Synthesis and in vitro stability of amino acid prodrugs of 6-β-naltrexol for microneedle-enhanced transdermal delivery

Article abstract of DOI:10.1016/j.bmcl.2014.09.072

A small library of amino acid ester prodrugs of 6-β-naltrexol (NTXOL, 1) was prepared in order to investigate the candidacy of these prodrugs for microneedle-enhanced transdermal delivery. Six amino acid ester prodrugs were synthesized (6a-f). 6b, 6d, and 6e were stable enough at skin pH (pH 5.0) to move forward to studies in 50% human plasma. The lead compound (6e) exhibited the most rapid bioconversion to NTXOL in human plasma (t_1/2 = 2.2 ± 0.1 h).

Full text of DOI:10.1016/j.bmcl.2014.09.072

Bioorganic & Medicinal Chemistry Letters 24 (2014) 5212–5215
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro stability of amino acid prodrugs of
6-b-naltrexol for microneedle-enhanced transdermal delivery
Joshua A. Eldridge a,c, Mikolaj Milewski a, Audra L. Stinchcomb b,c, Peter A. Crooks d,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA
b Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201, USA
c AllTranz, Lexington, KY 40505, USA
d Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 July 2014
Accepted 24 September 2014
Available online 2 October 2014
A small library of amino acid ester prodrugs of 6-b-naltrexol (NTXOL, 1) was prepared in order to
investigate the candidacy of these prodrugs for microneedle-enhanced transdermal delivery. Six amino
acid ester prodrugs were synthesized (6a–f). 6b, 6d, and 6e were stable enough at skin pH (pH 5.0)
to move forward to studies in 50% human plasma. The lead compound (6e) exhibited the most rapid
bioconversion to NTXOL in human plasma (t1/2 = 2.2 0.1 h).
Keywords:
Prodrugs
Ó 2014 Elsevier Ltd. All rights reserved.
Amino acid esters
Microneedles
Stability
Transdermal
A recent surge in research interest has focused on the design of
amino acid prodrugs as delivery systems. The strategy has been
applied to oral,1 intraocular,2 intranasal3 and intravenous4 thera-
peutic agents which exhibit suboptimal physiochemical properties
that limit drugability. Generally, the goal of amino acid prodrug
design is to target nutrient transporters at various biological barri-
ers; however, at skin-relevant pH 5.0, the free amine of an amino
acid promoiety can also enhance aqueous solubility. Indeed, amino
acid promoieties have already been explored as solubility-enhanc-
ing agents in the design of injectable metronidazole prodrugs.5,6 In
passive transdermal delivery systems, where intact stratum cor-
neum (SC) limits the permeability of hydrophilic molecules, ioniz-
able amine promoieties would be expected to diminish molecular
permeability of a prodrug. However, the use of microneedles to
are currently few examples of amino acid prodrugs in the literature
which are intended for MN.9
MN is a technique in which small microchannels are created in
the skin with micrometer-sized needles. SC is bypassed in this way,
and as a consequence of this, MN has expanded the pool of mole-
cules that can be delivered by the percutaneous route. Improved
skin transport of hydrophilic or charged compounds,10–12 and mol-
ecules with large molecular weights in the kilodalton range13 has
been observed utilizing MN. For instance, transdermal delivery of
large proteins14 and hydrophilic compounds such as calcein8 have
been enhanced by MN. Previously, work in our labs demonstrated
that the FDA-approved opioid antagonist naltrexone (NTX, 1) and
its active metabolite, 6-b-naltrexol (NTXOL, 2) could not be deliv-
ered in therapeutic levels by passive transdermal delivery tech-
niques.15 Accordingly, attempts were first made to manipulate
the physiochemical properties of NTX by the prodrug approach.
Straight-chain and branched-chain alkyl esters and carbonates of
NTX were prepared, but these prodrugs achieved suboptimal skin
diffusion for therapeutic delivery in man.16–20 Therefore, it was
envisaged that a switch to MN, and the use of more hydrophilic
amino acid prodrugs of 2, might be an appropriate strategy. NTXOL
is attractive for this research, because it is thought to be instru-
mental in the therapeutic effects of orally dosed NTX. Also, 2 has
an aliphatic hydroxyl group, which is not present in the NTX mol-
ecule that is more suitable for the design of ester prodrugs. We
postulated that identification of a lead NTXOL prodrug for further
MN studies could eventually be advantageous to improve the
create skin microchannels followed by the application of
a
drug-containing transdermal formulation allows for delivery of
hydrophilic species.7 This method is referred to in this article as
microneedle-enhanced transdermal delivery (MN) and is more
specifically known as the ‘poke and patch’ technique. Therefore,
our goal in this study was to synthesize amino acid prodrugs of
NTXOL (2) and to investigate their stability properties in buffers
and in human plasma (HP), in order to predict MN candidacy.
