Incorporation of Phosphonate into Benzonaphthyridine Toll-like Receptor 7 Agonists for Adsorption to Aluminum Hydroxide

Article abstract of DOI:10.1021/acs.jmedchem.6b00489

Small molecule Toll-like receptor 7 (TLR7) agonists have been used as vaccine adjuvants by enhancing innate immune activation to afford better adaptive response. Localized TLR7 agonists without systemic exposure can afford good adjuvanticity, suggesting peripheral innate activation (non-antigen-specific) is not required for immune priming. To enhance colocalization of antigen and adjuvant, benzonaphthyridine (BZN) TLR7 agonists are chemically modified with phosphonates to allow adsorption onto aluminum hydroxide (alum), a formulation commonly used in vaccines for antigen stabilization and injection site deposition. The adsorption process is facilitated by enhancing aqueous solubility of BZN analogs to avoid physical mixture of two insoluble particulates. These BZN-phosphonates are highly adsorbed onto alum, which significantly reduced systemic exposure and increased local retention post injection. This report demonstrates a novel approach in vaccine adjuvant design using phosphonate modification to afford adsorption of small molecule immune potentiator (SMIP) onto alum, thereby enhancing co-delivery with antigen.

Full text of DOI:10.1021/acs.jmedchem.6b00489

Article
pubs.acs.org/jmc
Incorporation of Phosphonate into Benzonaphthyridine Toll-like
Receptor 7 Agonists for Adsorption to Aluminum Hydroxide
Alex Cortez,† Yongkai Li,† Andrew T. Miller,† Xiaoyue Zhang,† Kathy Yue,† Jillian Maginnis,†
Janice Hampton,† De Shon Hall,† Michael Shapiro,† Bishnu Nayak,†,∥ Ugo D’Oro,§ Chun Li,†
David Skibinski,§ M. Lamine Mbow,‡,⊥ Manmohan Singh,‡ Derek T. O’Hagan,‡ Michael P. Cooke,†
Nicholas M. Valiante,‡,# and Tom Y.-H. Wu*,†
†Genomics Institute of Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, United States
‡GSK Vaccines, 45 Sydney Street, Cambridge, Massachusetts 02139, United States
§GSK Vaccines, Via Florentina, 1, 53100, Siena, Italy
S
* Supporting Information
ABSTRACT: Small molecule Toll-like receptor 7 (TLR7) agonists have been used as vaccine adjuvants by enhancing innate
immune activation to afford better adaptive response. Localized TLR7 agonists without systemic exposure can afford good
adjuvanticity, suggesting peripheral innate activation (non-antigen-specific) is not required for immune priming. To enhance
colocalization of antigen and adjuvant, benzonaphthyridine (BZN) TLR7 agonists are chemically modified with phosphonates to
allow adsorption onto aluminum hydroxide (alum), a formulation commonly used in vaccines for antigen stabilization and
injection site deposition. The adsorption process is facilitated by enhancing aqueous solubility of BZN analogs to avoid physical
mixture of two insoluble particulates. These BZN-phosphonates are highly adsorbed onto alum, which significantly reduced
systemic exposure and increased local retention post injection. This report demonstrates a novel approach in vaccine adjuvant
design using phosphonate modification to afford adsorption of small molecule immune potentiator (SMIP) onto alum, thereby
enhancing co-delivery with antigen.
these interactions, ligand exchange through phosphates has
been determined to afford the strongest adsorption. We
thought to leverage this physical interaction to create a small
molecule adjuvant that can be adsorbed onto alum, as well as
the antigen, to maximize antigen/adjuvant co-delivery.
Recently, we have described the rational design of a series of
BZN TLR7 agonists for use as vaccine adjuvants.7 In this
report, we disclose the BZN pharmacophore modified with
phosphonate group for adsorption onto aluminum hydroxide
Al(OH)3 (Figure 1). Structure−activity relationship, alum
adsorption study, and impact on pharmacokinetics (PK) are
described.
INTRODUCTION
■
Currently, several adjuvants have been approved for use in
human vaccines. According to O’Hagan and De Gregorio,
vaccine adjuvants can be classified into particulates/emulsions
(generation 1) or synthetic component that activates the
immune system (generation 2).1 Alum is a mineral salt used in
many commercial vaccines.2 It is believed that alum particulates
increase the effectiveness of vaccines by inducing local
inflammation to help recruit antigen-presenting cells to the
injection site, in addition to stabilizing the antigen and creating
a depot at the injection site.3 MF59 is a water-in-oil emulsion
licensed for use in flu vaccine. Its mechanism of action appears
to be the creation of a transient “immunocompetent” local
environment at the injection site, resulting in the recruitment of
key immune cells for antigen uptake.4 AS04 is composed of
alum and monophosphoryl lipid A (MPLA) and is used in
vaccines against hepatitis B virus and human papilloma virus.5
MPLA is a detoxified TLR4 agonist derived from cell wall
lipopolysaccharide (LPS) of Gram-negative bacteria.6
CHEMISTRY
■
On the basis of our understanding of BZN TLR7 agonist
structure−activity relationship, chemical modification was
focused at the C8 position on the core (R1) and at the C4′
position on the phenethyl ring (R2). Preparation of
Proteins can adsorb onto alum through hydrophobic
Received: April 1, 2016
interaction, electrostatic attraction, and ligand exchange.3 Of
© XXXX American Chemical Society
A
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bond and deprotection of the MOM group at R2 to afford
phenol 30, which was subjected to further derivatization with
various phosphonate-containing alkyl halides. Alkylation
generally proceeded smoothly with either sodium hydride or
cesium carbonate as the base. Phosphonate ester deprotection
with trimethylsilyl bromide, followed by carboxylic ester
hydrolysis with aqueous sodium hydroxide, afforded phos-
phonic acids 31a−d. The high polarity of these final
phosphonates generally required the use of reverse-phase
HPLC for purification.
Figure 1. Benzonaphthyridine TLR7 pharmacophore modified with
phosphonate group for adsorption onto aluminum hydroxide through
ligand exchange.
phosphonates at R1 was described in Schemes 1 and 2. The
previously described benzyl alcohol 1b8 was oxidized to the
aldehyde 2, which then underwent an Abramov reaction with
tris(trimethylsilyl) phosphite followed by another oxidation to
yield keto-phosphonate 3 (Scheme 1). Aldehyde 2 can also
react with lithiated difluoromethyl phosphonate ester to
generate 4, which is further oxidized and deprotected to give
α-difluoro keto-phosphonate 6. To prepare alkyl phosphonates
(Scheme 2), Horner−Wadsworth−Emmons reactions were
carried out using either methylene bis-phosphonate ester or
fluoromethylene bis-phosphonate ester with aldehyde 2 to yield
exclusively the (E)-alkenes (7 and 8). These intermediates can
be deprotected to give the unsaturated phosphonic acids 9 and
10 or hydrogenated and deprotected to give saturated
phosphonic acid 12.
The BZN core was modified at R2 by alkylation of the
previously described phenol 138 with various alkyl/benzyl
halides containing phosphonate or phosphonate precursor
(Scheme 3). The alkylation reaction generally tolerated internal
ethylene glycol units and the presence of α-fluorine atoms next
to the phosphonate. Phosphonate on 15 was introduced via a
Pd-mediated cross-coupling of triethyl phosphite with aryl
iodide 14.9 Deprotection using trimethylsilyl bromide in
dichloromethane served as a general condition to afford the
final phosphonic acids 15, 16a−d.
RESULTS AND DISCUSSION
■
Although proteins bearing phosphate groups (OPO3H2) were
known to have ligand exchange interaction with aluminum
hydroxide,3 we hypothesized that phosphonate (CPO3H2)
would retain high affinity toward alum but that C−P bond was
more stable than O−P bond toward biological phosphatase
hydrolysis. SAR studies were carried out around 2-(phenethyl)-
8-methylbenzonaphthyridine (1a), a TLR7 agonist pharmaco-
phore previously described by our lab.7,8 Phosphonate groups
were introduced on either R1 or R2 and tested in human
embryonic kidney (HEK) TLR7 reporter, IL-6 mouse
splenocyte, and IL-6 human peripheral blood mononuclear
cell (hPBMC) assays. For R1 substitution, carbonyl (3, 6),
alkenyl (9, 10), and alkyl phosphonates (12) all showed
appreciable activity across the assays (Table 1). Compound 12
suggested that a two-carbon alkyl chain is an optimal spacer
between the pharmacophore and the R1 phosphonate.
Phosphonate installation at R2 position generally involved O-
alkylation of intermediate 13 or 30. Different methylene units
(CH2) or ethylene glycol units [(CH2)2O] were explored as
linkers between the benznonaphthyridine pharmacophore and
R2 phosphonate groups. Switching from R1 = methyl to
propionic acid generally boosted activity of corresponding R2
analogs (31a vs 16a, 31b vs 16b, 31c vs 16c, 31d vs 16d). It is
unclear whether the length of R2 linkers has a direct impact on
the in vitro activity. Since phosphonate analogs were more
soluble in water as the sodium salt than in DMSO, all in vitro
assays were performed using 10 mM aqueous stock solutions of
the monosodium salt (prepared in situ by adding 1 equiv of 1 N
NaOH). In general, there is a 2- to 10-fold upward EC50 shift
from HEK-TLR7 reporter cell line to primary cell assays, but
most analogs still exhibit activities in the micromolar range
(Table 1). The percentage of maximum IL-6 induction is much
To improve the aqueous solubility of BZN-phosphonate, a
propionic acid side chain was installed at R1. This was achieved
by first preparing the boronic ester 22 in three steps starting
from Boc-protected 5-bromo-2-chloroaniline 17 (Scheme 4).
Chemoselective hydrogenation of the alkene on 19 without
reducing the aryl chloride was achieved using Wilkinson’s
catalyst. Boronic ester 22 was coupled with functionalized 3-
chloropicolinonitrile 27 using Pd-mediated cross-coupling
(Scheme 5). This was followed by hydrogenation of the triple
a
Scheme 1. Preparation of Keto-phosphonates at R1
a
Reagents and conditions: (a) IBX, DMSO; (b) (TMSO)3P, Tol, 80 °C; (c) IBX, DMSO; (d) (EtO)2P(O)CF2H, LDA, THF, − 78 °C; (e) IBX,
DMSO/EtOAc, 80 °C; (f) TMSI, DCM, 0 °C.
