a-containing LNPs results in
the rapid appearance of FIX protein (within 4−6 h) that
remains stable for up to 4−6 days and is therapeutically
effective. Additionally, hFIX protein production with the 10a
LNP formulation (2 mg/kg) was noted to be ca. 2× of that
found with the MC3 LNP formulation (2 mg/kg), 2500 ng/
mL vs 1250 ng/mL. These data provide an initial correlation
between siRNA (10a FVII knockdown, 63% at 0.03 mg/kg)
and mRNA delivery for the lipids of the current study. The
application of additional selected lipids of this study to the
formulation and delivery of mRNA is underway and will be
reported in due course.
Some lipids of the current study have been examined for
their biodegradability in a mouse plasma assay, while a
tolerability assay has been utilized to gain a preliminary reading
on treatment toleration by intravenous injection of increasing
doses of FVII LNPs. Mouse plasma stability was measured
with time points at 0, 15, 30, 45, 60, and 120 min with a
selected lipid used as an internal standard. Mouse plasma
stability is listed as either T1/2 or % remaining at the 2 h time
point. In the tolerability assessment, nontargeting siRNA-
containing LNPs are introduced via a single bolus injection. In
life, clinical observations are made at 2, 6, 24, and 72 h post
dose. Body weight and activity levels were measured/
considered in life. After necropsy, the liver and spleen were
examined for morphology changes. A lipid exemplar was
selected from the bis-linear esters of Table 4 (10a), one from
the mono-branched esters of Table 6 (10f) and a bis-branched
ester congener from Table 6 (10p) to provide a preliminary
look at biodegradability and response in the tolerability assay.
Table 8 presents the cLogD, measured pKa, FVII knockdown
data, and mouse plasma stability measurement for lipids 10a,
10f, and 10p. Bis-linear ester 10a is rapidly cleaved by mouse
plasma esterases, with T1/2 = 6.9 min, not a surprising
observation given the unhindered nature of the ester carbonyl
moieties. As branched chain esters are added, one in the case
of 10f and two in 10p, plasma esterase activity cleaves these
These data suggest the potential to tune calculated and
measured physicochemical parameters to achieve desired levels
of lipophilicity and measured basicity by a combination of bis-
acid chain length/number and size of branched chain ester
moieties/S-to-N carbon chain length/N-substitution. The
ability to bring targeted lipids presented in Table 6 into
desirable physicochemical space and achieve good in vivo
activity (FVII knockdown, >90%) is noteworthy and could not
have been anticipated based on the literature data of Table 1.
As we move forward with further changes in lipid design, this
multivariable approach should allow us to realize a target
lipophilicity and measured basicity with iterative structural
alteration(s). Further studies of the impact of structure on
calculated and measured physicochemical properties are
underway and will be reported in due course.
Table 8. Biodegradability in the Mouse Plasma Stability
Assessment and Lipid Levels in In Vivo Studies
FVII knockdown
(%) (0.03 mg/kg)
mouse plasma stability T1/2
or % remaining at 2 h
lipid cLogD
pKa
10a
10f
10p
11.32
10.91
13.70
6.03
6.04
6.38
63
82
99
6.9 min
73%
100%
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with 0.109 M sodium citrate buffer (BD Biosciences, San Diego, CA)
and processed to plasma. Plasma was tested immediately or stored at
−80 °C for later analysis for factor VII levels. Measurement of FVII
protein in plasma was determined using the colorimetric Biophen VII
assay kit (Aniara Diagnostica, USA). Absorbance was measured at 405
nm and a calibration curve was generated using the serially diluted
control plasma to determine levels of factor VII in plasma from
treated animals, relative to the saline-treated control animals.
EXPERIMENTAL SECTION
■
Biology. siRNA Formulation into Lipid Nanoparticles. The LNPs
were prepared, in a manner similar to that reported in reference 27, by
mixing appropriate volumes of lipids in ethanol with an aqueous phase
containing siRNA duplexes using a Nanoassemblr microfluidic device,
followed by downstream processing. For the encapsulation of siRNA,
∼0.2 mg/mL siRNA was dissolved in 5 mM citrate buffer (pH 3.5).
Lipids at the desired molar ratio were dissolved in ethanol. The molar
percentage ratio for the constituent lipids is 58% ionizable amino
lipids, 7% DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) (Avan-
ti Polar Lipids), 33.5% cholesterol (Avanti Polar Lipids), and 1.5%
DMG-PEG (1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol;
PEG chain molecular weight: 2000) (NOF America Corporation). At
a flow ratio of 1:3 ethanol/aqueous phases, the solutions were
combined in the microfluidic device using Nanoassemblr (Precision
NanoSystems). The total combined flow rate was 12 mL/min per
microfluidics chip and the total lipid-to-RNA weight ratio was ∼30:1.
The mixed material was then diluted three times with Tris buffer (pH
7.4) containing 50 mM sodium chloride and 9% sucrose after leaving
the micromixer outlet, reducing the ethanol content to 6.25%. The
diluted LNP formulation was concentrated and diafiltered by
tangential flow filtration using hollow fiber membranes (mPES Kros
membranes, Spectrum Laboratories) and Tris buffer (pH 7.4)
containing 50 mM sodium chloride and 9% sucrose. A total of 10
diavolumes were exchanged, effectively removing the ethanol. The
particle size and polydispersity index (PDI) were characterized using a
Zen3600 (Malvern Instruments, with Zetasizer 7.1 software, Malvern,
U.K.). A volume of 50 μL of formulations were diluted into 950 μL of
Tris buffer at pH = 7.4 and equilibrated to 25 °C prior to analysis in a
1 mL cuvette using the following settings for measurement: material
refractive index of 1.5, dispersant viscosity of 1.1 cP, and refractive
index of 1.3. Each sample was analyzed for up to 30 runs.
Encapsulation efficiency was calculated by determining the
unencapsulated siRNA content by measuring the fluorescence
intensity (Fi) upon the addition of RiboGreen (Molecular Probes)
to the LNP and comparing this value to the total fluorescence
intensity (Ft) of the RNA content that is obtained upon lysis of the
LNPs by 1% Triton X-100, where % encapsulation = (Ft − Fi)/Ft ×
100). All of the lipids of these studies were associated with
encapsulation efficiencies of >90%.
Mouse Plasma Stability. Lipid stock solution was prepared by
dissolution of the lipid in isopropanol at the concentration of 5 mg/
mL. A requisite volume of the lipid-isopropanol solution was then
diluted to 100 μM concentration at a total volume of 1.0 mL with in
50:50 (v/v) ethanol/water. Ten microliters of this 100 μM solution
was spiked into 1.0 mL of mouse plasma (BioIVT, cat. no.
MSE00PLNHUNN, CD-1 mouse, anticoagulant: sodium heparin,
not filtered) that was prewarmed to 37 °C and stirred at 50 rpm with
a magnetic stir bar. The starting concentration of lipids in plasma was
thus 1 μM. At time points 0, 15, 30, 45, 60, and 120 min, 0.1 mL of
the plasma was withdrawn from the reaction mixture and the protein
was precipitated by adding 0.9 mL of ice-cold 4:1 (v/v) acetonitrile/
methanol with 1 μg/mL of a selected internal standard lipid added.
After filtration through a 0.45 μm 96-well filtering plate, the filtrates
were analyzed by LC−MS (Thermo Fisher’s Vanquish UHPLC−
LTQ XL linear ion trap mass spectrometer; Waters XBridge BEH
Shield RP18 2.5 μm (2.1 × 100 mm) column with its matching guard
column). Mobile phase A was 0.1% formic acid in water, and mobile
phase B was 0.1% formic acid in 1:1 (v/v) acetonitrile/methanol. The
flow rate was 0.5 min/min. Elution gradient was as follows: time, 0−1
min: 10% B; 1−6 min: 10−95% B; 6−8.5 min: 95% B; 8.5−9 min:
95−10% B; 9−10 min: 10% B. Mass spectrometry was in positive
scanning mode from 600−1100 m/z. The peak of the molecular ion
of the lipids was integrated in extracted ion chromatography (XIC)
using Xcalibur software (Thermo Fisher). The relative peak area
compared to T = 0, after normalization by the peak area of the
internal standard, was used as the percentage of the lipid remaining at
each time point. T1/2 values were calculated using the first-order decay
model.
Mouse Tolerability. Six to eight week-old, CD-1 female mice
(Charles River Labs) were used for tolerability studies. All procedures
used in animal studies were approved by the Institutional Animal Care
and Use Committee. Lipid nanoparticles containing nontargeting
siRNA (proprietary sequence based on a commercially available
proprietary siRNA from Thermo Fisher Scientific) were administered
to CD-1 females via intravenous injection at 1, 3, 10, and 30 mg/kg
siRNA/kg dose. The general health condition of each animal was
monitored for 72 h. Changes in life of >10% weight loss, and after
necropsy (72 h), observations of gross liver and morphology changes
(e.g., liver color change, liver fibrosis, etc.) and/or spleen enlargement,
would result in designating the run at that dose as not tolerated.
Chemistry. General Methods. Starting materials and other
reagents were purchased from commercial suppliers and were used
without further purification, unless otherwise indicated. All reactions
were performed under a positive pressure of nitrogen or argon or with
a drying tube, at ambient temperature (unless otherwise stated), in
anhydrous solvents, unless otherwise indicated. The reactions were
assayed by high-performance liquid chromatography (HPLC) and
terminated as judged by the consumption of starting material. The
1H-NMR spectra were recorded on Varian or Bruker instruments
Measurement of pKa in Formulated Lipid Nanoparticles Using
the TNS Assay. The apparent pKa of ionizable lipid in the lipid
nanoparticle was determined using 6-(p-toluidino)-2-naphthalenesul-
fonic acid sodium salt (TNS reagent, Sigma-Aldrich, St. Louis, MO).
Lipid nanoparticles were diluted in PBS to a concentration of 1 mM
total lipid. TNS was prepared as a 1 mg/mL stock solution in DMSO
and then further diluted using distilled water to a working solution of
60 μg/mL (179 mM). Lipid nanoparticle samples were diluted to 90
μM lipid in 165 μL of buffered solutions containing 10 mM HEPES,
10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, and final
TNS working solution of 1.33 μg/mL (4 μM) where the pH ranged
from 3.8 to 12. Following pipette mixing and incubation at room
temperature in the dark for 15 min, fluorescence intensity was
measured at room temperature in a BioTek Cytation3 imaging reader
using excitation and emission wavelengths of 321 and 445 nm,
respectively. The fluorescence signal was plotted as a function of the
pH and analyzed using a nonlinear (Boltzmann) regression analysis
with the apparent pKa determined as the pH giving rise to half-
maximal fluorescence intensity. Amino lipid apparent pKa values were
calculated using the Henderson−Hasselbalch equation.
1
operating at the field strength indicated. the H-NMR spectra are
obtained as DMSO-d6 or CDCl3 solutions as indicated (reported in
ppm) using TMS as the reference. Other NMR solvents were used as
needed. When peak multiplicities are reported, the following
abbreviations are used: s = singlet, d = doublet, t = triplet, m =
multiplet, br = broadened, dd = doublet of doublets, dt = doublet of
triplets. Coupling constants, when given, are reported in hertz. The
NMR spectra of individual test compounds can be found in the
Supporting Information. The mass spectra were obtained using liquid
chromatography−mass spectrometry (LC−MS) on a Shimadzu
instrument using atmospheric pressure chemical ionization (APCI)
or electrospray ionization (ESI). The mass spectra were also
In Vivo ActivityFactor VII Knockdown.15,21 Seven to eight week-
old, female Balb/C mice were purchased from Charles River
Laboratories (Hollister, CA). Mice were held in a pathogen-free
environment and all procedures involving animals were performed in
accordance with guidelines established by the Institutional Animal
Care and Use Committee (IACUC). Lipid nanoparticles containing
factor VII siRNA15 were administered intravenously at a dosing
volume of 10 mL/kg. After 48 h, mice were anesthetized via isoflurane
and blood was collected retro-orbitally into microtainer tubes coated
K
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Article
measured by direct injection on a PerkinElmer PE-SCIEX API-150
instrument or Agilent-TRAP XCT instrument using electrospray
(ESI) ionization. All test compounds showed >95% purity as
determined by high-performance liquid chromatography (HPLC).
HPLC conditions were as follows: Agilent 1290 Infinity; Halo RP-
Amide, 3.00 mm × 100 mm, 2.7 μm, 30 °C; 10% → 90% CH3CN,
0.05% perchloric acid/H2O to 90% → 10% CH3CN, 0.05% perchloric
acid/H2O, 30 min run, flow rate of 1.0 mL/min, UV detection (λ =
205 nm) or 10% → 90% CH3CN, 0.05% perchloric acid/H2O to 90%
→ 10% CH3CN, 0.05% perchloric acid/H2O, 30 min run, flow rate of
1.0 mL/min, ELSD detection. HPLC analyses of test compounds can
be found in the Supporting Information.
8,8′-[(Phenylmethyl)imino]bis-octanoate 1,1′-Dimethyl Ester
(5a). To a solution of benzyl amine (50 g, 0.46 mol) in acetonitrile
(750 mL) was added powdered potassium carbonate (148.3 g, 1.07
mol), followed by the addition of a solution of methyl-8-
bromooctanoate (250.9 g ,1.058 mol, 2.3 equiv) in acetonitrile (250
mL) over a period of 10 min. The mixture was then placed at 80 °C
and was stirred, under nitrogen, for 16 h. The mixture was cooled
down to room temperature and water (1.0 L) was added. Stirring was
continued for 10 min, then stirring was stopped, and the layers were
allowed to separate. The organic phase was separated and
concentrated in vacuo (bath temperature, 40 °C) and the residue
was dissolved in ethyl acetate (1.0 L). The organic phase was washed
with water (1.0 L) and brine (1.0 L) and dried over anhydrous
sodium sulfate. Filtration and concentration in vacuo afforded crude
5a as a pale yellow oil (201.3 g), which was utilized in the next
reaction without further purification. 1H-NMR (400 MHz, CDCl3): δ
= 7.20−7.36 (5), 3.67 (s, 6), 3.52 (s, 2), 2.25−2.42 (8), 1.20−1.70
(20).
brine (1.5 L). The organic phase was separated, dried (Na2SO4),
filtered, and concentrated in vacuo to provide crude 8a as a yellow oil.
The crude product was purified by chromatography (750 g, 100−200
mesh), packed in petroleum ether, and eluted with a gradient of
petroleum ether/ethyl acetate from 100:0 to 90:10. Fractions
containing 8a were combined and concentrated in vacuo to give 8a
(167 g, 0.257 mol, 72%) as a clear, colorless oil. 1H-NMR (400 MHz,
CDCl3): δ = 5.63 (m, 2), 5.53 (m, 2), 4.61 (d, J = 6.4 Hz, 4), 3.12 (t,
J = 7.2 Hz, 4), 2.28 (t, J = 7.6 Hz, 4), 2.09 (m, 4), 1.44 (s, 9), 1.15−
1.70 (36), 0.85 (t, J = 7.2 Hz, 6); MS (ESI, Agilent-TRAP XCT):
594.9 (M+ − BOC).