The field of MN is relatively new,8 and to our knowledge, there
⇑
Corresponding author. Tel.: +1 501 686 6495; fax: +1 501 686 6057.
E-mail address: pacrooks@uams.edu (P.A. Crooks).
http://dx.doi.org/10.1016/j.bmcl.2014.09.072
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

J. A. Eldridge et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5212–5215
5213
therapeutic outcomes of patients during protracted alcohol and/or
opioid cessation efforts, since current forms of NTX therapy are
associated with adverse events that lead to noncompliance.21 In
this respect, we have recently demonstrated that pegylated pro-
drugs of NTX, while more soluble in 0.3 M acetate buffer than
NTX, unfortunately exhibit problematic viscosity properties that
limit microchannel transport when compared to NTX itself.22
Amino acid ester prodrug design has been approached in this
study as an alternative strategy to improve molecular hydrophilicity
while simultaneously avoiding the creation of a viscous and oily pro-
drug material. Herein we describe the synthesis and stability of
amino acid ester prodrugs of 2. Among the compounds mentioned,
6e is the established lead compound for further MN developmental
work.
It is known that amino acid ester prodrugs can vary widely in
their stability properties in various hydrolytic media (enzymatic
and non-enzymatic), depending on molecular factors and on the
pH of the hydrolysis media. Skin pH is around 5.0 on average. Thus,
we set the criterion that no produg could degrade more than ten
percent within the intended forty-eight hour time course of future
skin diffusion studies that would be conducted in 0.3 M acetate
buffer vehicle (i.e. t90 P48 h at pH 5.0). Also, since a prodrug must
hydrolyze in vivo to release parent drug, rapid hydrolysis at pH 7.4,
with or without enzyme catalysis, was established as an important
physiochemical parameter. Prodrugs exhibiting a combination of
these features were considered to be appropriate molecules for fur-
ther drug development.
Amino acid esters of 2 were prepared as depicted in Scheme 1.
NTXOL (2) was afforded in 85–92.7% yield (depending on scale)
from NTX using the synthetic methodology of de Costa et al.23 3-
O-allyl-NTXOL (3) was prepared by treating 2 with allyl bromide
under reflux in acetone in the presence of potassium carbonate.
Excess allyl bromide was washed out with hexanes. For each cou-
pling reaction, 3 and DMAP were dissolved at room temperature in
DCM. The appropriate Fmoc-protected amino acid promoiety was
converted to an activated acid chloride in a separate flask by son-
icating it in DCM with thionyl chloride under an argon stream, as
described by Sureshbabu et al.,24 and the residual HCl that had
not been blown off in the argon stream was quenched with DMAP
base. The cocktail containing 3 and DMAP was then added drop-
wise to the acid chloride solution over the course of two minutes
at 0 °C, and the reaction mixture was warmed to room temperature
and left to react for four hours.
In every case, the reactions were worked up with DI water and
brine. Final purification was achieved by chromatography over sil-
ica that had been pretreated with 1% TEA in hexanes. Isolated pre-
cursors (4a–f) were used without further purification. Estimated
isolation yields were 75–90%.
Deallylation was performed utilizing the methods of Chandrase-
khar et al.25 with modifications. The fully protected precursor was
dissolved in THF and treated with tetrakis(triphenylphosphine)pal-
ladium ((PPh3)4Pd) and poly-methylhydrosiloxane (PMHS). Zinc
chloride (2 M solution in diethyl ether) was added with rapid stir-
ring, and the reaction was run for 24 h. The THF was evaporated
under an argon stream, and the gummy residue was reconstituted
in DCM and worked up with a 1% sodium bicarbonate solution and
brine. The combined organic fractions were concentrated and chro-
matographed as before to afford the deallylated prodrug product in
estimated yields ranging from 50% to 85%. Compounds 5a–f were
advanced to the next step without further purification.