B
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a
Scheme 2. Preparation of Alkyl Phosphonates at R1
a
Reagents and conditions: (a) NaH, [(OEt)2P(O)]2CH2, THF 0 °C (for 7) or LDA, [(OEt)2P(O)]2CFH, THF − 78 °C (for 8); (b) TMSBr,
DCM, 0 °C; (c) H2, Pd/C, EtOH, DCM; (d) TMSBr, DCM, 0 °C.
a
Scheme 3. Preparation of Alkoxyphosphonates at R2
a
Reagents and conditions: (a) Cs2CO3, 1-(bromomethyl)-3-iodobenzene, DMF; (b) (EtO)3P, Pd(OAc)2, 90 °C; (c) TMSBr, DCM, 0 °C; (d) NaH,
Br(CH2)3PO(OEt)2, DMF; (e) NaH, Br(CH2)3CF2PO(OEt)2, DMF; (f) NaH, I(CH2)2O(CH2)2O(CH2)2PO(OEt)2, DMF; (g) NaH,
I(CH2)2O(CH2)2O(CH2)2CF2PO(OEt)2, DMF.
a
Scheme 4. Preparation of Boronic Ester Suzuki Cross-Coupling Partner 22
a
Reagents and conditions: (a) 18, Pd(PPh3)4, K2CO3, Tol/EtOH, 100 °C; (b) Wilkinson’s catalyst, H2, EtOAc/EtOH; (c) 21, Pd2(dba)3, Xphos,
KOAc, dioxane, 100 °C.
lower in hPBMC compared to mouse splenocytes likely
because the reference compound resiquimod is a TLR7/8
agonist and can activate more cell types compared to TLR7-
selective agonists within human PBMC mixed cell population.
On the contrary, mouse lacks functional TLR8; therefore,
mouse splenocytes respond to TLR7 agonists similarly to
TLR7/8 agonist, as indicated by the similar percentage max
activation.10 All of the BZN-phosphonates were tested to be
TLR7-selective agonists, with no appreciable TLR8 activity
(<5% relative to resiquimod) tested up to 30 μM. (For
comparison, resiquimod HEK-TLR8 EC50 = 4.0 μM, set to
100%.)
The process of alum adsorption involved first the preparation
of a solution of BZN-phosphonate in an appropriate buffer
(e.g., histidine, pH 6.5) and then adding to a suspension of
aluminum hydroxide particulate. To avoid creating a physical
comixture of two insoluble particulates, BZN-phosphonates
needed to be completely soluble. Since antigens are expected to
be adsorbed onto the same alum formulation, the pH range
should not be detrimental to the fragile proteins. Hence, BZN-
phosphonates must show high aqueous solubility within pH
C
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a
Scheme 5. Preparation of Propionic Acid at R1 and Alkoxyphosphonates at R2
a
Reagents and conditions: (a) NaH, MOMCl, DMF; (b) CuI, Pd(PPh3)2Cl2, TES-acetylene, Et3N, DMF, 60 °C; (c) TBAF, THF; (d) CuI,
Pd(PPh3)2Cl2, Et3N, DMF, 60 °C; (e) 22, Pd2(dba)3, NaHCO3, dicyclohexyl(2′,6′-dimethoxybiphenyl-2-yl)phosphine, n-BuOH/H2O, 100 °C; (f)
H2, Pd/C, EtOH/THF; (g) HCl/EtOH; (h) Cs2CO3, Br(CH2)3PO(OEt)2, DMF; (i) TMSBr, DCM, 0 °C; (j) NaOH, EtOH, 80 °C; (k) Cs2CO3,
Br(CH2)3CF2PO(OEt)2, DMF; (l) Cs2CO3, I(CH2)2O(CH2)2O(CH2)2PO(OEt)2, DMF; (m) Cs2CO3, I(CH2)2O(CH2)2O(CH2)2CF2PO(OEt)2,
DMF.
Table 1. In Vitro Activities of Benzonaphthyridines Phosphonates
a
a
a
compd
R1
R2
HEK-TLR7
IL-6 mouse spleno
IL-6 human PBMC
3
C(O)PO3H2
C(O)CF2PO3H2
(E)-CHCHPO3H2
(E)-CHCFPO3H2
CH2CH2PO3H2
Me
OMe
5.6 (112%)
0.39 (87%)
14 (117%)
3.4 (100%)
2.5 (81%)
not active
7.8 (56%)
1.7 (90%)
3.4 (40%)
0.40 (107%)
30 (26%)
6
OMe
9
OMe
0.15 (148%)
0.20 (100%)
0.090 (146%)
0.23 (94%)
0.31 (97%)
3.1 (94%)
10
OMe
0.57 (220%)
0.13 (124%)
1.1 (98%)
12
OMe
16a
16b
16c
16d
31a
31b
31c
31d
O(CH2)3PO3H2
Me
O(CH2)3CF2PO3H2
3.2 (47%)
1.6 (31%)
8.2 (55%)
0.98 (36%)
0.29 (46%)
0.36 (35%)
0.49 (58%)
2.1 (35%)
Me
O(CH2)2O(CH2)2O(CH2)2PO3H2
O(CH2)2O(CH2)2O(CH2)2CF2PO3H2
O(CH2)3PO3H2
3.6 (83%)
Me
1.2 (95%)
0.81 (100%)
0.31 (100%)
2.4 (74%)
CH2CH2CO2H
CH2CH2CO2H
CH2CH2CO2H
CH2CH2CO2H
0.065 (107%)
0.24 (95%)
0.35 (110%)
0.039 (100%)
O(CH2)3CF2PO3H2
O(CH2)2O(CH2)2O(CH2)2PO3H2
O(CH2)2O(CH2)2O(CH2)2CF2PO3H2
0.19 (140%)
1.2 (80%)
a
EC50 in μM. Shown in parentheses are % of max activation in reference to resiquimod (internal standard), which is set at 100%. Resiquimod
typically produced 600−1200 pg/mL IL-6 in mSpleno assays and 20 000−12 500 pg/mL IL-6 in hPBMC assays. Data represent the median of at
least two experiments performed in triplicate.
range 6.5−7.2 in order to be efficiently coadsorbed onto alum,
together with the antigens. The initial phosphonate analog 16a
with hydrocarbon linker at R2 showed poor solubility at neutral
pH (Figure 2). It did, however, show increasing solubility with
increasing pH, suggesting fraction of ionization (FI) is greatly
enhanced when the phosphonate is fully deprotonated. We
hypothesized that placement of fluorine atoms α to the
phosphonate group can decrease the pKa, resulting in higher
fraction of ionization (FI) at more neutral pH (16b). Insertion
of polyethylene glycol linker also provided added solubility
(16d). Finally, installation of another ionizable group
(propionic acid) at R1 further increased solubility within the
D
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Figure 2. pH-dependent solubility of selected BZN-phosphonate
analogs. The acceptable pH range for general protein antigen stability
is 6.5−7.2. Each line represented one compound.
desired pH range (31c). The fraction of BZN-phosphonate
bound to alum was determined indirectly from unbound
fraction in the supernatant after centrifugation removal of
insoluble alum particulate (Table 2). Alum adsorption of
Figure 3. PK of BZN-phosphonate 16d. Balb/C mice (3 per group)
were injected intramuscularly with 100 μg of compounds with or
without alum. (a) Compound concentrations in serum were measured
at the indicated time points after injection. (b) Compound
concentration in the injected muscles were measured 24 h after
injection. Data represent the median SD.
Table 2. Benzonaphthyridine Phosphonate Adsorption
Efficiency onto Alum
compd
% adsorbed onto alum
a
16a
16d
31b
31c
31d
nd (insoluble)
98
95
97
97
other reports of similar concept utilizing different delivery
methods. Local retention can be enhanced by manipulating
physicochemical properties,13 attaching phospholipid,14 and
forming dendrimers.15 Tissue restriction can be achieved by
conjugation of SMIP onto macromolecules such as antigen,16
albumin,17 or antibody.18 Finally, small molecule adjuvants can
be delivered by polymer encapsulation technologies.19,20 All
these modalities aim to facilitate antigen-specific immune
response (exogenous or cancer) and reduce unnecessary
systemic immune activation. It remains to be seen which will
have significant translational impact in clinical settings.
a
Adsorption efficiency cannot be determined using protocols
described in Experimental Section due to poor compound solubility
at pH range tested (6.5−7.2).
phosphonate 16a could not be accurately determined due to
insolubility. All soluble BZN-phosphonates tested show high
degree of adsorption (>95%) in the conditions described.
Addition of inorganic phosphate to the alum-bound for-
mulations can desorb the TLR7 agonist, suggesting the
adsorption is a reversible process (data not shown).
Previously, we have reported that benzonaphthyridine TLR7
agonists can exhibit different PK profiles with different
physicochemical properties.7 As expected, intramuscular
injection of soluble BZN-phosphonate 16d in aqueous buffer
afforded high systemic exposure and low muscle concentration
at 24 h (Figure 3). These are properties not desired for safe and
effective adjuvants. Consistent with our design, when 16d is
adsorbed onto aluminum hydroxide, the formulation gave
significantly reduced systemic Cmax and increased injection site
muscle retention. Alum created a local deposition for the
adsorbed BZN-phosphonate, which is slowly released over
time, likely from competition by small organic ions within the
interstitial fluid (e.g., citrate).3
The BZN-phosphonates described here were extensively
studied in mouse vaccine models and discussed in separate
publications.7,11 Alum adsorption offers one way to increase
injection site local retention, reduce systemic exposure, and co-
deliver antigen and adjuvant. Lu et al. have described a similar
method of modifying antigen with phosphonate linker to
enhance alum adsorption, which resulted in more efficient
(antigen-sparing) vaccination.12 Recently, there have been
CONCLUSION
■
Localized innate immune activation is a key attribute for safe
and effective adjuvant. Here, low molecular weight TLR7
agonist is locally retained at the injection site not by its
nonpolarity or insolubility but by physical adsorption onto
alum, a particulate used to deliver protein antigens. Adsorption
of antigens to alum is commonly found in many commercial
human vaccines. Phosphonate modification of BZN TLR7
agonists allows coadsorption and colocalization of antigen and
adjuvant to potentially elicit greater antigen-specific immune
response.
EXPERIMENTAL SECTION
■
Chemistry. All reagents were obtained from commercial vendors
unless specified. Nuclear magnetic resonance (1H and 13C NMR)
spectra were obtained with a Bruker spectrometer operating at 400
MHz. Chemical shifts are given in parts per million (δ, ppm) and are
referenced to the solvent in which they were run. Carbon, hydrogen,
and nitrogen analyses were performed by Midwest Microlab, LLC,
Indianapolis, IN, and were within 0.4% of the theoretical values unless
otherwise indicated. Flash column chromatography was performed on
an ISCO CombiFlash system using Redisep normal phase disposable
columns (Teledyne ISCO, Lincoln, NE). For compound elution, a
gradient of either methanol in dichloromethane or ethyl acetate in
E
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hexanes was used as the mobile phase. The structures of all
compounds were consistent with their spectral data. Unless noted
otherwise, the reported yields were not optimized. All biologically
evaluated compounds were determined to have purity of >95% using
an Agilent LCMS system (C18 column; eluting gradient 10−90%
acetonitrile in water).