8,8′-Iminobis-octanoic Acid, 1,1′-Di-(2Z)-2-nonen-1-yl Ester
(9a). To 8a (20 g, 30.8 mmol) in CH2Cl2 (80 mL), cooled in an
ice-water bath under nitrogen, was added trifluoroacetic acid (44.7 g,
0.39 mol, 30 mL) over 10 min. The mixture was then allowed to
warm to room temperature and stir for 5 h. The mixture was
concentrated in vacuo, diluted with ethyl acetate (300 mL), and
washed with saturated aq. Na2CO3 (300 mL). The organic phase was
separated, washed with brine (300 mL), and dried (Na2SO4).
Concentration in vacuo afforded crude 9a (15 g, 89%) as a pale
yellow, viscous oil. This material was utilized in subsequent reactions
without further purification. LC−MS (Shimadzu 2020; ELSD A:
water/0.05% TFA: B: CH3CN/0.05% TFA 95:5 to 5:95 A/B at 2.00
min, hold 0.7 min): RT 2.335 min, m/e = 550.5 (M + H+); 1H-NMR
(400 mHz, CDCl3): δ = 5.64 (m, 2), 5.54 (m, 2), 4.63 (d, J = 6.4 Hz,
4), 2.61 (m, 4), 2.31 (m, 4), 2.10 (m, 4), 1.64 (m, 4), 1.53 (m, 4),
1.20−1.45 (28), 0.90 (m, 6).
8,8′-[[[[2-(Dimethylamino)ethyl]thio]carbonyl]imino]bis-octa-
noic Acid, 1,1′-Di-(2Z)-2-nonen-1-yl Ester (10a). To a solution of 9a
(3.00 g, 5.40 mmol) in CH2Cl2 (60 mL), cooled in an ice water bath
under nitrogen, was added triphosgene (1.62 g, 5.40 mmol) over 5
min followed by the addition of pyridine (2.16 g, 27.3 mmol, 5 equiv)
over a period of 5 min. The mixture was allowed to warm to room
temperature and stir for 4 h and then concentrated in vacuo (bath
temperature, 23 °C). The residue was dissolved in pyridine (20 mL)
and cooled in an ice-water bath under nitrogen, and then 2-
(dimethylamino)-ethanethiol-HCl (Sigma-Aldrich, D141003) (3.87 g,
27 mmol) was added in one portion. The solution was allowed to
warm to room temperature and stir for 20 h. The solvent was
removed in vacuo and the residue was dissolved in ethyl acetate (200
mL). The solution was washed with 10% aq. citric acid (2 × 200 mL),
saturated aq. NaHCO3 (3 × 200 mL), and brine (200 mL) and dried
(Na2SO4). Filtration and concentration in vacuo provided 3.1 g of
crude 10a as an orange oil. The orange oil was dissolved in heptane
(60 mL) and to the resulting solution were added methanol (45 mL)
and water (15 mL). The resulting two-phase mixture was stirred for
30 min and then allowed to stand to 10 min to separate. The aq.
MeOH lower phase was removed and this procedure was repeated
two additional times. The heptane layer was then treated with
activated charcoal (DARCO, 100 mesh, 1.0 g) and the mixture was
allowed to stir for 16 h. The charcoal was removed by filtration
through a pad of Celite, the filter cake was rinsed with heptane (50
mL), and the solvent was removed in vacuo to give 10a (2.9 g, 4.25
mmol, 79%) as a pale yellow, viscous oil. LC−MS (Shimadzu 2020;
ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to 0:100
A/B at 2.00 min, hold 3.00 min): RT 1.039 min, m/e = 681.6 (M +
H+); 1H-NMR (400 mHz, CDCl3): δ = 5.63 (m, 2), 5.56 (m, 2), 4.64
(d, J = 8.0 Hz, 4), 3.30 (brm, 2), 3.07 (t, J = 6.4 Hz, 2), 2.61 (brm, 2),
2.35 (s, 6), 2.28−2.34 (4), 2.12 (m, 4), 1.64 (m, 4), 1.20−1.40 (28),
0.91 (m, 6).
8,8′-[[(1,1-Dimethylethoxy)carbonyl]imino]bis-octanoate 1,1′-
Dimethyl Ester (6a). A solution of crude 5a (201 g) in ethanol
(1.7 L) in a Parr reactor was sparged with nitrogen for 10 min. Then,
di-t-butyl-dicarbonate (115.7 g, 0.53 mol) and 10% Pd/C (15 g) were
added and the vessel atmosphere was replaced with hydrogen (70 psi)
and the mixture was stirred for 15 h at room temperature. The
pressure was released, the mixture was again sparged with nitrogen for
20 min and then filtered through a pad of Celite, the filter cake was
rinsed with ethanol (2 × 200 mL), and the combined filtrates were
concentrated in vacuo to furnish crude 6a as a pale yellow oil. Crude
6a was purified by chromatography on a column of silica gel (1000 g,
100−200 mesh), packed with petroleum ether, and eluted with a
gradient of petroleum ether/heptane (100:0 to 90:10) using the flash
technique. Fractions containing 6a were combined and concentrated
in vacuo to afford 6a as a clear, colorless oil (159 g, 0.37 mol, 81%
1
from benzyl amine). H-NMR (400 MHz, CDCl3): δ = 3.63 (s, 6),
3.11 (m, 4), 2.28 (m, 4), 1.40 (s, 9), 1.23−1.74 (20).
8,8′-[[(1,1-Dimethylethoxy)carbonyl]imino]bis-octanoic Acid
(7a). To a solution of 6a (150 g, 0.349 mol) in THF (90 mL) and
methanol (200 mL), cooled in an ice-water bath under nitrogen, was
added a solution of NaOH (48.8 g, 1.2 mol) in water (300 mL) over
20 min. The mixture was allowed to warm to room temperature and
stirred for 14 h. The solution was diluted with water (1.0 L) and
washed with MTBE (0.75 L). The aqueous phase was separated and
acidified to pH 4−5 with 1.5 N aq. HCl and then extracted with
CH2Cl2 (2 × 1.0 L). The combined organic layers were washed with
brine (2 × 1.0 L) and dried (Na2SO4). Filtration and concentration in
vacuo afforded 148 g of crude 7a as a pale yellow viscous liquid. This
material was utilized in the subsequent reaction without further
1
purification. H-NMR (400 MHz, DMSO-d6): δ = 3.06 (m, 4), 2.17
(t, J = 7.2 Hz, 4), 1.47 (s, 9), 1.10−1.60 (20).
8,8′-[[[[2-(Diethylamino)ethyl]thio]carbonyl]imino]bis-octanoic
Acid, 1,1′-Di-(2Z)-2-nonen-1-yl Ester (10b). On an identical scale as
that reported for the synthesis of 10a, utilization of 2-(diethylamino)-
ethanethiol-HCl (Sigma-Aldrich, D86604) in place of the dimethyl
variant led to the isolation of 10b (2.72 g, 3.83 mmol, 71%) as a clear,
pale yellow viscous liquid. ESI-MS (+ mode): 709.5 (M + H+), 731.6
(M + Na+); 1H-NMR (400 mHz, CDCl3): δ = 5.64 (m, 2), 5.53 (m,
2), 4.62 (d, J = 6.4 Hz, 4), 3.30 (brm, 4), 2.98 (m, 2), 2.64 (m, 2),
2.57 (m, 4), 2.29 (m, 4), 2.09 (m, 4), 1.57 (brm ,4), 1.20−1.40 (28),
1.04 (m, 6), 0.88 (m, 6).
8,8′-[[(1,1-Dimethylethoxy)carbonyl]imino]bis-octanoic Acid,
1,1′-Di-(2Z)-2-nonen-1-yl Ester (8a). To a solution of crude 7a
(145 g, 0.357 mol) in CH2Cl2 (1.5 L), stirring in an ice-water bath
under nitrogen, were added DMAP (22 g, 0.18 mol) and (Z)-2-
nonenol (101.56 g, 0.714 mol). Then, EDC-HCl (209 g, 1.09 mol)
was added in portions over 2 h. The resulting solution was then
allowed to warm to room temperature and stir for 16 h. The reaction
was quenched by the addition of 10% aq. citric acid (250 mL) and the
resulting mixture was washed with 10% aq. citric acid (1.5 L) and
L
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8,8′-[[[[3-(Dimethylamino)propyl]thio]carbonyl]imino]bis-octa-
noic Acid, 1,1′-Di-(2Z)-2-nonen-1-yl Ester (10c). On an identical
scale as that reported for the synthesis of 10a, utilization of 3-
(dimethylamino)-propanethiol-HCl16 in place of the dimethyl-variant
led to the isolation of 10c (2.78 g, 3.99 mmol, 74%) as a clear, yellow
allowed to warm to room temperature and stir for 20 h. The solvent
was removed in vacuo and the residue was dissolved in ethyl acetate
(200 mL). The solution was washed with 10% aq. citric acid (2 × 200
mL), saturated aq. NaHCO3 (3 × 200 mL), and brine (200 mL) and
dried (Na2SO4). Filtration and concentration in vacuo provided crude
15a as an orange oil. The orange oil was dissolved in heptane (50 mL)
and to the resulting solution were added methanol (37.5 mL) and
water (12.5 mL). The resulting two-phase mixture was stirred for 30
min and then was allowed to stand to 10 min to separate. The aq.
MeOH lower phase was removed and this procedure was repeated
two additional times. The heptane layer was then treated with
activated charcoal (DARCO, 100 mesh, 1.0 g) and the mixture was
allowed to stir for 16 h. The charcoal was removed by filtration
through a pad of Celite, the filter cake was rinsed with heptane (50
mL), and the solvent was removed in vacuo to give 15a (1.03 g, 1.51
mmol, 69%) as a pale yellow, viscous oil. ESI-MS (+ mode): 684.4
1
viscous liquid. ESI-MS (+ mode): 696.1 (M + H+); H-NMR (400
mHz, CDCl3): δ = 5.63 (m, 2), 5.52 (m, 2), 4.61 (d, J = 6.4 Hz, 4),
3.27 (brm, 4), 2.91 (t, J = 6.0 Hz, 2), 2.27−2.37 (6), 2.22 (s, 6), 2.10
(m, 4), 1.78 (m, 2), 1.61 (brm, 4), 1.22−1.40 (30), 0.87 (m, 6).
8,8′-[[[[3-(Diethylamino)propyl]thio]carbonyl]imino]bis-octa-
noic Acid, 1,1′-Di-(2Z)-2-nonen-1-yl Ester (10d). On an identical
scale as that reported for the synthesis of 10a, utilization of 3-
(diethylamino)-propanethiol-HCl17 in place of the dimethyl variant
led to the isolation of 10d (3.05 g, 4.21 mmol, 78%) as a clear, yellow
viscous liquid. LC−MS (Shimadzu 2020; ELSD A: water/0.05% TFA:
B: CH3CN/0.05% TFA 40:60 to 5:95 A/B at 6.00 min, hold 2.00
1
min): RT 1.039 min, m/e = 723.6 (M + H+); H-NMR (400 mHz,
1
(M + H+); H-NMR (500 mHz, CDCl3): δ = 5.65 (m, 1), 5.52 (m,
CDCl3): δ = 5.65 (m, 2), 5.53 (m, 2), 4.61 (d, J = 6.4 Hz, 4), 3.27
(brs, 4), 2.90 (t, J = 6.0 Hz, 2), 2.44−2.51 (m, 6), 2.29 (m, 4), 2.06
(m, 4), 1.75 (m, 2), 1.45−1.70 (8), 1.20−1.40 (28), 1.00 (m, 6), 0.86
(m, 6).
1), 4.87 (m, 1), 4.62 (d, J = 6.4 Hz, 2), 3.27 (brm, 4), 3.02 (t, J = 7.0
Hz, 2), 2.53 (t, J = 7.0 Hz, 2), 2.26−2.34 (4), 2.30 (s, 6), 1.46−1.66
(10), 1.22−1.40 (32), 0.89 (m, 9).
8-((tert-Butoxycarbonyl)amino)octanoate Undecan-6-yl Ester
(12b). To a solution of 11 (15.0 g, 58 mmol) in CH2Cl2 (200
mL), cooled in an ice-water bath under nitrogen, were added in order
EDC-HCl (16.6 g, 86 mmol, 1.5 equiv), DMAP (0.706 g, 5 mmol),
and Et3N (16 mL, 11.61 g, 0.115 mol, 2.0 equiv). After stirring for 5
min, 6-undecananol (CombiBlocks, #QF-4463) (10.0 g, 58 mmol) in
CH2Cl2 (50 mL) was added over a period of 15 min. The mixture was
allowed to warm to room temperature and stir for 16 h. The reaction
diluted with CH2Cl2 (200 mL), washed with water (250 mL),
saturated aq. NaHCO3 (400 mL), and brine (2 × 500 mL), and dried
(Na2SO4). Concentration in vacuo afforded crude 12b, which was
utilized in the next step without further purification.
8-((tert-Butoxycarbonyl)amino)octanoate Nonan-5-yl Ester
(12a). To a solution of 11 (16.1 g, 62 mmol) in CH2Cl2 (200
mL), cooled in an ice-water bath under nitrogen, were added in order
EDC-HCl (17.8 g, 93 mmol, 1.5 equiv), DMAP (0.76 g, 6 mmol),
and Et3N (17.2 mL, 12.48 g, 0.123 mol, 2.0 equiv). After stirring for 5
min, 5-nonanol (TCI America, #N0337) (9.0 g, 62 mmol) in CH2Cl2
(50 mL) was added over a period of 15 min. The mixture was allowed
to warm to room temperature and stir for 16 h. The reaction was
diluted with CH2Cl2 (200 mL), washed with water (250 mL),
saturated aq. NaHCO3 (400 mL), and brine (2 × 500 mL), and dried
(Na2SO4). Concentration in vacuo afforded crude 12a, which was
utilized in the next step without further purification.
8-Amino-octanoate Nonan-5-yl Ester (13a). To a solution of
crude 12a in CH2Cl2 (250 mL), cooled in an ice-water bath under
nitrogen, was added TFA (45.9 mL) over a period of 10 min. The
mixture was allowed to warm to room temperature and then stir for 3
h. The solvent and TFA were then removed in vacuo and the residue
was dissolved in EtOAc (250 mL). The resulting solution was washed
with saturated aq. NaHCO3 (250 mL) and brine (250 mL) and dried
(Na2SO4). Filtration and concentration in vacuo gave crude 13a as a
viscous, yellow liquid. Crude 13a was purified by chromatography on
a column of silica gel (300 g, 60−120 mesh), packed, and eluted with
CH2Cl2/MeOH/Et3N (95:4:1) using the flash technique. Fractions
containing 13a were combined and concentrated in vacuo to afford
13a (8.5 g, 49%) as a pale yellow viscous liquid.