N
OH
O
O
c
HO
R
Cl
R
O
1
HO
O
a
N
N
OH
OH
d
b
O
2
O
HO
OH
O
OH
3
N
OH
N
f
e
OH
O
R
O
O
O
O
O
HO
R= (S-configuration)
5a, CH(CH3)NHFmoc
5b, CH[CH(CH3)2]NHFmoc
5c, CH[CH2CH(CH3)2]NHFmoc
O
R
R= (S-configuration)
4a, CH(CH3)NHFmoc
4b, CH[CH(CH3)2]NHFmoc
4c, CH[CH2CH(CH3)2]NHFmoc
4d, CH[CH(CH3)(CH2CH3)]NHFmoc
4e, CH2CH2NHFmoc
5d, CH[CH(CH3)(CH2CH3)]NHFmoc
5e, CH2CH2NHFmoc
5f,
4f,
N
N
Fmoc
Fmoc
R= (S-configuration)
6a, CH(CH3)NH2
N
6b, CH[CH(CH3)2]NH2
6c, CH[CH2CH(CH3)2]NH2
6d,CH[CH(CH3)(CH2CH3)]NH2
6e, CH2CH2NH2
OH
O
O
6f,
HO
O
R
N
H
Scheme 1. Synthesis of amino acid ester prodrugs of 6-b-naltrexol: (a) forma-
midinesulfinic acid (4.0 equiv), aqueous NaOH (0.53 M), 80 °C, 1.5 h;23 (b) allyl
bromide (1.1 equiv), K2CO3 (4 equiv), acetone, reflux, 5.5 h; (c) Fmoc-protected
amino acids (2.0 equiv), SOCl2 (1.99 equiv), sonicate 30 min, DMAP (2.0 equiv),
argon stream; (d) DMAP (2.0 equiv), DCM, 0 °C to rt, 4 h, argon atmosphere; (e)
PMHS (3.0 equiv), 2 M ZnCl2 in Et2O (52 drops via syringe/100 mg 2), (PPh3)4Pd
(5 mol %), THF, rt, 24 h, argon atmosphere; (f) octanethiol (10 equiv), DBU
(25 mol %), THF, rt, 24 h, argon atmosphere.
and the final prodrugs were eluted with copious column washes
using ethyl acetate. Compounds 6a and 6f required acetone and
TEA to be eluted from silica, and hydrolytic degradation of these pro-
drugs resulted. The range of yields at this stage was ꢀ20% (6a) to 80%
(6d). The final prodrugs were characterized by 1H NMR and 13C NMR
spectrometry, and ESI-MS analysis.31 Final yields of prodrugs are
reported along with the spectral data. Despite some degradation
duringpurification, compounds6aand6fwereobtainedinsufficient
purity for stability comparison to the other prodrugs.
For prodrugs 6a–f, stability studies were conducted in donor
vehicle (0.3 M acetate buffer, pH 5.0). For prodrugs 6b and 6e, sta-
bility studies were also conducted in receiver solution (25 mM
HEPES-buffered Hanks’ balanced salt solution, pH 7.4). These donor
Fmoc removal was accomplished by 24 h treatment with DBU
base (25 mol %) and octanethiol (10 equiv) in dry THF, as described
by Sheppeck et al.26 In the case of 6e, the final prodrug was isolated
by precipitation and triturationfromdiethyl ether. The a-aminoacid
ester prodrugs were recovered via aqueous workup and column
chromatography, utilizing silica deactivated with 1% TEA in hexanes,

5214
J. A. Eldridge et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5212–5215
and receiver solutions have been used routinely in our skin diffu-
sion studies. Reactions were initiated by charging a 10 mL volume
of hydrolysis media thermostated at 32 0.5 °C (pH 5.0 samples)
and 37 0.5 °C (pH 7.4 samples) with approximately 1 mg of pro-
drug followed by vortex stirring for one minute. The suspension
10 min at 10,000 rpm. Supernatants were removed into individual
culture tubes and dried under N2 gas in a bath maintained at
37 0.5 °C. Residues were reconstituted in 150.0 lL methanol con-
taining 0.04% HFBA by vortexing for 30 s. Samples were immedi-
ately transferred to low-volume inserts and injected onto the
guard column/Waters Phenyl column described above for HPLC
analysis. Gradient programs and flow rates varied between 6b,
6d and 6e in the plasma stability studies. This was necessary to
avoid matrix peak interference with the analytes. Data were ana-
lyzed with an eight-point standard curve in each case. The system
consisted of an Agilent 1100 series HPLC instrument equipped with
a G1322A Degasser, a G1311A Quat Pump, a G1313A autosampler,
and a photodiode array detector set at 280 nm.