(s, 1H), 6.68 (d, 1H, J = 8.3 Hz), 4.39−4.28 (m, 4H), 3.73 (s, 3H),
3.21 (t, 2H, J = 8.5 Hz), 3.06 (t, 2H, J = 8.5 Hz), 2.30 (s, 3H), 1.37−
1.34 (m, 6H). LRMS [M + H] = 558.2
To a solution of 5 (29.0 mg, 0.0521 mmol, 1.0 equiv) in DCM (1
mL, 0.05 M) at 0 °C was added TMSI (37 μL, 0.26 mmol, 5.0 equiv).
The reaction was warmed to room temperature over 2 h, and more
TMSI was added (19 μL, 0.13 mmol, 2.5 equiv). The reaction was
stirred for another 30 min and then quenched with small amounts of
water. The DCM was removed by evaporation, and then DMSO/
water was added. The mixture was adjusted to pH 9 and directly
purified on RP-HPLC using a C18 column, eluting with 10−40% 95:5
(MeCN/5 mM NH4OAc) in 10 mM NH4OAc (pH 9) gradient. The
fractions containing the product were combined and concentrated in
vacuo to give 6 (28 mg, 88%) as a solid. 1H NMR (dimethylsulfoxide-
d6): δ 8.82 (s, 1H), 8.5 (br, 1H), 8.44 (s, 1H), 8.2 (br, 1H), 7.98 (d,
1H, J = 8.2 Hz), 7.2 (br, 2H), 7.05 (d, 1H, J = 8.3 Hz), 6.73 (s, 1H),
6.67 (d, 1H, J = 8.3 Hz), 3.70 (s, 3H), 2.99−2.87 (m, 4H), 2.25 (s,
3H). LRMS [M + H] = 502.2
(E)-2-(5-Amino-2-(4-methoxy-2-methylphenethyl)benzo[f ]-
[1,7]naphthyridin-8-yl)vinylphosphonic Acid (9). Scheme 2a,b.
To a stirred suspension of NaH (8 mg, 60% in mineral oil, 0.19 mmol,
1.2 equiv) in THF (2 mL, 0.1 M) cooled at 0 °C was added a solution
of tetraethyl methylenediphosphonate (61 mg, 0.21 mmol, 1.3 equiv)
in THF (1 mL, 0.21 M). To the resulting reaction mixture was added a
solution of 5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f ][1,7]-
naphthyridine-8-carbaldehyde (2) (60 mg, 0.16 mmol, 1.0 equiv) in
THF (2 mL, 0.08 M). The reaction was stirred at rt for 30 min, then
solvents were removed in vacuo, and the resulting residue was purified
by a CombiFlash system (ISCO) using a gradient of 0−5% MeOH/
DCM to provide (E)-diethyl 2-(5-amino-2-(4-methoxy-2-
methylphenethyl)benzo[f ][1,7]naphthyridin-8-yl)vinylphosphonate
(7) as colorless solid (32 mg, 40%). To a solution of 7 (48 mg, 0.095
mmol, 1.0 equiv) in DCM (1 mL, 0.095 M) at 0 °C was added TMSBr
(145 mg, 0.95 mmol, 10 equiv). The reaction was warmed to room
temperature over 2 h and then quenched with small amounts of
MeOH. The DCM was removed by evaporation, and then DMSO/
water was added. The mixture was adjusted to pH 9 and directly
purified on RP-HPLC using a C18 column, eluting with 10−40% 95:5
(MeCN/5 mM NH4OAc) in 10 mM NH4OAc (pH 9) gradient. The
fractions containing the product were combined and concentrated in
vacuo to give 9 (25 mg, 59%) as a solid. 1H NMR (dimethylsulfoxide-
d6): δ 9.76 (s, 1H), 9.33 (s, 1H), 9.03 (s, 1H), 8.82 (s, 1H), 8.60 (d,
1H, J = 8.4 Hz), 7.87 (d, 1H, J = 8.4 Hz), 7.78 (s, 1H), 7.31 (dd, 1H, J
= 17.6, 21.6 Hz), 7.03 (d, 1H, J = 8.4 Hz), 6.69 (m, 2H), 6.61 (dd, 1H,
J = 2.8, 8.4 Hz), 3.64 (s, 3H), 3.14−3.06 (m, 2H), 2.97−2.91 (m, 2H),
2.23 (s, 3H). LRMS [M + H] = 450.2
(E)-2-(5-Amino-2-(4-methoxy-2-methylphenethyl)benzo[f ]-
[1,7]naphthyridin-8-yl)-1-fluorovinylphosphonic Acid (10).
Scheme 2a,b. To a stirred solution of tetraethyl fluoromethylene-
diphosphonate (412 mg, 1.35 mmol, 2.5 equiv) in THF (5 mL, 0.27
M) cooled at −78 °C was added LDA solution (1.8 M in
ethylbenzene/pentane/hexane, 0.60 mL, 1.08 mmol, 2.0 equiv). The
resulting reaction mixture was warmed up to rt and stirred for 30 min
before it was cooled back down to −78 °C. A solution of 5-amino-2-
(4-methoxy-2-methylphenethyl)benzo[f ][1,7]naphthyridine-8-carbal-
dehyde (2) (200 mg, 0.54 mmol, 1.0 equiv) in THF (3 mL, 0.18 M)
was added, and the reaction mixture was allowed to warm up to rt
slowly. The reaction was quenched with saturated aqueous NH4Cl
solution. Aqueous phase was extracted with DCM (3×). The
combined organic phases were combined and concentrated in vacuo.
The residue was purified by a CombiFlash system (ISCO) using a
gradient of 0−5% MeOH/DCM to provide (E)-diethyl 2-(5-amino-2-
(4-methoxy-2-methylphenethyl)benzo[f ][1,7]naphthyridin-8-yl)-1-
fluorovinylphosphonate (8) as a colorless solid (137 mg, 48%). To a
solution of 8 (137 mg, 0.26 mmol, 1.0 equiv) in DCM (5 mL, 0.05 M)
at 0 °C was added TMSBr (401 mg, 2.6 mmol, 10 equiv). The reaction
was warmed to room temperature over 2 h and then quenched with
small amounts of MeOH. The DCM was removed by evaporation, and
then DMSO/water was added. The mixture was adjusted to pH 9 and
directly purified on RP-HPLC using a C18 column, eluting with 10−
5-Amino-2-(4-methoxy-2-methylphenethyl)benzo[f ][1,7]-
naphthyridine-8-carbonylphosphonic Acid (3). Scheme 1a−c.
The synthesis of compound 1b is described in ref 8, example 108. To a
solution of (5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f ][1,7]-
naphthyridin-8-yl)methanol (1b) (0.560 g, 1.50 mmol, 1.0 equiv) in
DMSO (10 mL, 0.15 M) at room temperature was added IBX (0.630
g, 2.25 mmol, 1.5 equiv). The reaction was stirred for 2.5 h and then
diluted with water. The aqueous layer was extracted with 2% MeOH/
DCM (4×). The combined organic layers were dried over anhydrous
MgSO4 and concentrated in vacuo. The resulting residue was purified
by a CombiFlash system (ISCO) using a gradient of 0−5% MeOH/
DCM to provide 5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f ]-
[1,7]naphthyridine-8-carbaldehyde (2) (325 mg, 58%) as a solid. To a
stirred suspension of 2 (100 mg, 0.27 mmol, 1.0 equiv) in toluene (1
mL, 0.27 M) was added tris(trimethylsilyl) phosphite (81 mg, 0.27
mmol, 1.0 equiv). The reaction was stirred at 80 °C for 60 min, then
solvents were removed, and the resulting residue was taken up in
DMSO (1 mL, 0.27 M), and IBX (113 mg, 0.41 mmol, 1.5 equiv) was
added. The reaction was stirred at rt for 2.5 h, filtered, and directly
purified on RP-HPLC using a C18 column, eluting with 10−40% 95:5
(MeCN/5 mM NH4OAc) in 10 mM NH4OAc (pH 9) gradient. The
fractions containing the product were combined and concentrated in
1
vacuo to give 3 (12 mg, 9%) as a solid. H NMR (dimethylsulfoxide-
d6): δ 9.84 (s, 1H), 9.35 (s, 1H), 9.09 (s, 1 H), 8.89 (s, 1 H), 8.76 (d,
1H, J = 8.4 Hz), 8.60 (s, 1H), 8.19 (d, 1H, J = 8.8 Hz), 7.04 (d, J = 8.8
Hz, 1H), 6.70 (d, 1H, J = 2.8 Hz), 6.62 (dd, 1H, J = 2.8, 8.4 Hz), 3.64
(s, 3H), 3.15−3.09 (m, 2H), 2.97−2.91 (m, 2H), 2.23 (s, 3H). LRMS
[M + H] = 452.1.