8-Amino-octanoate Undecan-5-yl Ester (13b). To a solution of
crude 12b in CH2Cl2 (250 mL), cooled in an ice-water bath under
nitrogen, was added TFA (46.5 mL, 0.604 mol) over a period of 10
min. The mixture was allowed to warm to room temperature and then
stir for 3 h. The solvent and TFA were then removed in vacuo and the
residue was dissolved in EtOAc (250 mL). The resulting solution was
washed with saturated aq. NaHCO3 (250 mL) and brine (250 mL)
and dried (Na2SO4). Filtration and concentration in vacuo gave crude
13b as a viscous, yellow liquid. Crude 13b was purified by
chromatography on a column of silica gel (300 g, 60−120 mesh),
packed, and eluted with CH2Cl2/MeOH/Et3N (95:4:1) using the
flash technique. Fractions containing 13b were combined and
concentrated in vacuo to afford 13b (10.0 g, 56%) as a pale yellow
viscous liquid.
8-((8-(Nonan-5-yloxy)-8-oxooctyl)amino)octanoic Acid (Z)-Non-
2-en-1-yl Ester (14a). To 13a (8.5 g, 29 mmol) in CH3CN (60 mL)
were added 8-bromo-octanoic acid (Z)-2-nonenylester19 (10.3 g, 29
mmol) and K2CO3 (8.2 g, 59 mmol). The mixture was warmed in a
90 °C oil bath, under nitrogen, for 7 h. The mixture was cooled down
to room temperature, the solids were removed by filtration through a
pad of Celite, the filter cake was rinsed with CH3CN (2 × 50 mL),
and the combined filtrates were concentrated in vacuo to give crude
14a as a dark brown, viscous oil. The crude product was purified by
chromatography on a column of silica gel (250 g, 100−200 mesh),
packed, and eluted with EtOAc/hexanes (70:30). Fractions
containing 14a were combined and concentrated in vacuo, providing
14a (4.6 g, 28%) as a clear, pale yellow, viscous oil.
8-((8-(Undecan-6-yloxy)-8-oxooctyl)amino)octanoic Acid (Z)-
Non-2-en-1-yl Ester (14b). To 13b (10.0 g, 29 mmol) in CH3CN
(80 mL) were added 8-bromo-octanoic acid Z-2-nonenylester19 (11.0
g, 31 mmol) and K2CO3 (8.8 g, 62 mmol). The mixture was warmed
in a 90 °C oil bath, under nitrogen, for 7 h. The mixture was cooled
down to room temperature, the solids were removed by filtration
through a pad of Celite, the filter cake was rinsed with CH3CN (2 ×
60mL), and the combined filtrates were concentrated in vacuo to give
crude 14b as a dark brown, viscous oil. The crude product was
purified by chromatography on a column of silica gel (275 g, 100−200
mesh), packed, and eluted with EtOAc/hexanes (70:30). Fractions
containing 14b were combined and concentrated in vacuo, providing
14b (4.0 g, 22%) as a clear, pale yellow, viscous oil.
8-((((2-(Dimethylamino)ethyl)thio)carbonyl)(8-(nonan-5-yloxy)-
8-oxooctyl)amino)octanoic Acid (Z)-Non-2-en-1-yl Ester (15a). To
a solution of 13a (1.50 g, 2.19 mmol) in CH2Cl2 (25 mL), cooled in
an ice water bath under nitrogen, was added triphosgene (0.649 g,
2.19 mmol) over 5 min followed by the addition of pyridine (0.864 g,
10.9 mmol, 5 equiv) over a period of 5 min. The mixture was allowed
to warm to room temperature and stir for 4 h and then concentrated
in vacuo (bath temperature, 23 °C). The residue was dissolved in
pyridine (10 mL) and cooled in an ice-water bath under nitrogen, and
then 2-(dimethylamino)-ethanethiol-HCl (Sigma-Aldrich, D141003)
(1.55 g, 10.9 mmol) was added in one portion. The solution was
8-((((2-(Dimethylamino)ethyl)thio)carbonyl)(8-(undecan-6-
yloxy)-8-oxooctyl)amino)octanoic Acid (Z)-Non-2-en-1-yl Ester
(15b). To a solution of 13b (1.50 g, 2.00 mmol) in CH2Cl2 (20
mL), cooled in an ice water bath under nitrogen, was added
triphosgene (0.593 g, 2.00 mmol) over 5 min followed by the addition
of pyridine (0.790 g, 10.0 mmol, 5 equiv) over a period of 5 min. The
mixture was allowed to warm to room temperature and stir for 4 h
and then concentrated in vacuo (bath temperature, 23 °C). The
residue was dissolved in pyridine (10 mL) and cooled in an ice-water
bath under nitrogen, and then 2-(dimethylamino)-ethanethiol-HCl
(Sigma-Aldrich, D141003) (1.42 g, 10.0 mmol) was added in one
M
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portion. The solution was allowed to warm to room temperature and
stir for 20 h. The solvent was removed in vacuo and the residue was
dissolved in ethyl acetate (200 mL). The solution was washed with
10% aq. citric acid (2 × 200 mL), saturated aq. NaHCO3 (3 × 200
mL), and brine (200 mL) and dried (Na2SO4). Filtration and
concentration in vacuo provided crude 15b as an orange oil. The
orange oil was dissolved in heptane (50 mL) and to the resulting
solution were added methanol (37.5 mL) and water (12.5 mL). The
resulting two-phase mixture was stirred for 30 min and then allowed
to stand to 10 min to separate. The aq. MeOH lower phase was
removed and this procedure was repeated two additional times. The
heptane layer was then treated with activated charcoal (DARCO, 100
mesh, 1.0 g) and the mixture was allowed to stir for 16 h. The
charcoal was removed by filtration through a pad of Celite, the filter
cake was rinsed with heptane (50 mL) and the solvent was removed
in vacuo to give 15b (0.996 g, 1.40 mmol, 70%) as a pale yellow,
viscous oil. ESI-MS (+ mode): 711.5 (M + H+, base); 1H-NMR (500
mHz, CDCl3): δ = 5.63 (m, 1), 5.51 (m, 1), 4.85 (m, 1), 4.61 (d, J =
7.0 Hz, 2), 3.27 (brm, 4), 3.02 (t, J = 7.0 Hz, 2), 2.53 (t, J = 7.0 Hz,
2), 2.26−2.34 (4), 2.30 (s, 6), 2.09 (m, 2), 1.61 (brm, 4), 1.46−1.55
(6), 1.20−1.42 (34), 0.88 (m, 9).
4-((tert-Butoxycarbonyl)(4-(Z)-non-2-en-1-yloxy)-4-oxobutyl)-
amino)butanoic Acid (8b). To a suspension of 7b (21.70 g, 75.00
mmol) in dry dichloromethane (250 mL) were added in order EDC-
HCl (7.67 g, 40.00 mmol), Z-2-nonen-1-ol (5.84 g, 40.00 mmol),
diisopropylethyl amine (5.17 g, 40.0 mmol), and DMAP (0.490 g,
4.00 mmol). The mixture was allowed to stir at room temperature,
under nitrogen, overnight, then was cast into dichloromethane (250
mL) and water (500 mL). The organic layer was separated, washed
with brine (500 mL), and dried (Na2SO4). Filtration and
concentration in vacuo afforded crude 8b as a viscous semisolid,
which was purified by chromatography on a column of silica gel (750
g) using a gradient of dichloromethane/EtOAc (100:0 to 60:40).
Fractions containing 8b were pooled and concentrated in vacuo to
give 8b (16.44 g, 39.75 mmol, 53%) as a clear, viscous liquid. MS
1
(PerkinElmer): 412.0 (M − H+); H-NMR (500 mHz, CDCl3): δ =
5.63 (m, 1), 5.50 (m, 1), 4.61 (d, J = 7.0 Hz, 2), 3.14−3.25 (brm, 4),
2.33 (m, 2), 2.29 (m, 2), 2.08 (m, 2), 1.78−1.86 (4), 1.43 (s, 9), 1.33
(m, 2), 1.23−1.30 (6), 0.86 (t, J = 7.2 Hz, 3).
4-((tert-Butoxycarbonyl)(4-oxo-4-(tridecan-7-yloxy)butyl)-
amino)butanoic Acid (Z)-Non-2-en-1-yl Ester (8c). To a solution of
8b (3.10 g, 7.50 mmol) in anhydrous dichloromethane (50 mL) were
added in order EDC-HCl (2.15 g, 11.25 mmol), 7-tridecanol (1.50 g,
7.5 mmol), i-Pr2NEt (0.97 g, 7.5 mmol, 1.31 mL), and DMAP (92
mg, 0.75 mmol). The mixture was allowed to stir for 4 h at room
temperature under nitrogen and then cast into dichloromethane (50
mL) and water (100 mL). The organic phase was separated, washed
with brine (100 mL), and dried (Na2SO4). Filtration and
concentration in vacuo afforded crude 8c, which was purified by
chromatography on a column of silica gel (150 g, 230−400 mesh)
eluting with a gradient of hexanes/EtOAc from 95:5 to 50:50.
Fractions containing 8c were pooled and concentrated in vacuo to give
8c (3.17 g, 5.36 mmol, 71%) as a clear, pale yellow, viscous oil. MS
4,4′-[(Phenylmethyl)Imino]Bis-Butanoate-1, 1′-Dimethyl Ester
(5b). To a solution of methyl 4-bromobutanoate (100.0 g, 0.552
mol) in anhydrous CH3CN (600 mL) was added benzyl amine (29.5
g, 0.276 mol) followed by K2CO3 (152.6 g, 1.104 mol). The mixture
was warmed to 80 °C and stirred under nitrogen for 16 h. The
mixture was then cooled down to room temperature and diluted with
ice water (1.5 L). The resulting solution was extracted with EtOAc (3
× 350 mL), and the combined organic layers were dried (Na2SO4).
Filtration and concentration in vacuo provided crude 5b as a viscous,
yellow liquid. The crude product was purified by chromatography on
a column of silica gel (1.0 kg, 100−200 mesh) using a gradient of
EtOAc/hexanes (25:75 to 35:65). Fractions containing 5b were
pooled and concentrated in vacuo to give 5 (61.93 g, 0.201 mol, 73%)
1
(PerkinElmer): 596.4 (M + H+); H-NMR (500 mHz, CDCl3): δ =
5.63 (m, 1), 5.51 (m, 1), 4.86 (m, 1), 4.63 (d, J = 7.0 Hz, 2), 3.26−
3.25 (4), 2.24−2.33 (4), 2.09 (m, 2), 1.79−1.88 (4), 1.46−1.55 (4),
1.45 (s, 9), 1.36 (m, 2), 1.22−1.33 (22), 0.85−0.89 (9).
1
as a colorless, viscous oil. ESI-MS (+ mode): 308.2 (M + H+); H-
NMR (400 mHz, CDCl3): δ = 7.32 (m, 5), 3.65 (s, 6), 3.58 (s, 2),
2.80 (m, 4), 2.47 (m, 4).
4-((4-Oxo-4-(tridecan-7-yloxy)butyl)amino)butanoic Acid (Z)-
Non-2-en-1-yl Ester (9b). To a solution of 8c (3.10 g, 5.24 mmol)
in dry dichloromethane (50 mL) was added trifluoroacetic acid (12
mL). The mixture was stirred at room temperature, under nitrogen,
for 3 h. Concentration in vacuo resulted in crude 9b-TFA salt as a
viscous, yellow oil, which was dissolved in dichloromethane (150
mL), washed with saturated aq. NaHCO3 (3 × 150 mL), and dried
(Na2SO4). Filtration and concentration in vacuo gave crude 9b, which
was purified by chromatography on a column of silica gel (100 g,
230−400 mesh) using a gradient of hexanes/EtOAc (60:40 to 10:90).
Fractions containing 9b were pooled and concentrated in vacuo,
affording 9b (2.03 g, 4.09 mmol, 78%) as a clear, colorless, viscous
4,4′-[[(1,1-Dimethylethoxy)carbonyl]imino]bis-butanoate 1,1′-
Dimethyl Ester (6b). A solution of 5b (61.00 g, 0.198 mol) in
ethanol (600 mL), in a Paar vessel, was sparged with nitrogen for 15
min. Then, di-t-butyl-dicarbonate (47.6 g, 0.218 mol) and 10% Pd/C
(6 g) were added and the vessels atmosphere was replaced with
hydrogen (70 psi) and the mixture was stirred for 15 h at room
temperature. The pressure was released, the mixture was again
sparged with nitrogen for 20 min and then filtered through a pad of
Celite, the filter cake was rinsed with ethanol (2 × 100 mL), and the
combined filtrates were concentrated in vacuo to furnish crude 6b as a
pale yellow oil. Crude 6b was purified by chromatography on a
column of silica gel (250 g, 100−200 mesh), packed with petroleum
ether, and eluted with a gradient of petroleum ether/heptane (100:0
to 90:10) using the flash technique. Fractions containing 6b were
combined and concentrated in vacuo to afford 6b as a clear, colorless
oil (59.7 g, 0.188 mol, 95%). ESI-MS (+ mode): 318.2 (M + H+); 1H-
NMR (400 mHz, CDCl3): δ = 3.62, (s, 6), 3.29 (m, 4), 2.37 (m, 4),
1.85 (m, 4), 1.49 (s, 9).
4,4′-[[(1,1-Dimethylethoxy)carbonyl]imino]bis-butanoic Acid
(7b). To a solution of 6b (59.0 g, 0.185 mol) in THF (275 mL)
was added 6 N aq. NaOH (125 mL). The mixture was warmed in a 60
°C oil bath and allowed to stir under nitrogen for 4 h. The solution
was cooled down to room temperature and the THF was removed in
vacuo to afford a viscous, pale yellow oil, which was diluted with water
(300 mL) and extracted with EtOAc (3 × 250 mL). The aqueous
phase was carefully acidified with 2 N aq. HCl to ca. pH = 5, then the
mixture was extracted with EtOAc (2 × 300 mL), and the combined
organic phases were dried (Na2SO4). The mixture was filtered, the
filter cake was rinsed with EtOAc (100 mL), and the combined
filtrates were concentrated in vacuo to give 7b (47.64 g, 0.165 mol,
89%) as a white solid. ESI-MS (+ mode): 290.2 (M + H+); 1H-NMR
(400 mHz, CDCl3): δ = 3.05 (m, 4), 2.18 (m, 4), 1.67 (m, 4), 1.27 (s,
9).