was then filtered through a 0.45 l
m nylon syringe filter (AcrodiscÒ
Premium 25 mm Syringe Filter). Aliquots were withdrawn over a
period of approximately 3 half-lives. Samples were then diluted
with ACN-water 70:30 (v/v) for HPLC analysis. The HPLC system
consisted of a Waters 717 plus autosampler, a Waters 600 quater-
nary pump, and a Waters 2487 dual wavelength absorbance detec-
tor with Waters Empower™ software. A Brownlee (Wellesley, MA,
USA) C-8 reversed phase Spheri-5
lm column (220 Â 4.6 mm) with
a C-8 reversed phase guard column of the same type (15 Â 3.2 mm)
by Perkin ElmerÒ was used with the UV detector set at a wave-
length of 215 nm or 278 nm. The mobile phase consisted of
70:30 (v/v) ACN:(0.1% TFA with 0.065% 1-octane sulfonic acid
sodium salt, adjusted to pH 3.0 with TEA aqueous phase). Samples
were run at a flow rate of 1.5 ml/min with a run time of 4 min. The
amino acid ester prodrugs showed pseudo-first-order kinetic
behavior upon hydrolysis. Apparent pseudo-first-order hydrolysis
rate constants (Kapp) were estimated from the slope of the log-
transformed amount of prodrug remaining in the medium. All sta-
bility studies were carried out in duplicate (n = 2) except for the pH
7.4 stability study of 6d. Compound 6d was highly insoluble in pH
7.4 buffer, and had to be assayed a different way. At this time, it
was also prudent to develop a method for human plasma (HP) sta-
bility studies using gradient mobile phase methodology, so the pH
7.4 buffer stability of 6d was assayed alongside the HP hydrolysis
studies as described below (n = 3).
Compounds 6b, 6d and 6e showed pseudo-first-order kinetic
hydrolysis behavior in plasma, and 6d also hydrolyzed similarly
in 50% PBS buffer. Apparent pseudo-first-order hydrolysis rate con-
stants (Kapp) were calculated from the slope of log transformed
AUCprodrug/AUCIS as a function of time using the standard curves.
All plasma stability studies, and the pH 7.4 buffer stability study
of 6d, were carried out in triplicate (n = 3). Table 1 summarizes
the stability data which are reported as the mean SD.
It can be seen in Table 1 that 6a, 6c, and 6f were not suitable for
MN based on our minimum stability criterion; t90 P48 h at pH 5.0
and 32 °C. The latter cutoff was established for our studies, because
it becomes difficult to estimate the extent of bioconversion in the
viable skin following a diffusion experiment if a prodrug exten-
sively degrades during the experiment. Also, high levels of hydro-
lysis before skin transport do not allow a true estimation of drug
flux. Prodrug moieties with bulky side chains enhanced stability
in this series of prodrugs, with 6e also showing similar stability.
Overall, the best MN candidates were determined to be 6b, 6d
and 6e based on the pH 5.0 stability criteria. Prodrugs that did
not meet these minimum criteria were not pursued in further sta-
bility tests (Table 1).
Adequate stability at pH 5.0 was not enough to establish our
prodrugs as appropriate MN candidates. In a prodrug design, rapid
degradation under physiological conditions is desired, because the
prodrug itself is just a delivery system for the parent drug. As such,
an ideal prodrug for the MN paradigm should also exhibit rapid
hydrolysis at pH 7.4 (enzyme-assisted in plasma or in a chemical
buffer). This is true because viable skin bioconversion at pH 7.4
below the SC layer is expected to greatly enhance skin transport
of the parent drug based on previous studies.16–20,27–30 In Table 1,
it is obvious that significant hydrolysis rate enhancements of
approximately one order of magnitude or more were observed at
pH 7.4 as compared to pH 5.0; however, these rates were not rapid
enough to ensure enhanced skin transport of 2. Therefore, we
examined the hydrolysis behaviors of 6b, 6d, and 6e in 50% HP
to see if further rate enhancements could be expected in vivo.