2-(5-Amino-2-(4-methoxy-2-methylphenethyl)benzo[f ][1,7]-
naphthyridin-8-yl)-1,1-difluoro-2-oxoethylphosphonic Acid
(6). Scheme 1d−f. To a solution of diethyl difluoromethylphosph-
onate (244 mg, 1.30 mmol, 3.0 equiv) in THF (4 mL, 0.3 M) at −78
°C under nitrogen atmosphere was added dropwise 2 M LDA (0.65
mL, 1.3 mmol, 3.0 equiv, commercial grade). The reaction was stirred
at −78 °C for 25 min, and a solution of 5-amino-2-(4-methoxy-2-
methylphenethyl)benzo[f ][1,7]naphthyridine-8-carbaldehyde (2)
(0.160 g, 0.431 mmol, 1.0 equiv) in THF (4 mL, 0.1 M) was added
slowly. The reaction was stirred at −78 °C for 1 h, 0 °C for 1 h, and
then warmed to room temperature over 30 min. The reaction was
quenched with saturated aqueous ammonium chloride solution and
extracted twice with ethyl acetate. The combined organic layers were
washed with brine, dried over anhydrous MgSO4, and concentrated in
vacuo. The resulting residue was purified by a CombiFlash system
(ISCO) using a gradient of 0−5% MeOH/DCM to provide diethyl 2-
(5-amino-2-(4-methoxy-2-methylphenethyl)benzo[f ][1,7]-
naphthyridin-8-yl)-1,1-difluoro-2-hydroxyethylphosphonate (4) (79
1
mg, 33%) as a solid. H NMR (acetone-d6): δ 8.79 (s, 1H), 8.74 (s,
1H), 8.37 (d, 1H, J = 8.4 Hz), 7.94 (s, 1H), 7.45 (d, 1H, J = 8.4 Hz),
7.08 (d, 1H, J = 8.4 Hz), 6.8 (br, 2H), 6.73 (s, 1H), 6.68 (d, 1H, J =
8.4 Hz), 5.36 (dt, 1H, J = 21.4, 6.1 Hz), 4.41−4.31 (m, 4H), 3.73 (s,
3H), 3.19−3.02 (m, 4H), 2.28 (s, 3H), 1.37−1.26 (m, 6H). LRMS [M
+ H] = 560.2
To a solution of 4 (79.0 mg, 0.141 mmol, 1.0 equiv) in 1:1 DMSO/
ethyl acetate (2 mL, 0.07 M) was added IBX (60.0 mg, 0.214 mmol,
1.5 equiv). The reaction was heated to 80 °C for 1 h and then cooled
to room temperature. The mixture was diluted with ethyl acetate and
filtered through Celite. The filtrate was washed with water (2×), brine,
dried over anhydrous MgSO4, and concentrated in vacuo. The
resulting residue was purified by a CombiFlash system (ISCO) using a
gradient of 0−5% MeOH/DCM to provide diethyl 2-(5-amino-2-(4-
methoxy-2-methylphenethyl)benzo[f ][1,7]naphthyridin-8-yl)-1,1-di-
1
fluoro-2-oxoethylphosphonate (5) (29 mg, 37%) as a solid. H NMR
(acetone-d6): δ 8.92 (s, 1H), 8.85 (s, 1H), 8.59 (d, 1H, J = 8.5 Hz),
8.44 (s, 1H), 7.96 (d, 1H, J = 8.5 Hz), 7.09 (d, 1H, J = 8.5 Hz), 6.75
F
DOI: 10.1021/acs.jmedchem.6b00489
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Journal of Medicinal Chemistry
Article
40% 95:5 (MeCN/5 mM NH4OAc) in 10 mM NH4OAc (pH 9)
gradient. The fractions containing the product were combined and
concentrated in vacuo to give 10 (60 mg, 49%) as a solid. H NMR
A representative example for the synthesis of compounds 16a−d is
illustrated with the synthesis of 16d.
1
3-(2-(2-(4-(2-(5-Amino-8-methylbenzo[f ][1,7]naphthyridin-
2-yl)ethyl)-3-methylphenoxy)ethoxy)ethoxy)-1,1-
difluoropropylphosphonic Acid (16d). Scheme 3g,c. Repre-
sentative Preparation of Fluorophosphonate Electrophiles:
Diethyl 1,1-difluoro-3-(2-(2-iodoethoxy)ethoxy)-
propylphosphonate. To a solution of diethyl difluoromethyl-
phosphonate (2.00 mL, 12.7 mmol, 1.0 equiv) in THF (16 mL, 0.8
M) at −78 °C was slowly added a solution of LDA (7.00 mL, 14.0
mmol, 2.0 M, 1.1 equiv) in heptane/THF/ethylbenzene, and the
mixture was vigorously stirred for 30 min. In a separate reaction flask, a
solution of 1,2-bis(2-iodoethoxy)ethane (4.70 g, 12.7 mmol, 1.0 equiv)
in THF (16 mL, 0.8 M) was cooled to −78 °C. To this solution was
transferred, by cannula, the freshly prepared alkyllithium solution, and
the reaction mixture was allowed to stir for 1 h at −78 °C. At this
point, the cooling bath was removed and the reaction mixture was
allowed to warm to room temperature. The reaction mixture was then
quenched with a 1 M aqueous solution of HCl. The resulting mixture
was transferred to a separatory funnel and washed with CH2Cl2 three
times. The combined organic layers were dried over anhydrous
Na2SO4, and the volatiles were removed in vacuo. The resulting
residue was purified by a CombiFlash system (ISCO) using CH2Cl2 to
provide diethyl 1,1-difluoro-3-(2-(2-iodoethoxy)ethoxy)-
(dimethylsulfoxide-d6): δ 9.80 (s, 1H), 9.41 (s, 1H), 9.05 (s, 1H), 8.87
(s, 1H), 8.65 (d, 1H, J = 8.8 Hz), 8.08 (s, 1H), 7.76 (d, 1H, J = 8.4
Hz), 7.08 (d, 1H, J = 8.4 Hz), 7.02 (d, 1H, J = 8.4 Hz), 6.83−6.65 (m,
2H), 3.69 (s, 3H), 3.18−3.12 (m, 2H), 3.02−2.96 (m, 2H), 2.28 (s,
3H). LRMS [M + H] = 468.1
2-(5-Amino-2-(4-methoxy-2-methylphenethyl)benzo[f ][1,7]-
naphthyridin-8-yl)ethylphosphonic Acid (12). Scheme 2c,d. To
a
solution of (E)-diethyl 2-(5-amino-2-(4-methoxy-2-
methylphenethyl)benzo[f ][1,7]naphthyridin-8-yl)vinylphosphonate
(7) (120 mg, 0.24 mmol, 1.0 equiv) in DCM (5 mL, 0.05 M) and
EtOH (3 mL, 0.08 M) was added 10% palladium on carbon (24 mg,
0.022 mmol, 0.09 equiv). The reaction vessel was charged with a
hydrogen balloon and stirred at rt overnight. After the reaction was
complete as monitored by LCMS, solvents were removed, and the
resulting residue was purified by a CombiFlash system (ISCO) using a
gradient of 0−5% MeOH/DCM to provide diethyl 2-(5-amino-2-(4-
methoxy-2-methylphenethyl)benzo[f ][1,7]naphthyridin-8-yl)-
ethylphosphonate (11) (60 mg, 50%). To a solution of 11 (60 mg,
0.118 mmol, 1.0 equiv) in DCM (5 mL, 0.02 M) at 0 °C was added
TMSBr (180 mg, 1.18 mmol, 10 equiv). The reaction was warmed to
room temperature over 2 h and then quenched with small amounts of
MeOH. The DCM was removed by evaporation, and then DMSO/
water was added. The mixture was adjusted to pH 9 and directly
purified on RP-HPLC using a C18 column, eluting with 10−40% 95:5
(MeCN/5 mM NH4OAc) in 10 mM NH4OAc (pH 9) gradient. The
fractions containing the product were combined and concentrated in
vacuo to give 12 (33 mg, 62%) as a solid. 1H NMR
(dimethylsulfoxide-d6): δ 9.66 (s, 1H), 9.30 (s, 1H), 8.95 (s, 1H),
8.78 (s, 1H), 8.50 (d, 1H, J = 8.4 Hz), 7.54 (s, 1H), 7.45 (d, 1H, J =
8.4 Hz), 7.02 (d, 1H, J = 8.4 Hz), 6.69 (d, 1H, J = 2.8 Hz), 6.61 (dd,
1H, J = 2.8, 8.4 Hz), 3.64 (s, 3H), 3.14−3.06 (m, 2H), 3.00−2.90 (m,
4H), 2.22 (s, 3H), 2.02−1.92 (m, 2H). LRMS [M + H] = 452.2
3-((4-(2-(5-Amino-8-methylbenzo[f ][1,7]naphthyridin-2-yl)-
ethyl)-3-methylphenoxy)methyl)phenylphosphonic Acid (15).
Scheme 3a−c. The synthesis of compound 13 is described in ref 8,
example 50. To a solution of 4-(2-(5-amino-8-methylbenzo[f ][1,7]-
naphthyridin-2-yl)ethyl)-3-methylphenol (13) (100 mg, 0.29 mmol,
1.0 equiv) in dimethylformamide (3 mL, 0.10 M) at 22 °C was added
cesium carbonate (142 mg, 0.44 mmol, 1.5 equiv), and the resulting
mixture was allowed to stir for 30 min. At this point, 1-
(bromomethyl)-3-iodobenzene (131 mg, 0.44 mmol, 1.5 equiv) was
added to this mixture. The reaction mixture was allowed to stir at 55
°C for 18 h, after which it was diluted with ethyl acetate and water.
The biphasic layers were separated, and the organic layer was washed
twice with water. The organic layer was dried over anhydrous Na2SO4,
and the volatiles were removed in vacuo. The resulting residue was
purified by a CombiFlash system (ISCO) using 0−50% ethyl acetate in
hexanes gradient to provide 2-(4-(3-iodobenzyloxy)-2-methylpheneth-
yl)-8-methylbenzo[f ][1,7]naphthyridin-5-amine (14) (151 mg, 93%)
as a solid. To a stirred solution of 14 (151 mg, 0.27 mmol, 1.0 equiv)
in triethyl phosphate (47 mg, 0.28 mmol, 1.05 equiv) was added
palladium acetate (4.8 mg, 0.022 mmol, 0.08 equiv). The resulting
reaction mixture was heated at 90 °C overnight. After the reaction was
cooled down to rt, the residue was taken up in DCM (1 mL, 0.27 M)
at 0 °C and was treated with TMSBr (0.4 mL, 3.0 mmol, 11 equiv).