1
liquid. MS (PerkinElmer): 495.9 (M + H+); H-NMR (500 mHz,
CDCl3): δ = 5.63 (m, 1), 5.48 (m, 1), 4.84 (m, 1), 4.62 (d, J = 7.0 Hz,
2), 3.60 (brs, 1), 3.01−3.09 (4), 2.40−2.48 (4), 2.08 (m, 2), 1.98−
2.04 (4), 1.46−1.54 (4), 1.35 (m, 2), 1.20−1.31 (22), 0.84−0.88 (9).
4-((((2-(Dimethylamino)ethyl)thio)carbonyl)(4-oxo-4-(tridecan-
7-yloxy)butyl)amino)butanoate (Z)-Non-2-en-1-yl Ester (10e). To a
solution of 9b (2.00 g, 4.00 mmol) in CH2Cl2 (50 mL), cooled in an
ice water bath under nitrogen, was added triphosgene (1.19 g, 4.00
mmol) over 5 min followed by the addition of pyridine (1.58 g, 20.0
mmol, 5 equiv) over a period of 5 min. The mixture was allowed to
warm to room temperature and stir for 4 h and then concentrated in
vacuo (bath temperature, 23 °C). The residue was dissolved in
pyridine (15 mL) and cooled in an ice-water bath under nitrogen, and
then 2-(dimethylamino)-ethanethiol-HCl (Sigma-Aldrich, D141003)
(2.83 g, 20 mmol) was added in one portion. The solution was
allowed to warm to room temperature and stir for 20 h. The solvent
was removed in vacuo and the residue was dissolved in ethyl acetate
(175 mL). The solution was washed with 10% aq. citric acid (2 × 175
mL), saturated aq. NaHCO3 (3 × 175 mL), and brine (175 mL) and
dried (Na2SO4). Filtration and concentration in vacuo provided crude
10e as an orange oil. The orange oil was dissolved in heptane (60
mL) and to the resulting solution was added methanol (45 mL) and
N
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water (15 mL). The resulting two-phase mixture was stirred for 30
min and then allowed to stand to 10 min to separate. The aq. MeOH
lower phase was removed and this procedure was repeated two
additional times. The heptane layer was then treated with activated
charcoal (DARCO, 100 mesh, 0.75 g) and the mixture was allowed to
stir for 16 h. The charcoal was removed by filtration through a pad of
Celite, the filter cake was rinsed with heptane (50 mL), and the
solvent was removed in vacuo to give 10e (1.73 g, 2.76 mmol, 69%) as
a pale yellow, viscous oil. LC−MS (Shimadzu 2020; ELSD A: water/
0.05% TFA: B: CH3CN/0.05% TFA 80:20 to 0:100 A/B at 2.00 min,
(brm, 4), 2.90 (t, J = 7.2 Hz, 2), 2.42−2.55 (6), 2.33 (m, 4), 2.10 (m,
2), 3.86 (brm, 4), 1.73 (m, 2), 1.50 (m, 4), 1.15−1.40 (28), 0.98 (t, J
= 7.0 Hz, 6), 0.86 (m, 9).
4-((tert-Butoxycarbonyl)(4-oxo-4-(heptadecan-9-yloxy)butyl)-
amino)butanoic Acid (Z)-Non-2-en-1-yl Ester (8e). As described for
the preparation of 8c, 8b (6.20 g, 15.00 mmol) in dry dichloro-
methane (100 mL) was treated with EDC-HCl (4.30 g, 22.50 mmol),
9-heptadecanol (3.85 g, 15.00 mmol), i-Pr2NEt (1.94 g, 15.00 mmol,
2.62 mL), and DMAP (0.184 g, 1.50 mmol), leading to 8e (6.55 g,
10.05 mmol, 67%) as a clear, pale yellow, viscous oil after work-up
and purification by column chromatography. MS (PerkinElmer):
674.5 (M + Na+); 1H-NMR (500 mHz, CDCl3): δ = 5.63 (m, 1), 5.51
(m, 1), 4.86 (m, 1), 4.62 (d, J = 7.0 Hz, 2), 3.16−3.24 (4), 2.25−2.34
(4), 2.09 (m, 2), 1.80−1.87 (4), 1.46−1.54 (4), 1.45 (s, 9), 1.36 (m,
2), 1.21−1.32 (30), 0.85−0.90 (9).
1
hold 3.00 min): RT 1.045 min, m/e = 627.5 (M + H+); H-NMR
(500 mHz, CDCl3): δ = 5.63 (m, 1), 5.53 (m, 1), 4.87 (m, 1), 4.63
(d, J = 7.0 Hz, 2), 3.38 (brm, 4), 3.02 (t, J = 7.0 Hz, 2), 2.52 (t, J = 7.0
Hz, 2), 2.28−2.37 (4), 2.27 (s, 6), 2.09 (m, 2), 1.90 (brm, 4), 1.51
(brm, 4), 1.36 (m, 2), 1.22−1.34 (22), 0.88 (m, 9).
4-((4-Oxo-4-(heptadecan-9-yloxy)butyl)amino)butanoic Acid
(Z)-Non-2-en-1-yl Ester (9d). As described for the preparation of
9b, a solution of 8e (6.52 g, 10.00 mmol) in dry dichloromethane
(100 mL) was treated with trifluoroacetic acid (24 mL) to give 9d
(4.36 g, 7.90 mmol, 79%) as a clear, colorless viscous liquid after
work-up and purification by chromatography on a column of silica gel.
MS (PerkinElmer): 552.2 (M + H+); 1H-NMR (500 mHz, CDCl3): δ
= 5.65 (m, 1), 5.51 (m, 1), 4.84 (m, 1), 4.63 (d, J = 7.0 Hz, 2), 3.02−
3.10 (brm, 4), 2.93 (brs, 1), 2.40−2.48 (4), 2.08 (m, 2), 1.97−2.05
(4), 1.46−1.54 (4), 1.35 (m, 2), 1.22−1.33 (30), 0.85−0.89 (9).
4-((((2-(Dimethylamino)ethyl)thio)carbonyl)(4-oxo-4-(heptde-
can-9-yloxy)butyl)amino)butanoate (Z)-Non-2-en-1-yl Ester (10h).
As described for the synthesis of 10e, 9d (2.00 g, 3.62 mmol) in
CH2Cl2 (50 mL) was treated with triphosgene (1.08 g, 3.62 mmol)
and pyridine (1.58 g, 20.0 mmol), the mixture was concentrated in
vacuo and diluted with pyridine (15 mL), and to the resulting solution
(ice-water bath under nitrogen) was added 2-(dimethylamino)-
ethanethiol-HCl (Sigma-Aldrich, D141003) (2.83 g, 20.0 mmol) in
one portion. The solution was allowed to warm to room temperature
and stir for 20 h. The reaction was worked up and purified as
described for the preparation of 10e to afford 10h (1.78 g, 2.61 mmol,
72%) as a pale yellow, viscous oil. LC−MS (Shimadzu 2020; ELSD A:
water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to 0:100 A/B at
2.00 min, hold 3.00 min): RT 1.064 min, m/e = 683.5 (M + H+); 1H-
NMR (500 mHz, CDCl3): δ = 5.64 (m, 1), 5.52 (m, 1), 4.87 (m, 1),
4.63 (d, J = 6.9 Hz, 2), 3.38 (brm, 4), 3.02 (t, J = 7.2 Hz, 2), 2.52 (t, J
= 7.2 Hz, 2), 2.32 (m, 4), 2.27 (s, 6), 2.10 (m, 2), 1.90 (brm, 4), 1.51
(m, 4), 1.36 (m, 2), 1.20−1.33 (30), 0.87 (m, 9).
4-((((2-(Diethylamino)propyl)thio)carbonyl)(4-oxo-4-(heptde-
can-9-yloxy)butyl)amino)butanoate (Z)-Non-2-en-1-yl Ester (10i).
As described for the synthesis of 10e, 9d (2.00 g, 3.62 mmol) in
CH2Cl2 (50 mL) was treated with triphosgene (1.08 g, 3.62 mmol)
and pyridine (1.58 g, 20.0 mmol), the mixture was concentrated in
vacuo and diluted with pyridine (15 mL), and to the resulting solution
(ice-water bath under nitrogen) was added 3-(diethylamino)-
propanethiol-HCl17 (3.67 g, 20.0 mmol) in one portion. The solution
was allowed to warm to room temperature and stir for 20 h. The
reaction was worked up and purified as described for the preparation
of 10e to afford 10i (1.75 g, 2.42 mmol, 67%) as a pale yellow, viscous
oil. LC−MS (Shimadzu 2020; ELSD A: water/0.05% TFA: B:
CH3CN/0.05% TFA 80:20 to 0:100 A/B at 2.00 min, hold 3.00 min):
RT 1.065 min, m/e = 725.5 (M + H+); 1H-NMR (300 mHz, CDCl3):
δ = 5.62 (m, 1), 5.53 (m, 1), 4.86 (m, 1), 4.62 (d, J = 6.6 Hz, 2), 3.69
(brm, 4), 2.90 (t, J = 7.2 Hz, 2), 2.54 (m, 6), 2.31 (m, 4), 2.09 (m, 2),
1.78 (m, 2), 1.50 (m, 4), 1.20−1.40 (36), 1.04 (t, J = 7.2 Hz, 6), 0.88
(m, 9).
4-((tert-Butoxycarbonyl)(4-oxo-4-(pentadecan-8-yloxy)butyl)-
amino)butanoic Acid (Z)-Non-2-en-1-yl Ester (8d). As described for
the preparation of 8c, 8b (6.20 g, 15.00 mmol) in dry dichloro-
methane (100 mL) was treated with EDC-HCl (4.30 g, 22.50 mmol),
8-pentadecanol (3.43 g, 15.00 mmol), i-Pr2NEt (1.94 g, 15.00 mmol,
2.62 mL), and DMAP (0.184 g, 1.50 mmol), leading to 8d (6.36 g,
10.20 mmol, 68%) as a clear, pale yellow, viscous oil after work-up
and purification by column chromatography. MS (PerkinElmer):
646.4 (M + Na+); 1H-NMR (500 mHz, CDCl3): δ = 5.64 (m, 1), 5.52
(m, 1), 4.86 (m, 1), 4.62 (d, J = 7.0 Hz, 2), 3.15−3.24 (4), 2.25−2.34
(4), 2.10 (m, 2), 1.80−1.86 (4), 1.47−1.54 (4), 1.45 (s, 9), 1.36 (m,
2), 1.22−1.32 (26), 0.86−0.89 (9).
4-((4-Oxo-4-(pentadecan-8-yloxy)butyl)amino)butanoic Acid
(Z)-Non-2-en-1-yl Ester (9c). As described for the preparation of
9b, a solution of 8d (6.23 g, 10.00 mmol) in dry dichloromethane
(100 mL) was treated with trifluoroacetic acid (24 mL) to give 9c
(4.14 g, 7.90 mmol, 79%) as a clear, colorless viscous liquid after
work-up and purification by chromatography on a column of silica gel.
MS (PerkinElmer): 524.1 (M + H+); 1H-NMR (500 mHz, CDCl3): δ
= 5.64 (m, 1), 5.49 (m, 1), 4.84 (m, 1), 4.62 (d, J = 7.0 Hz, 2), 3.36
(brs, 1), 3.00−3.06 (brm, 4), 2.40−2.48 (4), 2.09 (m, 2), 1.97−2.04
(4), 1.46−1.53 (4), 1.37 (m, 2), 1.21−1.32 (28), 0.85−0.89 (9).
4-((((2-(Dimethylamino)ethyl)thio)carbonyl)(4-oxo-4-(pentade-
can-8-yloxy)butyl)amino)butanoate (Z)-Non-2-en-1-yl Ester (10f).
As described for the synthesis of 10e, 9b (2.10 g, 4.00 mmol) in
CH2Cl2 (50 mL) was treated with triphosgene (1.19 g, 4.00 mmol)
and pyridine (1.58 g, 20.0 mmol), the mixture was concentrated in
vacuo and diluted with pyridine (15 mL), and to the resulting solution
(ice-water bath under nitrogen) was added 2-(dimethylamino)-
ethanethiol-HCl (Sigma-Aldrich, D141003) (2.83 g, 20.0 mmol) in
one portion. The solution was allowed to warm to room temperature
and stir for 20 h. The reaction was worked up and purified as
described for the preparation of 10e to afford 10f (1.93 g, 2.96 mmol,
74%) as a pale yellow, viscous oil. LC−MS (Shimadzu 2020; ELSD A:
water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to 0:100 A/B at
2.00 min, hold 3.00 min): RT 1.052 min, m/e = 655.5 (M + H+); 1H-
NMR (400 mHz, CDCl3): δ = 5.56 (m, 1), 5.44 (m, 1), 4.77 (m, 1),
4.54 (d, J = 6.8 Hz, 2), 3.27 (brm, 4), 2.89 (t, J = 7.2 Hz, 2), 2.38 (t, J
= 7.2 Hz, 2), 2.19 (m, 4), 2.14 (s, 6), 2.02 (m, 2), 1.77 (brm, 4), 1.45
(m, 4), 1.10−1.30 (28), 0.80 (m, 9).
4-((((2-(Diethylamino)propyl)thio)carbonyl)(4-oxo-4-(pentade-
can-8-yloxy)butyl)amino)butanoate (Z)-Non-2-en-1-yl Ester (10g).
As described for the synthesis of 10e, 9c (2.10 g, 4.00 mmol) in
CH2Cl2 (50 mL) was treated with triphosgene (1.19 g, 4.00 mmol)
and pyridine (1.58 g, 20.0 mmol), the mixture was concentrated in
vacuo and diluted with pyridine (15 mL), and to the resulting solution
(ice-water bath under nitrogen) was added 3-(diethylamino)-
propanethiol-HCl17 (3.67 g, 20.0 mmol) in one portion. The solution
was allowed to warm to room temperature and stir for 20 h. The
reaction was worked up and purified as described for the preparation
of 10e to afford 10g (1.98 g, 2.84 mmol, 71%) as a pale yellow,
viscous oil. LC−MS (Shimadzu 2020; ELSD A: water/0.05% TFA: B:
CH3CN/0.05% TFA 80:20 to 0:100 A/B at 2.00 min, hold 3.00 min):
RT 1.067 min, m/e = 697.5 (M + H+); 1H-NMR (300 mHz, CDCl3):
δ = 5.60 (m, 1), 5.52 (m, 1), 4.86 (m, 1), 4.63 (d, J = 6.6 Hz, 2), 3.37
4,4′-((tert-Butoxycarbonyl)azanediyl)dibutyric Acid Bis-6-unde-
canoyl Ester (8f). To a suspension of 7b (14.46 g, 50 mmol) in
anhydrous dichloromethane (500 mL) were added in order EDC-HCl
(23.96 g, 125.0 mmol), 6-undecanol (17.24 g, 100.0 mmol), i-Pr2NEt
(12.92 g, 100.0 mmol, 8.71 mL), and DMAP (1.22 g, 10.00 mmol).