Interestingly, 6b and 6d had significantly increased half-lives in
plasma compared to those observed in pH 7.4 buffers which was
likely to be due to protein binding. In contrast, the half-life of 6e
was reduced by approximately 5.4-fold in HP compared to pH
7.4 PBS buffer. Therefore, 6e was the lead compound of this series
A stock solution of 1.6 mM 6d was prepared in DMSO. A stock
internal standard (IS) solution of salicylamide (0.55 mM) in meth-
anol containing 0.04% heptafluorobutyric acid ion pairing agent
(HFBA) was also prepared. An eight-point calibration curve was
generated from working solutions of 6d and the IS solution using
the same sample workup as described below. Reactions were initi-
ated by adding 40.0
a pre-warmed (37 0.5 °C) solution of 50% PBS (initial reaction
concentration of 82 M). More concentrated reactions were impos-
sible due to extensive precipitation of 6d at higher levels. Sampling
over the course of time was done by the following method. 50.0
aliquots of the reaction mixture were removed at pre-determined
time points. 50.0 L of internal standard solution and 50.0 L of
lL of the prodrug stock solution to 760.0 lL of
l
lL
l
l
blank 0.04% HFBA in methanol were added to the reaction mixture
aliquots in low volume inserts, and the solutions were vortexed.
Samples were immediately injected onto a 4
l
m Waters Phenyl
column (3.9 Â 150 mm) attached to
a 4 lm
Nova-pakÒ C18
3.9 Â 20 mm guard column. The mobile phase consisted of an
aqueous solution of 0.04% (HFBA) (Solvent A) and 0.04% HFBA in
methanol (Solvent B). 6d was eluted with a gradient program at
0.3 mL/min.
HP stability studies (50% HP in 50% PBS buffer) of 6b, 6d and 6e
were conducted as follows. Stock solutions of the prodrugs were
prepared in DMSO. The stock concentrations were 2.3 mM (6b),
1.6 mM (6d), and 7.2 mM (6e). It was not possible to perform the
reactions at the same concentrations, because 6b and 6d exhibited
the tendency to precipitate at 7.2 mM concentration. Reactions
Table 1
In vitro stability of prodrugs 6a–f in buffers and in 50% human plasma (HP)
were initiated by adding 40.0
760.0 L of a pre-warmed (37 0.5 °C) solution of 50% HP in 50%
PBS buffer (pH 7.4). Sampling over the course of time was carried
out using the following method. 50.0 L aliquots of a reaction mix-
ture were removed at various time points and placed into 1000
Eppendorf tubes. 50.0 L of internal standard solution was added
to the samples, and 300.0 L of ice cold methanol containing
lL of the prodrug stock solution to
l
Prodrug
t90 (pH 5.0)
t1/2 (pH 7.4)
t1/2 (50% HP)
6a
6b
6c
6d
6e
6f
9.9 0.5 h
5.2 0.2 d
0.9 0.0 d
14 d
5.6 0.5 d
5.2 0.7 h
No data
0.48 0.03 d
No data
0.9 0.0 d
0.5 0.2 d
No data
No data
0.76 0.03 d
No data
1.3 0.1 d
2.2 0.1 h
No data
l
lL
l
l
0.04% HFBA was charged to the tubes to precipitate proteins. The
mixtures were vortexed for 30 s and subsequently centrifuged for
n = 3.

J. A. Eldridge et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5212–5215
5215
23. de Costa, B. R.; Iadarola, M. J.; Rothman, R. B.; Berman, K. F.; George, C.;
Newman, A. H.; Mahboubi, A.; Jacobson, A. E.; Rice, K. C. J. Med. Chem. 1992, 35,
2826.
24. Sureshbabu, V. V.; Hemantha, H. P. ARKIVOC (Gainesville, FL, U. S.) 2008, 243.
25. Chandrasekhar, S.; Raji, R. C.; Jagadeeshwar, R. R. Tetrahedron 2001, 57, 3435.
26. Sheppeck, J. E.; Kar, H.; Hong, H. Tetrahedron Lett. 2000, 41, 5329.
27. Hamad, M. O.; Kiptoo, P. K.; Stinchcomb, A. L.; Crooks, P. A. Bioorg. Med. Chem.
2006, 14, 7051.
28. Hammell, D. C.; Hamad, M.; Vaddi, H. K.; Crooks, P. A.; Stinchcomb, A. L. J.
Controlled Release 2004, 97, 283.
29. Kiptoo, P. K.; Hamad, M. O.; Crooks, P. A.; Stinchcomb, A. L. J. Control. Release
2006, 113, 137.
of prodrugs. Also, the tendency of 6b and 6d to precipitate at pH
7.4 in PBS buffer is not a desirable physiochemical property.
Although, 6e was the established lead compound, its plasma
half-life is still fairly long for an ideal MN candidate, and it would
be necessary to perform skin diffusion and disposition studies to
determine if faster rates of bioconversion could be achieved in via-
ble skin tissue. Alternatively, hydrolysis of the prodrug in vivo may
occur by numerous routes, including hepatic metabolism, follow-
ing systemic delivery. Overall, 6e appears to be a suitable prodrug
for future MN studies that will utilize a ‘poke and patch’ drug deliv-
ery paradigm.