The reaction was warmed to room temperature over 2 h and then
quenched with small amounts of MeOH. The DCM was removed by
evaporation, and then DMSO/water was added. The mixture was
adjusted to pH 9 and directly purified on RP-HPLC using a C18
column, eluting with 10−40% 95:5 (MeCN/5 mM NH4OAc) in 10
mM NH4OAc (pH 9) gradient. The fractions containing the product
were combined and concentrated in vacuo to give 15 (15 mg, 10%) as
a solid. 1H NMR (dimethylsulfoxide-d6): δ 8.84 (s, 1H), 8.72 (s, 1H),
8.35 (d, 1H, J = 8.4 Hz), 7.67 (d, 1H, J = 12 Hz), 7.60−7.54 (m, 1H),
7.30−7.20 (m, 2H), 7.15 (d, 1H, J = 8.4 Hz), 7.11 (d, 1H, J = 8.4 Hz),
7.04 (s, 1H), 6.84 (s, 1H), 6.77 (m, 1H), 4.99 (s, 2H), 3.12−2.92 (m,
4H), 2.44 (s, 3 H), 2.27 (s, 3H). LRMS [M + H] = 514.2
1
propylphosphonate (1.09 g, 20%) as yellow oil. H NMR (CDCl3):
δ 4.33−4.21 (m, 4H), 3.80−3.73 (m, 4H), 3.68−3.59 (m, 4H), 3.27 (t,
2H, J = 6.7 Hz), 2.51−2.31 (m, 2H), 1.39 (t, 6H, J = 7.1 Hz). LRMS
[M + H] = 431.1
The synthesis of compound 13 is described in ref 8, example 50. To
a solution of 13 (0.400 g, 1.17 mmol, 1.0 equiv) in dimethylformamide
(11.7 mL, 0.10 M) at 22 °C was added 60% dispersion of sodium
hydride in mineral oil (0.700 g, 1.75 mmol, 1.5 equiv), and the
resulting mixture was allowed to stir for 30 min. At this point, diethyl
1,1-difluoro-3-(2-(2-iodoethoxy)ethoxy)propylphosphonate (0.600
mg, 1.40 mmol, 1.2 equiv) was added to this mixture. The reaction
mixture was then allowed to stir for 18 h, after which it was diluted
with ethyl acetate and water. The biphasic layers were separated, and
the organic layer was washed twice with water. The organic layer was
dried over anhydrous Na2SO4, and the volatiles were removed in
vacuo. The resulting residue was purified by a CombiFlash system
(ISCO) using 0−50% ethyl acetate in hexanes gradient to provide
diethyl 3-(2-(2-(4-(2-(5-amino-8-methylbenzo[f ][1,7]naphthyridin-2-
yl)ethyl)-3-methylphenoxy)ethoxy)ethoxy)-1,1-difluoropropyl-
1
phosphonate (302 mg, 40%) as a solid. H NMR (CDCl3): δ 8.56 (s,
1H), 8.38 (s, 1H), 8.10 (d, 1H, J = 8.2 Hz), 7.51 (s, 1H), 7.19 (d, 1H, J
= 9.7 Hz), 6.98 (d, 1H, J = 8.4 Hz), 6.75 (s, 1H), 6.67 (d, 1H, J = 8.3
Hz), 6.07 (br, 2H), 4.30−4.23 (m, 4H), 4.10 (t, 2H, J = 5.0 Hz), 3.85
(t, 2H, J = 4.7 Hz), 3.79−3.71 (m, 4H), 3.66−3.64 (m, 2H), 3.09 (t,
2H, J = 8.5 Hz), 2.96 (t, 2H, J = 8.5 Hz), 2.51 (s, 3H), 2.48−2.35 (m,
2H), 2.26 (s, 3H), 1.37 (t, 6H, J = 7.1 Hz). LRMS [M + H] = 646.7
To a solution of diethyl 3-(2-(2-(4-(2-(5-amino-8-methylbenzo[f ]-
[1,7]naphthyridin-2-yl)ethyl)-3-methylphenoxy)ethoxy)ethoxy)-1,1-
difluoropropylphosphonate (0.300 g, 0.465 mmol, 1.0 equiv) in
CH2Cl2 (4.65 mL, 0.10 M) at 0 °C was slowly added trimethylsilyl
bromide (0.950 mL, 6.98 mmol, 15 equiv). After 1 h the ice bath was
removed and the reaction mixture was allowed to stir at 22 °C for 18
h. At this point, the volatiles were removed in vacuo and the resulting
residue was purified by reverse phase HPLC using a 20−90% 0.5 mM
NH4OAc (in MeCN) to 10 mM NH4OAc (in water) gradient to
1
deliver 16d (160 mg, 60%) as a solid. H NMR (dimethylsulfoxide-
d6): δ 8.83 (s, 1H), 8.68 (s, 1H), 8.32 (d, 1H, J = 8.3 Hz), 7.34 (s,
1H), 7.14 (d, 1H, J = 8.4 Hz), 7.09 (br, 2H), 7.08 (d, 1H, J = 8.4 Hz),
6.74 (s, 1H), 6.68 (d, 1H, J = 8.3 Hz), 4.01 (t, 2H, J = 4.5 Hz), 3.70 (t,
2H, J = 3.3 Hz), 3.61 (t, 2H, J = 7.6 Hz), 3.59−3.54 (m, 2H), 3.50−
3.48 (m, 2H), 3.07 (t, 2H, J = 6.8 Hz), 2.94 (t, 2H, J = 6.8 Hz), 2.43
(s, 3H), 2.25 (s, 3H), 2.21−2.06 (m, 2H). LRMS [M + H] = 590.2.
3-(4-(2-(5-Amino-8-methylbenzo[f ][1,7]naphthyridin-2-yl)-
ethyl)-3-methylphenoxy)propylphosphonic Acid (16a).
Scheme 3d,c. The title compound was prepared according to the
procedure described for 16d but first using commercially available
G
DOI: 10.1021/acs.jmedchem.6b00489
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Journal of Medicinal Chemistry
Article
diethyl 3-bromopropylphosphonate as the electrophile followed by
TMSBr hydrolysis of the diethyl phosphonate.
Diethyl (3-(4-(2-(5-Amino-8-methylbenzo[f ][1,7]naphthyridin-2-yl)-
4.14−4.05 (m, 6H), 3.85−3.82 (m, 2H), 3.77−3.70 (m, 4H), 3.66−
3.63 (m, 2H), 3.11 (t, 2H, J = 8.3 Hz), 2.97 (t, 2H, J = 8.3 Hz), 2.52
(s, 3H), 2.25 (s, 3H), 2.18−2.09 (m, 2H), 1.31 (t, 6H, J = 7.1 Hz).
LRMS [M + H] = 596.3.
ethyl)-3-methylphenoxy)propyl)phosphonate, the Product after Alkylation
1
1
Title Compound 16c. H NMR (MeOD-d4): δ 8.73 (s, 1H), 8.66
of 13 with Diethyl 3-bromopropylphosphonate. H NMR (dimethylsulf-
(s, 1H), 8.38 (d, 1H, J = 8.4 Hz), 7.52 (s, 1H), 7.47 (d, 1H, J = 8.3
Hz), 7.36 (s, 1H), 6.93 (d, 1H, J = 8.4 Hz), 6.75 (s, 2H), 6.64 (d, 1H, J
= 10.8 Hz), 4.09−4.06 (m, 2H), 3.80−3.76 (m, 2H), 3.69−3.64 (m,
2H), 3.64−3.59 (m, 2H), 3.53−3.49 (m, 2H), 3.25 (t, 2H, J = 7.5 Hz),
3.09 (t, 2H, J = 7.5 Hz), 2.58 (s, 3H), 2.28 (s, 3H), 2.13−2.01 (m,
2H). LRMS [M + H] = 540.2.
oxide-d6): δ 9.91 (s, 1H), 9.14 (s, 1H), 8.72 (d, 1H, J = 8.2 Hz), 7.86
(s, 1H), 7.59 (d, 1H, J = 9.8 Hz), 7.51 (d, 1H, J = 8.3 Hz), 7.20 (s,
1H), 7.12 (d, 1H, J = 8.3 Hz), 7.00 (br, 2H), 4.56−4.45 (m, 6H), 3.61
(t, 2H, J = 8.5 Hz), 3.49 (t, 2H, J = 8.5 Hz), 2.90 (s, 3H), 2.74 (s, 3H),
2.47−2.39 (m, 2H), 2.36−2.28 (m, 2H), 1.72 (t, 6H, J = 7.0 Hz).
LRMS [M + H] = 522.6.
1
Ethyl 3-(3-(tert-Butoxycarbonylamino)-4-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate (22). Scheme
4a−c. To a solution of tert-butyl 5-bromo-2-chlorophenylcarbamate
(17) (1.22 g, 4.0 mmol, 1.0 equiv) in acetonitrile (12 mL, 0.3 M) and
EtOH (8 mL, 0.5 M) was added K2CO3 (1.1 g, 8.0 mmol, 2.0 equiv).
The reaction was degassed and flushed with N2, then to it were added
(E)-ethyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)acrylate (18)
(1.1 g, 4.8 mmol, 1.2 equiv) and Pd(PPh3)4 (0.46 g, 0.4 mmol, 0.1
equiv). The reaction was flushed again with N2 and stirred at 100 °C
overnight. After cooling to room temperature, hexane was added, and
the mixture was filtered through a pad of silica, eluting with EA/Hex
(1:1) until the product was completely eluted. The filtrate was
concentrated and purified on CombiFlash, eluting with 0−15% EA in
Hex to give (E)-ethyl 3-(3-(tert-butoxycarbonylamino)-4-
Title Compound 16a. TFA was added to the H NMR sample to
solubilize the compound for analysis. 1H NMR (dimethylsulfoxide-d6):
δ 9.72 (br, 1H), 9.01 (s, 1H), 8.96 (br, 1H), 8.85 (s, 1H), 8.54 (d, 1H,
J = 8.4 Hz), 7.54 (s, 1H), 7.42 (d, 1H, J = 8.2 Hz), 7.08 (d, 1H, J = 8.4
Hz), 6.74 (s, 1H), 6.66 (d, 1H, J = 8.3 Hz), 3.95 (t, 2H, J = 6.4 Hz),
3.14 (t, 2H, J = 8.6 Hz), 2.97 (t, 2H, J = 8.6 Hz), 2.50 (s, 3H), 2.27 (s,
3H), 1.91−1.81 (m, 2H), 1.67−1.56 (m, 2H). LRMS [M + H] =
466.2.
4-(4-(2-(5-Amino-8-methylbenzo[f ][1,7]naphthyridin-2-yl)-
ethyl)-3-methylphenoxy)-1,1-difluorobutylphosphonic Acid
(16b). Scheme 3e,c. The title compound was prepared according
to the procedure described in 16d but using diethyl 4-bromo-1,1-
difluorobutylphosphonate as the electrophile which was prepared from
1,3-dibromopropane.
1
1
chlorophenyl)acrylate (19) as a white solid (0.8 g, 62%). H NMR
Diethyl 4-Bromo-1,1-difluorobutylphosphonate. H NMR (CDCl3): δ
(CDCl3): δ 8.40 (s, 1H), 7.60 (d, 1H, J = 16.0 Hz), 7.30 (s, 1H, J = 8.0
Hz), 7.08 (d, 1H, J = 8.0 Hz), 7.02 (s, 1H), 6.43 (d, 1H, J = 16.0 Hz),
4.23 (q, 2H, J = 8.0 Hz), 1.52 (s, 9H), 1.31 (t, 3H, J = 8.0 Hz). LRMS
[M + H] = 326.1.