The resulting mixture was stirred for 18 h at room temperature, under
nitrogen, and then cast into dichloromethane (300 mL) and water
(1.0 L). The organic phase was separated, washed with brine (1.0
mL), and dried (Na2SO4). Filtration and concentration in vacuo
O
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afforded the crude mixture as a pale yellow, viscous semisolid, which
was purified by chromatography on a column of silica gel (1000 g,
230−400 mesh) using a gradient of hexanes/EtOAc (90:10 to 50:50),
and fractions containing 8f were pooled and concentrated in vacuo to
provide 8f (14.36 g, 24.00 mmol, 48%) as a clear, colorless, viscous
40.0 mmol), i-Pr2NEt (5.16 g, 40.0 mmol, 6.96 mL), and DMAP
(0.488 g, 2.00 mmol) to provide 8g (8.90 g, 13.60 mmol, 68%), after
work-up and chromatography on a column of silica gel, as a clear, pale
1
yellow, viscous oil. MS (PerkinElmer): 676.4 (M + Na+); H-NMR
(500 mHz, CDCl3): δ = 4.86 (m, 2), 3.16−3.25 (brm, 4), 2.27 (m, 4),
1.83 (m, 4), 1.47−1.54 (8), 1.45 (s, 9), 1.22−1.34 (32), 0.88 (t, J =
7.2 Hz, 12).
1
liquid. MS (PerkinElmer): 620.5 (M + Na+); H-NMR (500 mHz,
CDCl3): δ = 4.86 (m, 2), 3.16−3.24 (brm, 4), 2.27 (m, 4), 1.83 (m,
4), 1.46−1.54 (4), 1.43 (s, 9), 1.35−1.41 (4), 1.22−1.34 (24), 0.85−
0.90 (12).
4,4′-Azanediyldibutyric Acid Bis-tridecan-7-yl Ester (9f). As
described for the preparation of 9b, a solution of 8g (8.50 g, 13.0
mmol) in dry dichloromethane (100 mL) was treated with
trifluoroacetic acid (20 mL) to give 9f (3.38 g, 6.12 mmol, 47%) as
a clear, colorless viscous liquid after work-up and purification by
chromatography on a column of silica gel. MS (PerkinElmer): 554.3
(M + H+); 1H-NMR (500 mHz, CDCl3): δ = 4.84 (m, 2), 3.00−3.08
(brm, 4), 2.42 (m, 4), 2.01 (m, 4), 1.46−1.53 (8), 1.19−1.32 (33),
0.87 (t, J = 7.2 Hz, 12).
4,4′-[[[[3-(Dimethylamino)propyl]thio]carbonyl]imino]bis-buta-
noic Acid Bis-7-tridecanoyl Ester (10n). As described for the
synthesis of 10e, 9f (2.21 g, 4.00 mmol) in CH2Cl2 (50 mL) was
treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g,
20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 3-(dimethylamino)-propanethiol-HCl16 (3.11 g,
20.0 mmol) in one portion. The solution was allowed to warm to
room temperature and stir for 20 h. The reaction was worked up and
purified as described for the preparation of 10e to afford 10n (1.84 g,
2.64 mmol, 66%) as a pale yellow, viscous oil. LC−MS (Shimadzu
2020; ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to
0:100 A/B at 2.00 min, hold 3.00 min): RT 1.054 min, m/e = 699.5
(M + H+); 1H-NMR (300 mHz, CDCl3): δ = 4.86 (m, 2), 3.37 (brm,
4), 2.92 (t, J = 7.2 Hz, 2), 2.42 (m, 2), 2.24−2.44 (10), 1.80−1.96
(6), 1.46−1.57 (8), 1.18−1.37 (32), 0.87 (m, 12).
4,4′-[[tert-Butoxycarbonyl]imino]bis-butanoic Acid Bis-8-penta-
decanoyl Ester (8h). To a solution of 7b (13.0 g, 44.9 mmol) in
dichloromethane (150 mL), cooled in an ice-water bath under
nitrogen, was added EDC-HCl (24.1 g, 125 mmol). The mixture was
allowed to stir for 15 min, and then Et3N (12.6 g, 125 mmol), DMAP
(2.7 g, 22.45 mmol), and 8-heptadecanol (20.3 g, 89.0 mmol) were
added in order. The resulting mixture was allowed to warm to room
temperature and stir for 12 h. The reaction was quenched with water
(150 mL) and the organic phase was separated. The aq. phase was
extracted with dichloromethane (3 × 100 mL) and the combined
organic phases were washed with 10% aq. NaHCO3 (250 mL) and
brine (250 mL) and dried (Na2SO4). Concentration in vacuo
provided crude 8h as a light brown, viscous oil. The crude product
was purified by chromatography on a column of silica gel (400 g,
100−200 mesh), packed with hexanes, and eluted with a gradient of
hexanes/ethyl acetate from 100:0 to 75:25 using the flash technique.
Fractions containing 8h were cooled down and concentrated in vacuo
to give 8h (19.0 g, 26.9 mmol, 60%) as a clear, pale yellow, viscous oil.
1H-NMR (400 mHz, CDCl3): δ = 4.87 (m, 2), 3.22 (brm, 4), 2.24
4,4′-Azanediyldibutyric Acid Bis-undecan-6-yl Ester (9e). As
described for the preparation of 9b, a solution of 8f (13.76 g, 23.00
mmol) in dry dichloromethane (250 mL) was treated with
trifluoroacetic acid (50 mL) to give 9e (7.67 g, 15.41 mmol, 67%)
as a clear, colorless viscous liquid after work-up and purification by
chromatography on a column of silica gel. MS (PerkinElmer): 498.0
(M + H+); 1H-NMR (500 mHz, CDCl3): δ = 4.85 (m, 2), 3.04−3.12
(brm, 4), 2.44 (m, 4), 2.02 (m, 4), 1.45−1.54 (5), 1.21−1.32 (28),
0.88 (t, J = 7.2 Hz, 12).
4,4′-[[[[3-(Dimethylamino)ethyl]thio]carbonyl]imino]bis-buta-
noic Acid Bis-6-undecanoyl Ester (10j). As described for the
synthesis of 10e, 9e (1.99 g, 4.00 mmol) in CH2Cl2 (50 mL) was
treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g,
20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 3-(dimethylamino)-ethanethiol-HCl (Sigma-
Aldrich, D141003) (2.83 g, 20.0 mmol) in one portion. The solution
was allowed to warm to room temperature and stir for 20 h. The
reaction was worked up and purified as described for the preparation
of 10e to afford 10j (1.78 g, 2.84 mmol, 71%) as a pale yellow, viscous
oil. MS (PerkinElmer): 629.4 (M + H+); 1H-NMR (500 mHz,
CDCl3): δ = 4.86 (m, 2), 3.33−3.41 (brm, 4), 3.01 (t, J = 6.8 Hz, 2),
2.51 (t, J = 6.8 Hz, 2), 2.27−2.33 (brm, 4), 2.26 (s, 6), 1.82−1.94 (4),
1.47−1.54 (8), 1.21−1.34 (24), 0.87 (t, J = 7.2 Hz, 12).
4,4′-[[[[3-(Dimethylamino)propyl]thio]carbonyl]imino]bis-buta-
noic Acid Bis-6-undecanoyl Ester (10k). As described for the
synthesis of 10e, 9e (1.99 g, 4.00 mmol) in CH2Cl2 (50 mL) was
treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g,
20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 3-(dimethylamino)-propanethiol-HCl16 (3.11 g,
20.0 mmol) in one portion. The solution was allowed to warm to
room temperature and stir for 20 h. The reaction was worked up and
purified as described for the preparation of 10e to afford 10k (1.80 g,
2.80 mmol, 70%) as a pale yellow, viscous oil. LC−MS (Shimadzu
2020; ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to
0:100 A/B at 2.00 min, hold 3.00 min): RT 1.048 min, m/e = 643.5
(M + H+); 1H-NMR (300 mHz, CDCl3): δ = 4.87 (m, 2), 3.37 (brm,
4), 2.91 (t, J = 7.2 Hz, 2), 2.26−2.40 (6), 2.22 (s, 6), 1.80 (m, 4),
1.43−1.56 (8), 1.20−1.37 (26), 0.88 (m, 12).
4,4′-[[[[3-(Diethylamino)propyl]thio]carbonyl]imino]bis-buta-
noic Acid Bis-6-undecanoyl Ester (10m). As described for the
synthesis of 10e, 9e (1.99 g, 4.00 mmol) in CH2Cl2 (50 mL) was
treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g,
20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 3-(diethylamino)-propanethiol-HCl17 (3.67 g,
20.0 mmol) in one portion. The solution was allowed to warm to
room temperature and stir for 20 h. The reaction was worked up and
purified as described for the preparation of 10e to afford 10m (1.85 g,
2.76 mmol, 69%) as a pale yellow, viscous oil. LC−MS (Shimadzu
2020; ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to
0:100 A/B at 2.00 min, hold 3.00 min): RT 1.061 min, m/e = 671.5
(M + H+); 1H-NMR (300 mHz, CDCl3): δ = 4.87 (m, 2), 3.37 (brm,
4), 2.90 (t, J = 7.2 Hz, 2), 2.51 (q, J = 7.2 Hz, 4), 2.31 (m, 4), 1.89
(brm, 4), 1.75 (m, 4), 1.45−1.58 (8), 1.20−1.46 (24), 1.01 (t, J = 7.2
Hz, 6), 0.88 (m, 12).
(m, 4), 1.85 (m, 4), 1.48−1.64 (8), 1.46 (s, 9), 1.13−1.32 (40), 0.88
(m, 12).
4,4′-[Imino]bis-butanoic Acid Bis-8-pentadecanoyl Ester (9g).
To a solution of 8h (18.9 g, 26.5 mmol) in dichloromethane (190
mL), cooled in an ice-water bath under nitrogen, was added
CF3CO2H (16.39 mL, 214.2 mmol, 8 equiv) at such a rate so as to
maintain the internal temperature of 0−5 °C. The mixture was then
allowed to warm to room temperature and stir for 6 h. The solvent
and excess CF3CO2H were removed in vacuo and the residue was
taken up in dichloromethane (150 mL), washed with 10% aq.
NaHCO3 (3 × 150 mL) and brine (250 mL), and dried (Na2SO4).
Filtration and concentration in vacuo provided crude 9g as a viscous
yellow oil, which was purified by chromatography on a column of
silica gel (400 g, 100−200 mesh), packed with hexanes/EtOAc
(75:25), and eluted with a gradient of hexanes/EtOAc (75:25 to
25:75) using the flash technique. Fractions containing 9g were pooled
and concentrated in vacuo to afford 9g as a pale yellow, viscous oil
(12,4 g, 20.2 mmol, 76%). LC−MS (Shimadzu 2020; ELSD A: water/
0.05% TFA: B: CH3CN/0.05% TFA 80:20 to 0:100 A/B at 2.00 min,
4,4′-((tert-Butoxycarbonyl)azanediyl)dibutyric Acid Bis-7-tride-
canoyl Ester (8g). A described for the preparation of 8f, a suspension
of 7b (5.78 g, 20 mmol) in anhydrous dichloromethane (200 mL)
was treated with EDC-HCl (9.58 g, 50.0 mmol), 7-tridecanol (8.00 g,
P
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Journal of Medicinal Chemistry
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1
hold 3.00 min): RT 1.756 min, m/e = 610.6 (M + H+); H-NMR
(400 mHz, CDCl3): δ = 4.86 (m, 2), 2.88 (m, 4), 2.40 (m, 4), 1.96
(m, 4), 1.46−1.55 (8), 1.20−1.34 (40), 0.86 (m, 12).
(s, 4.5), 1.42 (s, 4.5), 1.35 (m, 2), 1.20−1.32 (6), 0.86 (t, J = 7.2 Hz,
3).
(Z)-Non-2-en-1-yl N-(tert-butoxycarbonyl)-N-(2-oxo-2-(unde-
can-6-yloxy)ethyl)glycinate (8j). To a solution of 8i (5.36 g, 15.00
mmol) in anhydrous dichloromethane (100 mL) were added in order
EDC-HCl (2.18 g, 15.00 mmol), 6-undecanol (2.58 g, 15.00 mmol),
i-Pr2NEt (1.94 g, 15.00 mmol, 2.61 mL), and DMAP (0.183 g, 1.50
mmol). The mixture was allowed to stir for 5 h at room temperature,
under nitrogen, and then diluted with dichloromethane (100 mL).
The mixture was washed with water (200 mL) and brine (200 mL),
dried (Na2SO4), filtered, and concentrated in vacuo to provide 8j as a
yellow, viscous liquid. Crude 8j was purified by chromatography on a
column of silica gel (250 g, 230−400 mesh) using a gradient of
hexanes/ethyl acetate from 100:0 to 70:30. Fractions containing 8j
were pooled and concentrated in vacuo to yield 8j (4.38 g, 8.55 mmol,
57%) as a clear, pale yellow viscous oil. MS (PerkinElmer): 511.4 (M
4,4′-[[[[3-(Dimethylamino)ethyl]thio]carbonyl]imino]bis-buta-
noic Acid Bis-8-pentadecanoyl Ester (10o). As described for the
synthesis of 10e, 9g (2.44 g, 4.00 mmol) in CH2Cl2 (50 mL) was
treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g,
20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 2-(dimethylamino)-ethanethiol-HCl (Sigma-
Aldrich, D141003) (2.83 g, 20.0 mmol) in one portion. The solution
was allowed to warm to room temperature and stir for 20 h. The
reaction was worked up and purified as described for the preparation
of 10e to afford 10o (2.22 g, 3.00 mmol, 74%) as a pale yellow,
viscous oil. LC−MS (Shimadzu 2020; ELSD A: water/0.05% TFA: B:
CH3CN/0.05% TFA 80:20 to 0:100 A/B at 2.00 min, hold 3.00 min):
RT 1.077 min, m/e = 741.4 (M + H+); 1H-NMR (400 mHz, CDCl3):
δ = 4.87 (m, 2), 3.39 (brm, 4), 3.04 (t, J = 6.8 Hz, 2), 2.56 (t, J = 6.8
Hz, 2), 2.20−2.35 (10), 1.86 (brm, 4), 1.42−1.55 (8), 1.16−1.32
(40), 0.88 (m, 12).
1
− H); H-NMR (500 mHz, CDCl3): δ = 5.64 (m, 1), 5.50 (m, 1),
4.90 (m, 1), 4.68 (m, 2), 4.11 (s, 1), 4.09 (s, 1), 3.99 (s, 2), 2.09 (m,
2), 1.48−1.56 (6), 1.44 (s, 4.5), 1.43 (s, 4.5), 1.36 (m, 2), 1.22−1.32
(16), 0.84−0.90 (9).
4,4′-[[[[3-(Dimethylamino)propyl]thio]carbonyl]imino]bis-buta-
noic Acid Bis-8-pentadecanoyl Ester (10p). As described for the
synthesis of 10e, 9g (2.44 g, 4.00 mmol) in CH2Cl2 (50 mL) was
treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g,
20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 3-(dimethylamino)-propanethiol-HCl16 (3.11 g,
20.0 mmol) in one portion. The solution was allowed to warm to
room temperature and stir for 20 h. The reaction was worked up and
purified as described for the preparation of 10e to afford 10p (2.32 g,
3.08 mmol, 77%) as a pale yellow, viscous oil. LC−MS (Shimadzu
2020; ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to
0:100 A/B at 2.00 min, hold 3.00 min): RT 1.058 min, m/e = 755.5
(M + H+); 1H-NMR (400 mHz, CDCl3): δ = 4.89 (m, 2), 3.39 (brm,
4), 2.93 (t, J = 7.2 Hz, 2), 2.30−2.46 (6), 2.28 (s, 6), 1.75−1.93 (6),
1.45−1.60 (8), 1.20−1.35 (40), 0.92 (m, 12).
4,4′-[[[[3-(Diethylamino)propyl]thio]carbonyl]imino]bis-buta-
noic Acid Bis-8-pentadecanoyl Ester (10q). As described for the
synthesis of 10e, 9g (2.44 g, 4.00 mmol) in CH2Cl2 (50 mL) was
treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g,
20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 3-(diethylamino)-propanethiol-HCl17 (3.67 g,
20.0 mmol) in one portion. The solution was allowed to warm to
room temperature and stir for 20 h. The reaction was worked up and
purified as described for the preparation of 10e to afford 10q (2.29 g,
2.92 mmol, 73%) as a pale yellow, viscous oil. LC−MS (Shimadzu
2020; ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to
0:100 A/B at 2.00 min, hold 3.00 min): RT 1.052 min, m/e = 783.6
(M + H+); 1H-NMR (400 mHz, CDCl3): δ = 4.86 (m, 2), 3.36 (brm,
4), 2.90 (t, J = 7.2 Hz, 2), 2.47−2.55 (6), 2.31 (m, 4), 1.89 (brm, 4),
1.75 (m, 2), 1.45−1.56 (8), 1.20−1.40 (40), 1.01 (t, J = 7.2 Hz, 6),
0.87 (m, 12).
(Z)-Non-2-en-1-yl (2-Oxo-2-(undecan-6-yloxy)ethyl)glycinate
(9h). To a solution of 8j (4.09 g, 8.00 mmol) in anhydrous
dichloromethane (60 mL) was added trifluoroacetic acid (35 mL),
and the resulting mixture was stirred at room temperature under
nitrogen for 3 h. The solvent and excess CF3CO2H were removed in
vacuo and the residue was taken up in dichloromethane (75 mL),
washed with 10% aq. NaHCO3 (3 × 75mL) and brine (125 mL), and
dried (Na2SO4). Filtration and concentration in vacuo provided crude
9h as a viscous yellow oil, which was purified by chromatography on a
column of silica gel (75 g, 230−400 mesh) and eluted with a gradient
of hexanes/EtOAc (75:25 to 20:80). Fractions containing 9h were
pooled and concentrated in vacuo to provide 9h (2.70 g, 6.56 mmol,
82%) as a clear, pale yellow, viscous oil. MS (PerkinElmer): 412.4 (M
1
+ H+); H-NMR (500 mHz, CDCl3): δ = 5.67 (m, 1), 5.50 (m, 1),
4.96 (m, 1), 4.75 (d, J = 7.0 Hz, 2), 3.98 (s, 2), 3.96 (s, 2), 2.08 (m,
2), 1.52−1.58 (4), 1.36 (m, 2), 1.20−1.32 (19), 0.85−0.90 (9).
(Z)-Non-2-en-1-yl N-(((2-(Dimethylamino)ethyl)thio)carbonyl)-
N-(2-oxo-2-(undecan-6-yloxy)ethyl)glycinate (10r). As described
for the synthesis of 10e, 9h (1.64 g, 4.00 mmol) in CH2Cl2 (50
mL) was treated with triphosgene (1.19 g, 4.00 mmol) and pyridine
(1.58 g, 20.0 mmol), the mixture was concentrated in vacuo and
diluted with pyridine (15 mL), and to the resulting solution (ice-water
bath under nitrogen) was added 2-(dimethylamino)-ethanethiol-HCl
(Sigma-Aldrich, D141003) (2.83 g, 20.0 mmol) in one portion. The
solution was allowed to warm to room temperature and stir for 20 h.
The reaction was worked up and purified as described for the
preparation of 10e to afford 10r (1.25 g, 2.32 mmol, 58%) as a pale
yellow, viscous oil. LC−MS (Shimadzu 2020; ELSD A: water/0.05%
TFA: B: CH3CN/0.05% TFA 80:20 to 0:100 A/B at 2.00 min, hold
1
3.00 min): RT 1.041 min, m/e = 543.4 (M + H+); H-NMR (400
mHz, CDCl3): δ = 5.66 (m, 1), 5.50 (m, 1), 4.92 (m, 1), 4.70 (t, J =
7.2 Hz, 2), 4.19−4.24 (4), 3.05 (m, 2), 2.53 (m, 2), 2.27 (s, 6), 2.08
(m, 2), 1.43 (m, 4), 1.20−1.40 (20), 0.88 (m, 9).
(Z)-N-(tert-Butoxycarbonyl)-N-(2-(non-2-en-1-yloxy)-2-
oxoethyl)glycine (8i). To a solution of 2-[tert-butoxycarbonyl-
(carboxymethyl)amino]acetic acid (CombiBlocks, #SS-1100) (40.0
g, 0.172 mol) in dry dichloromethane (500 mL) were added in order
EDC-HCl (32.97 g, 0.172 mol), (Z)-non-2-en-1-ol (24.47 g, 0.172
mol), i-Pr2NEt (22.23 g, 0.172 mol, 29.96 mL), and DMAP (2.10 g,
17.2 mmol). The mixture was allowed to stir for 5 h at room
temperature under nitrogen, then diluted with dichloromethane (150
mL), washed with water (0.75 L) and brine (0.75 L), and dried
(Na2SO4). Filtration and concentration in vacuo afforded crude 8i as a
viscous liquid, which was purified by chromatography on a column of
silica gel (750 g, 230−400 mesh) using a gradient of dichloro-
methane/methanol from 100:0 to 95:5. Fractions containing 8i were
pooled and concentrated in vacuo to furnish 8i (17.78 g, 49.9 mmol,
29%) as a clear, pale yellow, viscous liquid. MS (PerkinElmer): 356.1
(M − H); 1H-NMR (500 mHz, CDCl3): δ = 10.10 (brs, 1), 5.66 (m,
1), 5.50 (m, 1), 4.70 (m, 2), 4.11 (s, 2), 4.00 (s, 2), 2.16 (m, 2), 1.43
(Z)-Non-2-en-1-yl N-(tert-Butoxycarbonyl)-N-(2-oxo-2-(pentade-
can-8-yloxy)ethyl)glycinate (8k). As described for the synthesis of 8j,
the reaction of 8i (5.36 g, 15.00 mmol) in anhydrous dichloro-
methane (100 mL) with 8-pentadecanol (3.43 g, 15.00 mmol), EDC-
HCl (2.18 g, 15.00 mmol), i-Pr2NEt (1.94 g, 15.00 mmol, 2.61 mL),
and DMAP (0.183 g, 1.50 mmol) led to 8k (4.68 g, 8.25 mmol, 55%)
as a clear, pale yellow, viscous oil after purification by chromatography
1
on a column of silica gel. MS (PerkinElmer): 590.3 (M + Na+); H-
NMR (500 mHz, CDCl3): δ = 5.65 (m, 1), 5.51 (m, 1), 4.90 (m, 1),
4.69 (m, 2), 4.11 (s, 1), 4.08 (s, 1), 3.99 (s, 2), 2.09 (m, 2), 1.46−1.56
(4), 1.44 (s, 4.5), 1.43 (s, 4.5), 1.37 (m, 2), 1.22−1.32 (26), 0.86−
0.90 (9).
(Z)-Non-2-en-1-yl (2-Oxo-2-(pentadecan-8-yloxy)ethyl)glycinate
(9i). As described for the synthesis of 9h, a solution of 8k (4.54 g, 8.00
mmol) in dry dichloromethane (60 mL) was treated with trifluoro-
acetic acid (35 mL), leading to 9i (2.77 g, 5.92 mmol, 74%) as a clear,
yellow, viscous oil after work-up and purification by column
Q
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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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Article
chromatography. MS (PerkinElmer): 468.2 (M + H+); 1H-NMR (500
mHz, CDCl3): δ = 5.69 (m, 1), 5.50 (m, 1), 4.96 (m, 1), 4.75 (d, J =
7.0 Hz, 2), 3.98 (s, 2), 3.95 (s, 2), 2.08 (m, 2), 1.52−1.58 (4), 1.36
(m, 2), 1.20−1.33 (27), 0.86−0.89 (9).
LC−MS (Shimadzu 2020; ELSD A: water/0.05% TFA: B: CH3CN/
0.05% TFA 80:20 to 0:100 A/B at 2.00 min, hold 3.00 min): RT
1.047 min, m/e = 641.5 (M + H+); 1H-NMR (300 mHz, CDCl3): δ =
5.66 (m, 1), 5.50 (m, 1), 4.92 (m, 1), 4.70 (m, 2), 4.15−4.27 (4), 2.96
(t, J = 6.9 Hz, 2), 2.38 (t, J = 7.2 Hz, 2), 2.46 (s, 6), 2.08 (m, 2), 1.81
(m, 2), 1.52 (brm, 4), 1.18−1.42 (32), 0.88 (m, 9).
(Z)-Non-2-en-1-yl N-(((2-(Dimethylamino)propyl)thio)carbonyl)-
N-(2-oxo-2-(pentadecan-8-yloxy)ethyl)glycinate (10s). As described
for the synthesis of 10e, 9i (1.87 g, 4.00 mmol) in CH2Cl2 (50 mL)
was treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58
g, 20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 3-(dimethylamino)-propanethiol-HCl16 (3.11 g,
20.0 mmol) in one portion. The solution was allowed to warm to
room temperature and stir for 20 h. The reaction was worked up and
purified as described for the preparation of 10e to afford 10s (1.54 g,
2.52 mmol, 63%) as a pale yellow, viscous oil. LC−MS (Shimadzu
2020; ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to
0:100 A/B at 2.00 min, hold 3.00 min): RT 1.049 min, m/e = 613.5
(Z)-Non-2-en-1-yl N-(((2-(Diethylamino)propyl)thio)carbonyl)-N-
(2-oxo-2-(heptadecan-9-yloxy)ethyl)glycinate (10v). As described
for the synthesis of 10e, 9j (1.98 g, 4.00 mmol) in CH2Cl2 (50 mL)
was treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58
g, 20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 3-(diethylamino)-propanethiol-HCl17 (3.67 g,
20.0 mmol) in one portion. The solution was allowed to warm to
room temperature and stir for 20 h. The reaction was worked up and
purified as described for the preparation of 10e to afford 10v (1.58 g,
2.36 mmol, 59%) as a pale yellow, viscous oil. LC−MS (Shimadzu
2020; ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to
0:100 A/B at 2.00 min, hold 3.00 min): RT 1.063 min, m/e = 669.5
1
(M + H+); H-NMR (300 mHz, CDCl3): δ = 5.65 (m, 1), 5.50 (m,
1), 4.92 (m, 1), 4.69 (m, 2), 4.13−4.30 (4), 2.95 (t, J = 7.2 Hz, 2),
2.32 (t, J = 7.2 Hz, 2), 2.20 (s, 6), 2.08 (m, 2), 1.78 (m, 2), 1.18−1.42
(32), 0.87 (m, 9).
1
(M + H+); H-NMR (300 mHz, CDCl3): δ = 5.64 (m, 1), 5.50 (m,
1), 4.92 (m, 1), 4.15−4.38 (4), 2.94 (t, J = 7.2 Hz, 2), 2.43−2.53 (6),
2.09 (m, 2), 1.73 (m, 4), 1.52 (brm, 4), 1.16−1.42 (32), 1.00 (t, J =
7.2 Hz, 6), 0.87 (m, 9).
(Z)-Non-2-en-1-yl N-(tert-Butoxycarbonyl)-N-(2-oxo-2-(heptade-
can-9-yloxy)ethyl)glycinate (8m). As described for the synthesis of
8j, the reaction of 8i (5.36 g, 15.00 mmol) in anhydrous
dichloromethane (100 mL) with 9-heptadecanol (3.85 g, 15.00
mmol), EDC-HCl (2.18 g, 15.00 mmol), i-Pr2NEt (1.94 g, 15.00
mmol, 2.61 mL), and DMAP (0.183 g, 1.50 mmol) led to 8m (5.00 g,
8.40 mmol, 56%) as a clear, pale yellow, viscous oil after purification
by chromatography on a column of silica gel. MS (PerkinElmer):
596.4 (M + H+); 1H-NMR (500 mHz, CDCl3): δ = 5.66 (m, 1), 5.50
(m, 1), 4.90 (m, 1), 4.69 (m, 2), 4.11 (s, 1), 4.08 (s, 1), 3.99 (s, 2),
2.09 (m, 2), 1.47−1.56 (4), 1.44 (s, 4.5), 1.43 (s, 4.5), 1.35 (m, 2),
1.21−1.33 (30), 0.85−0.89 (9).
(Z)-Non-2-en-1-yl (2-Oxo-2-(heptadecan-9-yloxy)ethyl)glycinate
(9j). As described for the synthesis of 9h, a solution of 8m (4.77 g,
8.00 mmol) in dry dichloromethane (60 mL) was treated with
trifluoroacetic acid (35 mL), leading to 9j (3.33 g, 6.72 mmol, 84%)
as a clear, yellow, viscous oil after work-up and purification by column
chromatography. MS (PerkinElmer): 496.2 (M + H+); 1H-NMR (500
mHz, CDCl3): δ = 5.69 (m, 1), 5.50 (m, 1), 4.97 (m, 1), 4.76 (d, J =
7.0 Hz, 2), 3.95 (s, 2), 3.93 (s, 2), 2.09 (m, 2), 1.51−1.58 (4), 1.35
(m, 2), 1.22−1.32 (31), 0.85−0.89 (9).