4.33−4.24 (m, 4H), 3.46 (t, 2H, J = 6.1 Hz), 2.31−2.13 (m, 4H), 1.39
(t, 6H, J = 7.1 Hz). LRMS [M + H] = 309.1
Diethyl (4-(4-(2-(5-Amino-8-methylbenzo[f ][1,7]naphthyridin-2-yl)-
ethyl)-3-methylphenoxy)-1,1-difluorobutyl)phosphonate, the Product
after Alkylation of 13 with Diethyl 4-Bromo-1,1-Difluorobutylphospho-
nate. 1H NMR (CDCl3): δ 8.61 (s, 1H), 8.38 (s, 1H), 8.08 (d, 1H, J =
8.2 Hz), 7.53 (s, 1H), 7.20 (d, 1H, J = 6.8 Hz), 7.00 (d, 1H, J = 8.2
Hz), 6.73 (s, 1H), 6.66 (d, 1H, J = 8.3 Hz), 6.02 (br, 2H), 4.33−4.26
(m, 4H), 3.98 (t, 2H, J = 6.1 Hz), 3.11 (t, 2H, J = 8.4 Hz), 2.98 (t, 2H,
J = 8.4 Hz), 2.52 (s, 3H), 2.35−2.28 (m, 2H), 2.27 (s, 3H), 2.13−2.08
(m, 2H), 1.40 (t, 6H, J = 7.1 Hz). LRMS [M + H] = 572.6
To a solution of 19 (5.00 g, 15.4 mmol, 1.0 equiv) in ethyl acetate/
ethanol (1:1, 0.3 M) was added Wilkinson’s catalyst (1.40 g, 1.54
mmol, 0.10 equiv). Hydrogen gas was introduced via a balloon, and
the reaction was stirred at room temperature for 24 h. The mixture was
filtered through a pad of Celite, washing with dichloromethane. The
filtrate was concentrated in vacuo and purified by CombiFlash using
0−10% ethyl acetate in hexane to give ethyl 3-(3-(tert-butoxycarbo-
nylamino)-4-chlorophenyl)propanoate (20) as a solid (4.80 g, 96%).
1H NMR (CDCl3): δ 8.05 (s, 1H), 7.23 (d, 1H, J = 8.0 Hz), 6.98 (s,
1H), 6.81 (d, 1H, J = 8.0 Hz), 4.13 (q, 2H, J = 8.0 Hz), 2.92 (t, 2H, J =
8.0 Hz), 2.61 (t, 2H, J = 8.0 Hz), 1.53 (s, 9H), 1.24 (t, 3H, J = 8.0 Hz).
LRMS [M + H] = 328.1.
1
Title Compound 16b. TFA was added to the H NMR sample to
solubilize the compound for analysis. 1H NMR (dimethylsulfoxide-d6):
δ 9.71 (br, 1H), 9.33 (br, 1H), 9.00 (s, 1H), 8.85 (s, 1H), 8.54 (d, 1H,
J = 8.4 Hz), 7.53 (s, 1H), 7.42 (d, 1H, J = 8.3 Hz), 7.08 (d, 1H, J = 8.4
Hz), 6.76 (s, 1H), 6.70 (d, 1H, J = 8.3 Hz), 3.97 (t, 2H, J = 6.2 Hz),
3.15 (t, 2H, J = 8.5 Hz), 2.98 (t, 2H, J = 8.5 Hz), 2.50 (s, 3H), 2.28 (s,
3H), 2.21−2.06 (m, 2H), 1.97−1.87 (m, 2H). LRMS [M + H] =
516.2.
A solution of 20 (6.56 g, 20 mmol, 1.0 equiv), 4,4,4′,4′,5,5,5′,5′-
octamethyl-2,2′-bi(1,3,2-dioxaborolane) (21) (10.16 g, 40.0 mmol, 2.0
equiv), tris(dibenzylideneacetone)dipalladium(0) (916 mg, 1.0 mmol,
0.05 equiv), 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (1.9
g, 4.0 mmol, 0.20 equiv), and potassium acetate (3.92 g, 40.0 mmol,
2.0 equiv) in 1,4-dioxane (0.2 M) was degassed and stirred at 100 °C
overnight. After cooling to ambient temperature, the reaction content
was concentrated in vacuo. The crude material was purified by
CombiFlash using 0−50% ethyl acetate in hexane to afford 22 (8.4 g,
>99% overestimate) as brown oil. The product was stored at −20 °C
2-(2-(2-(4-(2-(5-Amino-8-methylbenzo[f ][1,7]naphthyridin-
2-yl)ethyl)-3-methylphenoxy)ethoxy)ethoxy)ethylphosphonic
Acid (16c). Scheme 3f,c. Representative Preparation of
Phosphonate Electrophile: Diethyl 2-(2-(2-Iodoethoxy)-
ethoxy)ethylphosphonate. A microwave tube was charged with a
stirring bar, commercially available 1,2-bis(2-iodoethoxy)ethane (2.74
mL, 15 mmol, 1.0 equiv), and triethylphosphite (2.61 mL, 15 mmol,
1.0 equiv). The microwave tube was capped and then irradiated at 160
°C for 40 min with stirring. The reaction mixture was cooled down to
rt and was purified by CombiFlash using 0−75% EtOAc in hexanes, or
alternatively by RP-HPLC (0.035% TFA in ACN/0.05% TFA in H2O,
C18 column), to give diethyl 2-(2-(2-iodoethoxy)ethoxy)-
ethylphosphonate (2.85 g, 50%) as pale yellow oil. 1H NMR
(CDCl3): δ 4.14−4.02 (m, 4H), 3.76−3.70 (m, 4H), 3.65−3.60 (m,
4H), 3.25 (t, 2H, J = 7.0 Hz), 2.17−2.08 (m, 2H), 1.31 (t, 6H, J = 7.1
Hz). LRMS [M + H] = 381.1.
The title compound 16c was then prepared according to the
procedure described for 16d but utilizing diethyl 2-(2-(2-iodoethoxy)-
ethoxy)ethylphosphonate as the electrophile.
Diethyl (2-(2-(2-(4-(2-(5-Amino-8-methylbenzo[f ][1,7]naphthyridin-
2-yl)ethyl)-3-methylphenoxy)ethoxy)ethoxy)ethyl)phosphonate, the Prod-
uct after Alkylation of 13 with Diethyl 2-(2-(2-Iodoethoxy)ethoxy)-
ethylphosphonate. 1H NMR (CDCl3): δ 8.60 (s, 1H), 8.35 (s, 1H), 8.09
(d, 1H, J = 8.3 Hz), 7.57 (s, 1H), 7.23 (d, 1H, J = 8.3 Hz), 6.96 (d, 1H,
J = 8.3 Hz), 6.80 (br, 2H), 6.75 (s, 1H), 6.66 (d, 1H, J = 8.3 Hz),
1
and used within a month of synthesis. H NMR (CDCl3): δ 8.67 (s,
1H), 8.06 (s, 1H), 7.61 (d, 1H, J = 8.0 Hz), 6.81 (d, 1H, J = 8.0 Hz),
4.10 (t, 2H, J = 8.0 Hz), 2.91 (t, 2H, J = 8.0 Hz), 2.59 (t, 2H, J = 8.0
Hz), 1.50 (s, 9H), 1.23 (s, 12H), 1.22 (t, 3H, J = 8.0 Hz). LRMS [M +
H] = 420.2.
Ethyl 3-(5-Amino-2-(4-hydroxy-2-methylphenethyl)benzo-
[f][1,7]naphthyridin-8-yl)propanoate (30). Scheme 5a−g. To
a solution of 4-bromo-3-methylphenol (23) (25.0 g, 134 mmol, 1.0
equiv) in DMF (285 mL, 0.5 M) at 0 °C was added portionwise 60 wt
% NaH (8.06 g, 202 mmol, 1.5 equiv). The addition was controlled
such that internal reaction temperature never went above 10 °C. The
reaction was stirred at room temperature for 45 min, then a solution of
chloro(methoxy)methane (13.1 mL, 161 mmol, 1.2 equiv) in DMF
(50 mL, 3 M) was added dropwise via additional funnel. The reaction
was stirred at room temperature for 3.5 h and then quenched by
pouring into ice. The resulting mixture was stirred at room
temperature for 1 h. Ether was added, and the two layers were
separated. The aqueous layer was extracted (1×) with ether. The
H
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combined organic layers were washed with water (2×), brine, dried
over MgSO4, and concentrated to give colorless oil, 1-bromo-4-
(methoxymethoxy)-2-methylbenzene (24) (>100% yield due to
mineral oil in the NaH). The crude material was used in the next
step without further purification. A solution of 24 (23.4 g, 102 mmol
overestimate, 1.0 equiv), triethylamine (70 mL, 510 mmol, 5.0 equiv)
in DMF (200 mL, 0.5 M) was degassed and flushed with nitrogen. To
the reaction was added TES-acetylene (19.2 mL, 107 mmol, 1.05
equiv), CuI (1.9 g, 10 mmol, 0.098 equiv), and Pd(PPh3)2Cl2 (7.1 g,
10 mmol, 0.098 equiv). The reaction was heated to 60 °C and stirred
overnight. After cooling to room temperature, water and ether were
added. The layers were separated, and the organic layer was washed
with water (2×). The organic layer was separated and passed through
a pad of silica (packed with hexane). The silica was eluted with 10%
EA in Hex. The fractions were combined and concentrated to give
triethyl((4-(methoxymethoxy)-2-methylphenyl)ethynyl)silane as a
black oil (>100% yield). The crude material was used in the next
step without further purification. To a solution of triethyl((4-
(methoxymethoxy)-2-methylphenyl)ethynyl)silane (39.31 g, 135.6
mmol, 1.0 equiv) at 0 °C was slowly added tetrabutylammonium
fluoride (1 M solution in THF, 27 mL, 27.11 mmol, 0.20 equiv). At
this point, the ice bath was removed and the reaction mixture was
allowed to stir at room temperature for 45 min. The reaction mixture
was then passed through a pad of silica (packed with hexane) and
eluted with 20% EtOAc in hexanes to remove insoluble salts. The
crude product was then purified by CombiFlash using 0−10% EtOAc
in hexanes to give 1-ethynyl-4-(methoxymethoxy)-2-methylbenzene
(25) as slightly brown liquid (98% yield). A solution of 25 (11.0 g,
62.5 mmol, 1.0 equiv), 3,5-dichloropicolinonitrile (26) (9.73 g, 56.3
mmol, 0.90 equiv), CuI (1.19 g, 6.25 mmol, 0.10 equiv), Pd(PPh3)2Cl2
(4.40 g, 6.25 mmol, 0.10 equiv), and triethylamine (43 mL, 313 mmol,
5.0 equiv) in DMF (250 mL, 0.25 M) was degassed and flushed with
nitrogen. The reaction mixture was then heated to 60 °C and stirred
overnight. After cooling to room temperature, water was added. The
mixture was extracted with EtOAc (2×). The combined organic layers
were washed with 10% aq NH4OH (2×), brine and concentrated. The
crude material was filtered through a pad of silica (wetted with
hexane). The silica was eluted with 10% EtOAc in hexane. The
fractions were combined and concentrated. The resulting solids were
washed in hot ether and filtered to give a yellow solid, which was used
in the next step without further purification. The filtrate was
concentrated and purified by CombiFlash using 0−10% EtOAc in
hexanes to give 3-chloro-5-((4-(methoxymethoxy)-2-methylphenyl)-
ethynyl)picolinonitrile (27) as a yellow solid. Combined yield was
45%. 1H NMR (CDCl3): δ 8.64 (s, 1H), 7.90 (s, 1H), 7.45 (d, 1H, J =
8.0 Hz), 6.94 (s, 1H), 6.89 (d, 1H, J = 8.0 Hz), 5.20 (s, 2H), 3.49(s,
3H), 2.49 (s, 3H). LRMS [M + H] = 313.1.