(Z)-Non-2-en-1-yl N-(((2-(Dimethylamino)ethyl)thio)carbonyl)-
N-(2-oxo-2-(heptadecan-9-yloxy)ethyl)glycinate (10t). As described
for the synthesis of 10e, 9j (1.98 g, 4.00 mmol) in CH2Cl2 (50 mL)
was treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58
g, 20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 2-(dimethylamino)-ethanethiol-HCl (Sigma-
Aldrich, D141003) (2.83 g, 20.0 mmol) in one portion. The solution
was allowed to warm to room temperature and stir for 20 h. The
reaction was worked up and purified as described for the preparation
of 10e to afford 10t (1.38 g, 2.20 mmol, 55%) as a pale yellow, viscous
oil. LC−MS (Shimadzu 2020; ELSD A: water/0.05% TFA: B:
CH3CN/0.05% TFA 80:20 to 0:100 A/B at 2.00 min, hold 3.00 min):
RT 1.059 min, m/e = 627.5 (M + H+); 1H-NMR (400 mHz, CDCl3):
δ = 5.67 (m, 1), 5.51 (m, 1), 4.91 (m, 1), 4.70 (m, 2), 4.17−4.27 (4),
3.07 (m, 2), 2.52 (m, 2), 2.27 (s, 6), 2.08 (m, 2), 1.53 (brm, 4), 1.20−
1.41 (32), 0.88 (m, 9).
(Z)-Non-2-en-1-yl N-(((2-(Dimethylamino)propyl)thio)carbonyl)-
N-(2-oxo-2-(heptadecan-9-yloxy)ethyl)glycinate (10u). As de-
scribed for the synthesis of 10e, 9j (1.98 g, 4.00 mmol) in CH2Cl2
(50 mL) was treated with triphosgene (1.19 g, 4.00 mmol) and
pyridine (1.58 g, 20.0 mmol), the mixture was concentrated in vacuo
and diluted with pyridine (15 mL), and to the resulting solution (ice-
water bath under nitrogen) was added 3-(dimethylamino)-propane-
thiol-HCl16 (3.11 g, 20.0 mmol) in one portion. The solution was
allowed to warm to room temperature and stir for 20 h. The reaction
was worked up and purified as described for the preparation of 10e to
afford 10u (1.56 g, 2.44 mmol, 61%) as a pale yellow, viscous oil.
2,2′-((tert-Butoxycarbonyl)azanediyl)diacetic Acid Bis-undecan-
6-yl Ester (8n). To a solution of 2-[tert-butoxycarbonyl-
(carboxymethyl)amino]acetic acid (CombiBlocks, #SS-1100) (40.0
g, 0.172 mol) in dry dichloromethane (750 mL) were added in order
EDC-HCl (65.94 g, 0.344 mol), 6-undecanol (59.27 g, 0.344 mol), i-
Pr2NEt (44.46 g, 0.344 mol, 59.92 mL), and DMAP (4.20 g, 34.4
mmol). The mixture was allowed to stir for 5 h at room temperature
under nitrogen, then diluted with dichloromethane (250 mL), washed
with water (1.0 L) and brine (1.0 L), and dried (Na2SO4). Filtration
and concentration in vacuo afforded crude 8n as a yellow, viscous
liquid, which was purified by chromatography on a column of silica
gel (750 g, 230−400 mesh) using a gradient of dichloromethane/
methanol from 100:0 to 95:5. Fractions containing 8n were pooled
and concentrated in vacuo to furnish 8i (50.32 g, 92.9 mmol, 54%) as
a clear, pale yellow, viscous liquid. MS (PerkinElmer): 564.3 (M +
1
Na+); H-NMR (500 mHz, CDCl3): δ = 4.87−4.94 (2), 4.07 (s, 2),
3.97 (s, 2), 1.49−1.56 (8), 1.43 (s, 9), 1.22−1.34 (24), 0.84−0.90
(12).
2,2′-(Azanediyl)diacetic Acid Bis-undecan-6-yl Ester (9k). As
described for the synthesis of 9h, a solution of 8n (4.33 g, 8.00 mmol)
in dry dichloromethane (60 mL) was treated with trifluoroacetic acid
(35 mL), leading to 9k (3.33 g, 6.72 mmol, 84%) as a clear, yellow,
viscous oil after work-up and purification by column chromatography.
MS (PerkinElmer): 442.2 (M + H+); 1H-NMR (500 mHz, CDCl3): δ
= 4.98 (m, 2), 3.92 (s, 4), 1.52−1.60 (8), 1.20−1.35 (25), 0.88 (t, J =
7.2 Hz, 12).
2,2′-((((3-(Dimethylamino)propyl)thio)carbonyl)azanediyl)-
diacetic Acid Bis-undecan-6-yl Ester (10w). As described for the
synthesis of 10e, 9k (1.77 g, 4.00 mmol) in CH2Cl2 (50 mL) was
treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g,
20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 3-(dimethylamino)-propanethiol-HCl16 (3.11 g,
20.0 mmol) in one portion. The solution was allowed to warm to
room temperature and stir for 20 h. The reaction was worked up and
purified as described for the preparation of 10e to afford 10w (1.55 g,
2.64 mmol, 66%) as a pale yellow, viscous oil. LC−MS (Shimadzu
2020; ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to
0:100 A/B at 2.00 min, hold 3.00 min): RT 1.053 min, m/e = 587.4
(M + H+); 1H-NMR (300 mHz, CDCl3): δ = 4.92 (m, 2), 4.22 (s, 2),
4.16 (s, 2), 2.96 (t, J = 7.2 Hz, 2), 2.44 (m, 2), 2.30 (s, 6), 1.85 (m,
2), 1.47−1.62 (8), 1.17−1.40 (24), 0.87 (m, 12).
2,2′-((((3-(Diethylamino)propyl)thio)carbonyl)azanediyl)diacetic
Acid Bis-undecan-6-yl Ester (10x). As described for the synthesis of
10e, 9k (1.77 g, 4.00 mmol) in CH2Cl2 (50 mL) was treated with
triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g, 20.0 mmol),
the mixture was concentrated in vacuo and diluted with pyridine (15
R
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Article
mL), and to the resulting solution (ice-water bath under nitrogen)
was added 3-(diethylamino)-propanethiol-HCl17 (3.67 g, 20.0 mmol)
in one portion. The solution was allowed to warm to room
temperature and stir for 20 h. The reaction was worked up and
purified as described for the preparation of 10e to afford 10x (1.40 g,
2.28 mmol, 57%) as a pale yellow, viscous oil. LC−MS (Shimadzu
2020; ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to
0:100 A/B at 2.00 min, hold 3.00 min): RT 1.049 min, m/e = 615.5
(M + H+); 1H-NMR (300 mHz, CDCl3): δ = 4.92 (m, 2), 4.44 (s, 2),
4.16 (s, 2), 2.94 (t, J = 7.2 Hz, 2), 2.50−2.62 (6), 1.80 (m, 2), 1−47-
1.62 (8), 1.18−1.37 (24), 1.04 (t, J = 7.2 Hz, 6), 0.88 (m, 12).
2,2′-((tert-Butoxycarbonyl)azanediyl)diacetic Acid Bis-pentade-
can-8-yl Ester (8o). As described for the synthesis of 8n, a solution of
2-[tert-butoxycarbonyl(carboxymethyl)amino]acetic acid (Combi-
Blocks, #SS-1100) (40.0 g, 0.172 mol) in dry dichloromethane
(750 mL) was treated with EDC-HCl (65.94 g, 0.344 mol), 8-
pentadecanol (78.57 g, 0.344 mol), i-Pr2NEt (44.46 g, 0.344 mol,
59.92 mL), and DMAP (4.20 g, 34.4 mmol) to give, after work-up
and purification by column chromatography, 8o (65.24 g, 99.8 mmol,
58%) as a clear, pale yellow, viscous oil. MS (PerkinElmer): 652.6 (M
− H); 1H-NMR (500 mHz, CDCl3): δ = 4.90 (m, 2), 4.06 (s, 2), 3.97
(s, 2), 1.47−1.44 (8), 1.43 (s, 9), 1.20−1.32 (40), 0.84−0.88 (12).
2,2′-(Azanediyl)diacetic Acid Bis-pentadecan-8-yl Ester (9m). As
described for the synthesis of 9h, a solution of 8o (5.23 g, 8.00 mmol)
in dry dichloromethane (60 mL) was treated with trifluoroacetic acid
(35 mL), leading to 9m (3.33 g, 6.72 mmol, 84%) as a clear, yellow,
viscous oil after work-up and purification by column chromatography.
MS (PerkinElmer): 554.4 (M + H+); 1H-NMR (500 mHz, CDCl3): δ
= 4.97 (m, 2), 3.95 (s, 4), 1.50−1.60 (8), 1.20−1.32 (41), 0.88 (t, J =
7.2 Hz, 12).
2,2′-((((3-(Dimethylamino)propyl)thio)carbonyl)azanediyl)-
diacetic Acid Bis-pentadecan-8-yl Ester (10y). As described for the
synthesis of 10e, 9m (2.21 g, 4.00 mmol) in CH2Cl2 (50 mL) was
treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g,
20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 3-(dimethylamino)-propanethiol-HCl16 (3.11 g,
20.0 mmol) in one portion. The solution was allowed to warm to
room temperature and stir for 20 h. The reaction was worked up and
purified as described for the preparation of 10e to afford 10y (1.90 g,
2.72 mmol, 68%) as a pale yellow, viscous oil. LC−MS (Shimadzu
2020; ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to
0:100 A/B at 2.00 min, hold 3.00 min): RT 1.053 min, m/e = 699.5
(M + H+); 1H-NMR (300 mHz, CDCl3): δ = 4.91 (m, 2), 4.21 (s, 2),
4.16 (s, 2), 2.96 (t, J = 7.2 Hz, 2), 2.44 (m, 2), 2.29 (s, 6), 1.84 (m,
2), 1.45−1.60 (8), 1.17−1.40 (40), 0.87 (m, 12).
mmol, 60%) as a clear, pale yellow, viscous oil. MS (PerkinElmer):
732.4 (M + Na+); 1H-NMR (500 mHz, CDCl3): δ = 4.90 (m, 2), 4.07
(s, 2), 3.98 (s, 2), 1.47−1.55 (8), 1.44 (s, 9), 1.22−1.34 (48), 0.87 (t,
J = 7.2 Hz, 12).
2,2′-(Azanediyl)diacetic Acid Bis-heptadecan-9-yl Ester (9n). As
described for the synthesis of 9h, a solution of 8p (5.68 g, 8.00 mmol)
in dry dichloromethane (60 mL) was treated with trifluoroacetic acid
(35 mL), leading to 9n (3.71 g, 6.08 mmol, 76%) as a clear, yellow,
viscous oil after work-up and purification by column chromatography.
MS (PerkinElmer): 610.4 (M + H+); 1H-NMR (500 mHz, CDCl3): δ
= 4.98 (m, 2), 3.99 (s, 4), 1.51−1.59 (8), 1.20−1.32 (49), 0.88 (t, J =
7.2 Hz, 12).
2,2′-((((3-(Dimethylamino)ethyl)thio)carbonyl)azanediyl)-
diacetic Acid Bis-heptadecan-9-yl Ester (10aa). As described for the
synthesis of 10e, 9n (2.44 g, 4.00 mmol) in CH2Cl2 (50 mL) was
treated with triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g,
20.0 mmol), the mixture was concentrated in vacuo and diluted with
pyridine (15 mL), and to the resulting solution (ice-water bath under
nitrogen) was added 2-(dimethylamino)-ethanethiol-HCl (Sigma-
Aldrich, D141003) (2.83 g, 20.0 mmol) in one portion. The solution
was allowed to warm to room temperature and stir for 20 h. The
reaction was worked up and purified as described for the preparation
of 10e to afford 10aa (1.90 g, 2.56 mmol, 64%) as a pale yellow,
viscous oil. LC−MS (Shimadzu 2020; ELSD A: water/0.05% TFA: B:
CH3CN/0.05% TFA 80:20 to 0:100 A/B at 2.00 min, hold 3.00 min):
RT 1.071 min, m/e = 741.6 (M + H+); 1H-NMR (500 mHz, CDCl3):
δ = 4.91 (m, 2), 4.21 (s, 2), 4.17 (s, 2), 3.05 (t, J = 7.0 Hz, 2), 2.51 (t,
J = 7.0 Hz, 2), 2.26 (s, 6), 1.46−1.55 (8), 1.19−1.33 (48), 0.87 (t, J =
7.2 Hz, 12).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01407.
■
sı
Molecular formula strings (CSV)
HPLC traces and 1H-NMR spectra for final lipid
structures (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Kumar Rajappan − Arcturus Therapeutics Inc., San Diego,
California 92121, United States; orcid.org/0000-0002-
7140-0103; Phone: 858-900-2672; Email: kumar@
arcturusrx.com
2,2′-((((3-(Diethylamino)propyl)thio)carbonyl)azanediyl)diacetic
Acid Bis-pentadecan-8-yl Ester (10z). As described for the synthesis
of 10e, 9m (2.21 g, 4.00 mmol) in CH2Cl2 (50 mL) was treated with
triphosgene (1.19 g, 4.00 mmol) and pyridine (1.58 g, 20.0 mmol),
the mixture was concentrated in vacuo and diluted with pyridine (15
mL), and to the resulting solution (ice-water bath under nitrogen)
was added 3-(diethylamino)-propanethiol-HCl17 (3.67 g, 20.0 mmol)
in one portion. The solution was allowed to warm to room
temperature and stir for 20 h. The reaction was worked up and
purified as described for the preparation of 10e to afford 10z (1.74 g,
2.40 mmol, 68%) as a pale yellow, viscous oil. LC−MS (Shimadzu
2020; ELSD A: water/0.05% TFA: B: CH3CN/0.05% TFA 80:20 to
0:100 A/B at 2.00 min, hold 3.00 min): RT 1.051 min, m/e = 727.6
(M + H+); 1H-NMR (300 mHz, CDCl3): δ = 4.92 (m, 2), 4.21 (s, 2),
4.16 (s, 2), 2.95 (t, J = 7.2 Hz, 2), 2.50−2.62 (6), 1.79 (m, 2), 1.46−
1.58 (8), 1.20−1.37 (40), 1.04 (t, J = 7.2 Hz, 6), 0.87 (m, 12).
2,2′-((tert-Butoxycarbonyl)azanediyl)diacetic Acid Bis-heptade-
can-9-yl Ester (8p). As described for the synthesis of 8n, a solution of
2-[tert-butoxycarbonyl(carboxymethyl)amino]acetic acid (Combi-
Blocks, #SS-1100) (40.0 g, 0.172 mol) in dry dichloromethane
(750 mL) was treated with EDC-HCl (65.94 g, 0.344 mol), 9-
heptadecanol (88.22 g, 0.344 mol), i-Pr2NEt (44.46 g, 0.344 mol,
59.92 mL), and DMAP (4.20 g, 34.4 mmol) to give, after work-up
and purification by column chromatography, 8p (73.29 g, 0.103
Steven P. Tanis − Arcturus Therapeutics Inc., San Diego,
California 92121, United States; SPTanis PharmaChem
Consulting LLC, Carlsbad, California 92011, United States;
orcid.org/0000-0001-5917-8389; Phone: 760-421-4162;
Email: sptanis@sbcglobal.net, steve@arcturusrx.com
Authors
Rajesh Mukthavaram − Arcturus Therapeutics Inc., San Diego,
California 92121, United States
Scott Roberts − Arcturus Therapeutics Inc., San Diego,
California 92121, United States
Michelle Nguyen − Arcturus Therapeutics Inc., San Diego,
California 92121, United States
Kiyoshi Tachikawa − Arcturus Therapeutics Inc., San Diego,
California 92121, United States
Amit Sagi − Arcturus Therapeutics Inc., San Diego, California
92121, United States
Marciano Sablad − Arcturus Therapeutics Inc., San Diego,
California 92121, United States
Pattraranee Limphong − Arcturus Therapeutics Inc., San
Diego, California 92121, United States
S
https://dx.doi.org/10.1021/acs.jmedchem.0c01407
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
D.; Hope, M. J. Rational Design of Cationic Lipids for siRNA
Delivery. Nat. Biotechnol. 2010, 28, 172−176.
Angel Leu − Arcturus Therapeutics Inc., San Diego, California
92121, United States
(5) Jayaraman, M.; Ansell, S. M.; Mui, B. L.; Tam, Y. K.; Chen, J.;
Du, X.; Butler, D.; Eltepu, L.; Matsuda, S.; Narayanannair, J. K.;
Rajeev, K. G.; Hafez, I. M.; Akinc, A.; Maier, M. A.; Tracy, M. A.;
Cullis, P. R.; Madden, T. D.; Manoharan, M.; Hope, M. J. Maximizing
the Potency of siRNA Lipid Nanoparticles for Hepatic Gene Silencing
in vivo. Angew. Chem., Int. Ed. 2012, 51, 8529−8533.
Hailong Yu − Arcturus Therapeutics Inc., San Diego, California
92121, United States
Padmanabh Chivukula − Arcturus Therapeutics Inc., San
Diego, California 92121, United States
Joseph E. Payne − Arcturus Therapeutics Inc., San Diego,
California 92121, United States
Priya Karmali − Arcturus Therapeutics Inc., San Diego,
California 92121, United States
(6) Sabnis, S.; Kumarasinghe, E. S.; Salerno, T.; Mihai, C.; Ketova,
T.; Senn, J. J.; Lynn, A.; Bulychev, A.; McFadyen, I.; Chan, J.;
̈
Almarsson, O.; Stanton, M. G.; Benenato, K. E. A Novel Amino Lipid
Series for mRNA Delivery: Improved Endosomal Escape and
Sustained Pharmacology and Safety in Non-human Primates. Mol.
Ther. 2018, 26, 1509−1519.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01407
(7) Nabhan, J. F.; Wood, K. M.; Rao, V. P.; Morin, J.; Bhamidipaty,
S.; LaBranche, T. P.; Gooch, R. L.; Bozal, F.; Bulawa, C. E.; Guild, B.
C. Intrathecal Delivery of Frataxin mRNA Encapsulated in Lipid
Nanoparticles to Dorsal Root Ganglia as a Potential Therapeutic for
Friedreich’s Ataxia. Sci. Rep. 2016, 6, 1−10.
Notes
The authors declare the following competing financial
interest(s): KR and other Arcturus Therapeutics employees
have stock options some of which may have been exercised.
(8) Patel, S.; Ashwanikumar, N.; Robinson, E.; DuRoss, A.; Sun, C.;
̈
Murphy-Benenato, K. E.; Mihai, C.; Almarsson, O.; Sahay, G.
ABBREVIATIONS USED
■
Boosting Intracellular Delivery of Lipid Nanoparticle-encapsulated
mRNA. Nano Lett. 2017, 17, 5711−5718.
(9) Duff, R. J.; Deamond, S. F.; Roby, C.; Zhou, Y.; Ts’o, P. O. P.
Intrabody Tissue-Specific Delivery of Antisense Conjugates in
Animals: Ligand-Linker-Antisense Oligomer Conjugates. In Methods
in Enzymology, Phillips, M. I., Ed.; Academic Press: New York, 2000;
Vol. 313, pp. 297−321.
APCI, atmospheric pressure chemical ionization; BOC-
anhydride, di-tert-butyl dicarbonate; BOC, tert-butoxy-carbon-
yl; cLogD, calculated LogD, octanol/buffer (pH 7.4); CRISPR,
clustered regularly interspaced short palindromic repeats;
DLin-DMA, 1,2-dilinoleyoxy-3-dimethylaminopropane; DLin-
MC3-DMA, (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tet-
raene-19-yl 4-(dimethylamino)butanoate; DMAP, 4-dimethy-
lamino-pyridine; DMG-PEG, (1,2-dimyristoyl-sn-glycerol, me-
thoxypolyethylene glycol; DSPC, 1,2-distearoyl-sn-glycero-3-
phosphocholine; DMSO, dimethyl sulfoxide; ED50, median
effective dose; EDC-HCl, ethylcarbodimide hydrochloride;
ELSD, evaporative light scattering detection; ESI, electrospray
ionization; FIX, factor IX; FVII, factor VII; HEPES, 2-[4-(2-
hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; LC−MS,
liquid chromatography−mass spectroscopy; LDL-R, low-
density lipoprotein receptor; LNP, lipid nanoparticle;
LUNAR, Lipid-enabled and Unlocked Nucleomonomer
Agent-modified RNA; MES, 2-(N-morpholino)ethanesulfonic
acid; miRNA, microRNA; mRNA, messenger RNA; MS, mass
spectroscopy; MTBE, methyl tert-butyl ether; pKa, acid
dissociation constant; RNA, ribonucleic acid; siRNA, small
interfering RNA; TFA, trifluoroacetic acid; TNS, 6-(p-
toluidinyl)naphthalene-2-sulfonic acid; Tris, 2-amino-2-
(hydroxymethyl)propane-1,3-diol
(10) Nair, J. K.; Willoughby, J. L.; Chan, A.; Charisse, K.; Alam, M.
R.; Wang, Q.; Hoekstra, M.; Kandasamy, P.; Kel’in, A. V.; Milstein, S.;
Taneja, N.; O’Shea, J.; Shaikh, S.; Zhang, L.; van der Sluis, R. J.; Jung,
M. E.; Akinc, A.; Hutabarat, R.; Kuchimanchi, S.; Fitzgerald, K.;
Zimmermann, T.; van Berkel, T. J.; Maier, M. A.; Rajeev, K. G.;
Manoharan, M. Multivalent N-Acetylgalactosamine-Conjugated
siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated
Gene Silencing. J. Am. Chem. Soc. 2014, 136, 16958−16961.
(11) Prakash, T. P.; Graham, M. J.; Yu, J.; Carty, R.; Low, A.;
Chappell, A.; Schmidt, K.; Zhao, C.; Aghajan, M.; Murray, H. F.;
Riney, S.; Booten, S. L.; Murray, S. F.; Gaus, H.; Crosby, J.; Lima, W.
F.; Guo, S.; Monia, B. P.; Swayze, E. E.; Seth, P. P. Targeted Delivery
of Antisense Oligonucleotides to Hepatocytes Using Triantennary N-
Acetyl Galactosamine Improves Potency 10-Fold in Mice. Nucleic
Acids Res. 2014, 42, 8796−8807.
(12) (a)) Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I.
Cationic Lipid Saturation Influences Intracellular Delivery of
Encapsulated Nucelic Acids. J. Controlled Release 2005, 107, 276−
287. (b)) Dose escalation trial to evaluate the safety, tolerability,
pharmacokinetics, and pharmaco-dynamics of intravenous ALN-VSP02 in
patients with advanced solid tumors with liver involvement. Registry of
Federally and Privately Supported Clinical Trials, U.S. National
REFERENCES
■
Institutes of Health.
http://clinicaltrials.gov/ct2/show/
(1) Alnylam Pharmaceuticals. US prescribing information: ONPAT-
TRO (patisiran) lipid complex injection, for intravenous use. https://
www.accessdata.fda.gov/drugsatfda_docs/label/2018/210922s000lbl.
pdf ; accessed on September 29, 2020.
NCT00882180.
(13) Gollob, J. Phase I Dose Escalation Study of ALN-VSP02, a
Novel RNAi Therapeutic for Solid Tumors with Liver Involvement.
In ASCO Annual Meeting; American Society of Clinical Oncology:
2011, 4−8 June, Chicago, USA.
(14) Coelho, T.; Silva, A.; Suhr, O. B.; Adams, D.; Lozeron, P.;
Hawkins, P.; Mant, T.; et al. Final Phase I Safety, Pharmacokinetic
and Pharmacodynamic Results for ALN-TTR0l, a Novel RNAi
Therapeutic for the Treatment of Transthyretin Amyloidosis. In XIII
International Symposium on Amyloidosis; International Society of
Amyloidosis, 10 May 2012, Groningen, Netherlands.
(15) (a)) Maier, M. A.; Jayaraman, M.; Matsuda, S.; Liu, J.; Barros,
S.; Querbes, W.; Tam, Y. K.; Ansell, S. M.; Kumar, V.; Qin, J.; Zhang,
X.; Wang, Q.; Panesar, S.; Hutabarat, R.; Carioto, M.; Hettinger, J.;
Kandasamy, P.; Butler, D.; Rajeev, K. G.; Pang, B.; Charisse, K.;
Fitzgerald, K.; Mui, B. L.; Du, X.; Cullis, P.; Madden, T. D.; Hope, M.
J.; Manoharan, M.; Akinc, A. Biodegradable Lipids Enabling Rapidly
Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi
(2) European Medicines Agency. Summary of opinion (initial
authorization): Onpattro (patisiran) (2018). https://www.ema.
europa.eu/en/documents/smop-initial/chmp-summary-positive-
opinion-onpattro_en.pdf; accessed on September 29, 2020.
(3) MacLachlan, I.; Liposomal Formulations for Nucleic Acid
Delivery. Antisense Drug Technologies, 2nd Ed. Crooke, S. T., Ed.; CRC
Press: Boca Raton, FL, 2007; pp. 237−270.
(4) Sean, C. S.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.; Cho,
C. K.; Sah, D. W. Y.; Stebbing, D.; Crosley, E. J.; Yaworski, E.; Hafez,
I. M.; Dorkin, J. R.; Qin, J.; Lam, K.; Rajeev, K. G.; Wong, K. F.; Jeffs,
L. B.; Nechev, L.; Eiszenhardt, M. L.; Jayaraman, M.; Kazem, M.;
Maier, M. A.; Srinivasulu, M.; Weinstein, M. J.; Chen, Q.; Alvarez, A.;
Barros, S. A.; De, S.; Klimuk, S. K.; Borland, T.; Kosovrasti, V.;
Cantley, W. L.; Tam, Y. K.; Manoharan, M.; Ciufolini, M. A.; Tracy,
M. A.; de Fougerolles, A.; MacLachlan, I.; Cullis, P. R.; Madden, T.
T
https://dx.doi.org/10.1021/acs.jmedchem.0c01407
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Therapeutics. Mol. Ther. 2013, 21, 1570−1578. (b)) See the
Supporting Information for reference 15a).
(16) Harris, J.P.; Heal, D.J.; WO 1994026704A1, 1994.
(17) Prepared from 3-chloro-N,N-diethylpropanamine-HCl by the
method of reference 18. Crude 3-(diethylamino)-propanethiol-HCl
was purified by chromatography on a column of silica gel eluting with
a gradient from CH2Cl2/MeOH (100:0) to CH2Cl2/MeOH (90:10)
1
to give 3-(diethylamino)-propanethiol-HCl as an off-white solid. H-
NMR (400 mHz, CDCl3): δ = 12.20 (brs, 1), 3.15 (brs, 6), 2.69 (brs,
2), 2.20 (brs, 2), 1.26 (m, 6).
(18) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Supuran, C. T.
Carbonic Anhydrase Inhibitors. A General Approach for the
Preparation of Water-Soluble Sulfonamides Incorporatins Polyami-
no-Polycarboxylate Tails and of Their Metal Complexes Possessing
Long-Lasting, Topical Intraocular Pressure-Lowering Properties. J.
Med. Chem. 2002, 45, 1466−1476. Huang, Z.; Li, W.; Wang, R.;
Zhang, F.; Chi, Y.; Wang, D.; Liu, Z.; Zhang, Y.; Matsuura, E.; Liu, Q.
7-Ketocholesteryl-9-carboxynonanoate Induced Nuclear Factor-kappa
B Activation in J774A.1 Macrophages. Life Sci. 2010, 87, 651−657.
Borrelli, S.; Christodoulou, M. S.; Ficarra, I.; Silvani, A.; Cappelletti,
G.; Cartelli, D.; Damia, G.; Ricci, F.; Zucchetti, M.; Dosio, F.;
Passarella, D. New Class of Squalene-based Releasable Nano-
assemblies of Paclitaxel, Podophyllotoxin, Camptothecin and
Epothilone-A. Eur. J. Med. Chem. 2014, 85, 179−190. Jung, S.;
Kwon, W.; Wi, D.; Kim, J.; Ree, B. J.; Kim, Y. Y.; Kim, W. J.; Ree, M.
Hierarchical Self-Assembly and Digital Memory Characteristics of
Crystalline-Amorphous Brush Diblock Copolymers Bearing Electro-
active Moieties. Macromolecules 2016, 49, 1369−1382.
(19) Benenato, K. E.; Kumarasinghe, E. S.; Cornebise, M. WO
2018170306, 2018.
(20) Calculated using ACD Labs Structure Designer v12.0. cLogP
was calculated using ACD Labs version B; cLogD was calculated at
pH = 7.4.
(21) Whitehead, K. A.; Dorkin, J. R.; Vegas, A. J.; Chang, P. H.;
Veiseh, O.; Matthews, J.; Fenton, O. S.; Zhang, Y.; Olejnik, K. T.;
Yesilyurt, V.; Chen, D.; Barros, S.; Klebanov, B.; Novobrantseva, T.;
Langer, R.; Anderson, D. G. Degradable Lipid Nanoparticles with
Predictable in vivo siRNA Delivery Activity. Nat. Commun. 2014, 5,
4277.
(22) Ramaswamy, S.; Tonnu, N.; Tachikawa, K.; Limphong, P.;
Vega, J. B.; Karmali, P. P.; Chivukula, P.; Verma, I. M. Systemic
Delivery of Factor IX Messenger RNA for Protein Replacement
Therapy. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, E1941−E1950.
U
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J. Med. Chem. XXXX, XXX, XXX−XXX