A solution of 27 (1.87 g, 6.00 mmol, 1.0 equiv), 22 (3.14 g, 7.50
mmol, 1.25 equiv), tris(dibenzylideneacetone)dipalladium(0) (549
mg, 0.60 mmol, 0.10 equiv), dicyclohexyl(2′,6′-dimethoxybiphenyl-2-
yl)phosphine (493 mg, 1.20 mol, 0.20 equiv), and sodium bicarbonate
(1.5 g, 18 mmol, 3.0 equiv) in n-butanol/H2O (5:1, 0.2 M) was
degassed and stirred at 100 °C overnight. After cooling to ambient
temperature, the reaction content was diluted with ethyl acetate and
water. The two phases were separated, and the aqueous layer was
extracted twice with ethyl acetate. The combined organic layers were
washed with brine, dried over anhydrous MgSO4, and concentrated in
vacuo. The crude material was purified by flash chromatography on a
CombiFlash system (ISCO) using 0−40% ethyl acetate in DCM first
to remove the impurity, then 0−4% MeOH in DCM to give ethyl 3-
(5-amino-2-((4-(methoxymethoxy)-2-methylphenyl)ethynyl)benzo-
[f ][1,7]naphthyridin-8-yl)propanoate (28). The product can be
further purified by precipitating and washing in hot ether. Depending
on the purity of the product, the isolated yield ranges 40−60%. A
solution of 28 (3.0 g, 6.4 mmol, 1.0 equiv) in EtOH/THF (3:1, 40
mL, 0.16 M) was flushed with nitrogen. Then, 10 wt % Pd/C (0.6 g,
0.20 equiv by weight) was added. The reaction was flushed with
hydrogen (2×) and stirred under a hydrogen balloon. After 24 h, the
reaction was filtered through a pad of Celite, washing with 5% MeOH
in DCM. The filtrate was checked for presence of starting material
using LCMS. The hydrogenation reaction was repeated until no more
of the alkyne starting material or alkene intermediate can be detected.
The crude product was purified by CombiFlash using 0−4%MeOH in
DCM to give ethyl 3-(5-amino-2-(4-(methoxymethoxy)-2-
methylphenethyl)benzo[f ][1,7]naphthyridin-8-yl)propanoate (29) as
a white solid (>90%). 1H NMR (CDCl3): δ 8.63 (s, 1H), 8.37 (s, 1H),
8.11 (d, 1H, J = 8.0 Hz), 7.54 (s, 1H), 7.22 (d, 1H, J = 8.0 Hz), 7.01
(d, 1H, J = 8.0 Hz), 6.86 (s, 1H), 6.81 (d, 1H, J = 8.0 Hz), 6.22 (s,
2H), 5.15 (s, 2H), 4.15 (q, 2H, J = 8.0 Hz), 3.48(s, 3H), 3.13−3.08
(m, 4H), 2.97 (t, 2H, J = 8.0 Hz), 2.72 (t, 2H, J = 8.0 Hz), 2.27(s, 3H),
1.24 (t, 3H, J = 8.0 Hz). LRMS [M + H] = 474.2.
Compound 29 (2.04 g, 4.3 mmol, 1.0 equiv) was dissolved in EtOH
(21 mL, 0.2 M), and then to the mixture was added a solution of 4 M
HCl in dioxane (21 mL, 0.2 M). The product precipitated out as a
yellow salt. After stirring for 3 h, the reaction was poured into a stirring
solution of ether. The mixture was stirred for 10 min, then filtered and
washed with ether. The yellow solids were dried in vacuum overnight
to give ethyl 3-(5-amino-2-(4-hydroxy-2-methylphenethyl)benzo[f ]-
[1,7]naphthyridin-8-yl)propanoate (30) (2.08 g of bis-HCl salt, 97%).
Alternatively, the crude product can be purified by CombiFlash using
1
0−5% MeOH in DCM to give the free base. H NMR (CDCl3): δ
8.53 (s, 1H), 8.33 (s, 1H), 8.09 (d, 1H, J = 8.0 Hz), 7.56 (s, 1H), 7.24
(d, 1H, J = 8.0 Hz), 6.84 (d, 1H, J = 8.0 Hz), 6.69 (s, 1H), 6.60 (d, 1H,
J = 8.0 Hz), 4.14 (q, 2H, J = 8.0 Hz), 3.12−3.08 (m, 4H), 2.97 (t, 2H,
J = 8.0 Hz), 2.71 (t, 2H, J = 8.0 Hz), 2.21 (s, 3H), 1.25 (t, 3H, J = 8.0
Hz). LRMS [M + H] = 430.2
A representative example for the synthesis of compounds 31a−d is
illustrated with the synthesis of 31c.
3-(5-Amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)-
ethoxy)ethoxy)phenethyl)benzo[f ][1,7]naphthyridin-8-yl)-
propanoic Acid (31c). Scheme 5l,i,j. The title compound was
prepared utilizing the same phosphonate electrophile that was used to
produce 16c.
To a solution of 30 (108.0 mg, 0.252 mmol, 1.0 equiv) dissolved in
DMF (4.2 mL, 0.06 M) was added a solution of diethyl 2-(2-(2-
iodoethoxy)ethoxy)ethylphosphonate (190.0 mg, 0.50 mmol, 2.0
equiv) in DMF (0.36 mL, 0.7 M) and cesium carbonate (328.4 mg,
1.01 mmol, 4 equiv). The reaction was stirred at 60 °C. After 1.5 h (or
until reaction is complete by LCMS), DCM (2 vol equiv) was added
to the reaction. The solids (inorganic) were filtered, and the filtrate
was concentrated. The crude product was purified by CombiFlash
using 0−5%MeOH in DCM to give ethyl 3-(5-amino-2-(4-(2-(2-(2-
(diethoxyphosphoryl)ethoxy)ethoxy)ethoxy)-2-methylphenethyl)-
benzo[f][1,7]naphthyridin-8-yl)propanoate as an oil which upon
standing became a white solid (154.6 mg, 90%). To a solution of
ethyl 3-(5-amino-2-(4-(2-(2-(2-(diethoxyphosphoryl)ethoxy)ethoxy)-
ethoxy)-2-methylphenethyl)benzo[f ][1,7]naphthyridin-8-yl)-
propanoate (154.6 mg, 0.227 mmol, 1.0 equiv) in DCM (1.42 mL,
0.16 M) at 0 °C was added slowly TMSBr (347 mg, 2.27 mmol, 10
equiv). The reaction was stirred at room temperature overnight.
Additional TMSBr (173 mg, 1.13 mmol, 5.0 equiv) was added at 0 °C,
and the reaction was again stirred at room temperature overnight. The
solvent was removed by evaporation, and the crude orange solids were
dried in vacuum briefly. The solids were suspended in EtOH (0.454
mL, 0.5 M), and to the mixture was added 2.5 N NaOH (0.908 mL,
2.27 mmol, 10.0 equiv). The reaction was stirred at 80 °C for 3 h.
After cooling to room temperature, the mixture was adjusted to pH 9
and directly purified on RP-HPLC using a C18 column, eluting with
10−40% 95:5 (MeCN/5 mM NH4OAc) in 10 mM NH4OAc (pH 9)
gradient. The fractions containing the product were combined and
concentrated in vacuo to give 31c as a solid (105.4 mg, 70% yield from
1
30). H NMR (dimethylsulfoxide-d6): δ 9.02 (s, 1H), 8.82 (s, 1H),
8.55 (d, 1H, J = 8.0 Hz), 7.58 (s, 1 H), 7.49 (d, 1H, J = 8.4 Hz), 7.06
(d, 1H, J = 8.0 Hz), 6.76 (s, 1 H), 6.68 (d, 1H, J = 8.0 Hz), 4.03−4.00
(m, 2H), 3.71−3.69 (m, 2H), 3.60−3.54 (m, 4H), 3.51−3.49 (m, 2H),
3.16−3.12 (m, 2H), 3.03−2.96 (m, 4H), 2.67−2.66 (m, 2H), 2.33−
2.32 (m, 2H), 2.26 (s, 3H). LRMS [M + H] = 598.2.
3-(5-Amino-2-(2-methyl-4-(3-phosphonopropoxy)-
phenethyl)benzo[f ][1,7]naphthyridin-8-yl)propanoic Acid
(31a). Scheme 5h,i,j. The title compound was prepared according
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to the procedure described in example 31c but using commercially
available diethyl 3-bromopropylphosphonate as the electrophile. H
starting at 30 μM to obtain EC50 values. The compounds were then
added into the wells. After 18 h of incubation at 37 °C and 5% CO2,
supernatants were analyzed for different cytokines by Meso Scale
Discovery (MSD) multicytokine detection kit as described by the
manufacturer. Activity is reported as percentage of IL-6 over that
achieved by resiquimod (set to 100%), which is used as an internal
standard for every experiment.
1
NMR (MeOD-d4): δ 8.60 (s, 1H), 8.27 (s, 1H), 8.07 (d, 1H, J = 8.4
Hz), 7.52 (s, 1H), 7.30 (d, 1H, J = 8.4 Hz), 6.87 (d, 1H, J = 8.4 Hz),
6.67 (s, 1H), 6.60 (d, 1H, J = 8.4 Hz), 3.93 (t, J = 6.4 Hz, 2H), 3.49−
3.47 (m, 2H), 3.14−3.09 (m, 2H), 2.99−2.95 (m, 2H), 2.69−2.64 (m,
2H), 2.17 (s, 3H), 2.02−2.00 (m, 2H), 1.74−0.66 (m, 2H). LRMS [M
+ H] = 524.2.
Human PBMC Assay. Heparin anticoagulated whole blood was
obtained from healthy volunteers registered with The Scripps Research
Institute Normal Blood Donor Service. PBMCs were isolated by
density gradient centrifugation using Ficol-paque PLUS (GE Health-
care) as recommended by the manufacturer. The isolated PBMCs
were washed twice with PBS and resuspended in RPMI1640
supplemented with heat inactivated FBS (10% final), 100 U/mL
penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, 1 mM
MEM, 10 mM HEPES (Hyclone). The test compounds were
dissolved in endotoxin-free water, with addition of 1 mol equiv of 1
N NaOH to generate the sodium salt in situ. The resulting solutions
(10 mM) were serially diluted 3-fold for 10 points starting at 30 μM to
obtain EC50 values. The compounds were then added into the wells
containing 100 μL of culture media with hPBMCs ((1 × 106 cells)/
mL). After 18 h of incubation at 37 °C and 5% CO2, supernatants
were analyzed for different cytokines by Meso Scale Discovery (MSD)
multicytokine detection kit as described by the manufacturer. Activity
is reported as percentage of IL-6 over that achieved by resiquimod (set
to 100%), which is used as an internal standard for every experiment.
Representative SMIP-Alum Formulation Protocol. To a
suspension of SMIP (1 mg) in endotoxin-free water (0.25 mL) was
added 1 N NaOH solution (1 mol equiv) to obtain a clear solution. To
this solution was added 0.21 mL of alum suspension (9.0−11.0 mg/
mL, obtained as Alhydrogel from Invivogen), 0.1 mL of histidine
buffer (100 mM, pH 6.5), and 0.44 mL endotoxin-free water. The final
suspension (1 mg/mL SMIP and 3 mg/mL alum in 10 mM histidine)
was gently agitated for 1 h at room temperature. To determine fraction
of unbound SMIP, an aliquot (100 μL) was placed into an Eppendorf
tube and centrifuged (13 000 rpm for 10 min). The supernatant was
analyzed on LCMS using 0−90% MeCN in water gradient and
compared to the nonadsorbed SMIP reference.
Protocol for Acquisition of pH-Dependent Solubilities. The
solubility−pH profiles of 16d and 31c were determined by
potentiometric titration using Sirius T3 titrator (Sirius Analytical,
East Sussex, U.K.), whereas the solubility−pH profiles of 16a and 16b
were obtained by saturation shake-flask solubility assay as the
solubilities of these two compounds were poor and no aqueous pKa
values were able to be obtained. For the potentiometric titration
approach, the pKa values of each compound were determined using
the same Sirius T3 titrator prior to solubility determination using
MeOH as the cosolvent and data were processed with T3
accompanying software. In the potentiometric solubility assay, about
2 mg of the solid sample was weighed into a T3 vial and titration was
performed between pH 10 and pH 1.5. Precipitation occurred during
the titration range, causing pKa values to shift. The intrinsic solubility
was calculated according to the pKa shift based on Noyes−Withney
theory using T3 accompanying software, and solubility−pH profile
was extrapolated based on Henderson−Hasselbalch equation. In the
saturated shake-flask approach, saturated solubility samples were
prepared at different pHs and incubated for 24 h. The samples were
then filtered through 0.22 mm PVDF membranes, and the filtrates
were analyzed and quantified against a standard calibration curve of
each compound. The solubility−pH profiles were constructed by
connecting solubility data at different pH values.
3-(5-Amino-2-(4-(4,4-difluoro-4-phosphonobutoxy)-2-
methylphenethyl)benzo[f ][1,7]naphthyridin-8-yl)propanoic
Acid (31b). Scheme 5k,i,j. The title compound was prepared
according to the procedure described in example 31c and utilizing the
same fluorophosphonate electrophile that was used to produce 16b.
1H NMR (MeOD-d4): δ 8.69 (s, 1H), 8.45 (s, 1H), 8.22 (d, 1H, J =
8.4 Hz), 7.53 (s, 1H), 7.45 (d, 1H, J = 8.4 Hz), 6.89 (d, J = 8.4 Hz,
1H), 6.69 (s, 1H), 6.60 (d, 1H, J = 8.4 Hz), 3.95 (t, 2H, J = 6.4 Hz),
3.92−3.90 (m, 2H), 3.49−3.47 (m, 2H), 3.20−3.16 (m, 2H), 3.14−
3.10 (m, 2H), 3.03−2.99 (m, 2H), 2.74−2.70 (m, 2H), 2.22 (s, 3H).
LRMS [M + H] = 574.2.
3-(5-Amino-2-(4-(2-(2-(3,3-difluoro-3-phosphonopropoxy)-
ethoxy)ethoxy)-2-methylphenethyl)benzo[f ][1,7]-
naphthyridin-8-yl)propanoic Acid (31d). Scheme 5m,i,j. The
title compound was prepared according to the procedure described in
example 31c and utilizing the same fluorophosphonate electrophile
1
that was used to produce 16d. H NMR (DMSO-d6): δ 9.02 (s, 1H),
8.82 (s, 1H), 8.55 (d, 1H, J = 8.4 Hz), 7.58 (s, 1 H), 7.49 (d, 1H, J =
8.4 Hz), 7.07 (d, 1H, J = 8.4 Hz), 6.75 (s, 1H), 6.68 (d, 1H, J = 8.0
Hz), 4.03−4.00 (m, 2H), 3.72−3.70 (m, 2H), 3.66−3.62 (m, 2H),
3.58−3.56 (m, 2H), 3.53−3.52 (m, 2H), 3.16−3.12 (m, 2H), 3.03−
2.96 (m, 4H), 2.68−2.64 (m, 2H), 2.33−2.31 (m, 2H), 2.27 (s, 3H).
LRMS [M + H] = 648.2.
TLR7 Reporter Gene Assay. HEK293 cells were stably trans-
fected with a reporter vector expressing the firefly luciferase gene
under the control of an NF-κB dependent promoter and the resistance
gene to puromycin. Selected HEK293-NF-κB-luciferase cells were then
stably transfected with pUno plasmid expressing human TLR7 and the
resistance gene to blasticidin (Invivogen). Transfected cells were
cultured in the presence of selection antibiotics puromycin (2 μg/mL)
and blasticidin (5 μg/mL), and individual resistant clones were picked,
expanded, and tested for expression of luciferase upon stimulation with
the TLR7/8 agonist resiquimod. The best responding clone of
HEK293-hTLR7-NF-κB-luciferase cells was then selected for experi-
ments. The cell line was grown in DMEM (high glucose)
supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin,
100 μg/mL streptomycin, 2 μg/mL puromycin, and 5 μg/mL
blasticidin (Hyclone). For the assay, the cells were seeded in
DMEM media containing 3% FCS, 2 mM L-glutamine, 100 U
penicillin, 100 μg/mL streptomycin. 25 000 cells were plated onto
384-well plates in 50 μL volume of assay media. The test compounds
were dissolved in endotoxin-free water, with addition of 1 mol equiv of
1 N NaOH to generate the sodium salt in situ. The resulting solutions
(10 mM) were serially diluted 3-fold for 10 points starting at 30 μM to
obtain EC50 values. The compounds were then added into the wells.
After 6 h of incubation at 37 °C and 5% CO2, 30 μL of Bright-Glo
luciferase (Promega) detection reagent was added, and luminescence
intensity was measured by CLIPR (Molecular Devices). Activity is
reported as percentage of relative luminescence unit (RLU) over that
achieved by resiquimod (set to 100%), which is used as an internal
standard for every experiment.
Murine Splenocyte Assay. Spleens were removed aseptically
from female BALB/c mice (6−10 weeks old) and processed to single
cell suspension in PBS supplemented with 5% heat inactivated FCS
(Hyclone). Following RBC lysis, cells were resuspended ((5 × 106)/
mL) in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 U
penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate, 1 mM
MEM, 10 mM HEPES, and 50 μM 2-ME. 5 ×105 splenocytes in 100
μL volume ware plated onto 96-well flat-bottom plates. The test
compounds were dissolved in endotoxin-free water, with addition of 1
mol equiv of 1 N NaOH to generate the sodium salt in situ. The
resulting solutions (10 mM) were serially diluted 3-fold for 10 points
Animal Studies. For PK experiments female BALB/c mice (6−8
weeks old) were injected intramuscularly with a total of 100 μL of 1
mg/mL compound in alum (50 μL in each hind leg) and then bled
retro-orbitally at different time points postinjection. Blood was
processed into serum by centrifugation followed by protein
precipitation, reverse-phase gradient elution, and MRM detection via
ESI+ mass spectrometry to determine SMIP serum concentration
(PK). In some studies, animals were sacrificed after the final blood
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collection (24 h) and muscle was removed and flash frozen in liquid
nitrogen for tissue PK analyses.
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
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AUTHOR INFORMATION
Corresponding Author
*E-mail: tyhwu@yahoo.com. Phone: (858) 284-8839.
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Present Addresses
∥B.N.: HealthTell, San Ramon, CA 94583, U.S.
⊥M.L.M.: Boehringer Ingelheim Pharmaceuticals, Ridgefield,
CT 06877, U.S.
#N.M.V.: Moderna Therapeutics, Cambridge, MA 02139, U.S.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Liping Zhou for providing the solubility determi-
nation. T.Y.-H.W. thanks Xu Wu for help in initiating the
project. T.Y.-H.W. also thanks Peter G. Schultz for giving him
the opportunity and support to lead this project. This work was
supported in part by the Transformational Medical Tech-
nologies Program Contract HDTRA1-07-9-0001 from the
Department of Defense Chemical and Biological Defense
program through the Defense Threat Reduction Agency.
ABBREVIATIONS USED
■
TLR7, Toll-like receptor 7; BZN, benzonaphthyridine; SMIP,
small molecule immune potentiator; MPLA, monophosphoryl
lipid A; PK, pharmacokinetics; hPBMC, human peripheral
blood mononuclear cell; FI, fraction of ionization
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Huang, B.; Van Egeren, D. S.; Park, C.; Irvine, D. J. Structure-based
programming of lymph-node targeting in molecular vaccines. Nature
2014, 507, 519−522.
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DOI: 10.1021/acs.jmedchem.6b00489
J. Med. Chem. XXXX, XXX, XXX−XXX