30. Kiptoo, P. K.; Paudel, K. S.; Hammell, D. C.; Hamad, M. O.; Crooks, P. A.;
Stinchcomb, A. L. Eur. J. Pharm. Sci. 2008, 33, 371.
31. (2): 1HNMR spectral data were identical to de Costa et al.;21 Yield: 92.7%: (3):
1H NMR (CDCl3) 300 MHz: d 6.71 (d, J = 10 Hz, 1H), 6.56 (d, J = 10 Hz, 1H), 6.11–
6.02 (m, 1H), 5.39 (d, J = 10 Hz, 1H), 5.26 (d, J = 10 Hz, 1H), 4.70–4.60 (m, 2H),
4.50 (d, J = 10 Hz, 1H), 3.65–3.55 (m, 1H), 3.52 (d, J = 5 Hz, 1H), 3.10 (d, J = 5 Hz,
1H), 3.03 (d, J = 15 Hz, 1H), 2.68–2.52 (m, 2H), 2.36 (d, J = 5 Hz, 2H), 2.29–2.22
(m, 1H),), 2.18–2.11 (m, 2H), 2.04–1.94 (m, 1H), 1.68–1.59 (m, 1H), 1.52–1.47
(m, 1H), 1.41–1.34 (m, 2H), 0.88 -0.80 (m, 1H), 0.56–0.51 (m, 2H), 0.15–0.11
(m, 2H) ppm; Yield: 93.2%: (6b): 1H NMR 500 MHz (CDCl3): 7.72–7.63 (m,
<1H), 7.60–7.53 (m, <1H), 7.50–7.44 (m, <1H), 6.71 (d, J = 10 Hz, 1H), 6.59 (d,
J = 10 Hz, 1H), 4.95–4.82 (m, 1H), 4.53 (d, J = 10 Hz, 1H), 3.56 (d, J = 5 Hz, 1H),
3.11 (d, J = 5 Hz, 1H), 3.03 (d, J = 15 Hz, 1H), 2.69–2.55 (m, 2H), 2.39 (d, J = 5 Hz,
2H), 2.31–2.21 (m, 1H), 2.20–2.10 (m, 2H), 2.10–2.01 (m, 1H), 1.76–1.65 (m,
1H), 1.64–1.57 (m, 1H), 1.53–1.42 (m, 2H), 1.33–1.19 (m, 2H), 1.04 (d, J = 5 Hz,
½ H), 1.00 (d, J = 5 Hz, 3H), 0.97 (d, J = 5 Hz, ½ H) 0.93 (d, J = 5 Hz, 3 H), 0.89–
0.79 (m, 3H), 0.58–0.48 (m, 2H), 0.19–0.09 (m, 2H) ppm; 13C NMR (CDCl3)
300 MHz: d 175.48, 141.98, 141.01, 132.14, 130.82, 128.69, 123.01, 119.58,
118.70, 92.34, 70.09, 62.41, 59.42, 58.77, 48.30, 44.16, 31.98, 31.01, 29.59,
24.31, 22.85, 19.00, 17.96, 9.77, 4.30 ppm; MS (ESI) m/z: 443.9 (MH+); Yield:
36.2%: (6c): 1H NMR 500 MHz (CDCl3): d 7.70–7.65 (d, J = 10 Hz, 1H), 6.59 (d,
J = 10 Hz, 1H), 4.91–4.83 (m, 1H), 4.54 (d, J = 5 Hz, 1H), 3.68 (t, J = 15 Hz, 1H),
3.11 (d, J = 5 Hz, 1H), 3.03 (d, J = 20 Hz, 1H), 2.70–2.56 (m, 2H), 2.39 (d, J = 5 Hz,
2H), 2.30–2.22 (m, 1H), 2.21–2.12 (m, 1H), 2.12–2.02 (m, 1H), 1.87–1.75 (m,
1H), 1.71–1.62 (m, 2H), 1.62–1.58 (m, 1H), 1.58–1.52 (m, 1H), 1.52–1.44 (m,
3H), 0.92 (t, 7H, overlap with cyclopropyl methine), 0.59–0.50 (m, 2H), 0.19–
0.12 (m, 2H) ppm; 13C NMR (CDCl3) 300 MHz: d 176.91, 142.00, 141.15, 130.87,
119.66, 118.75, 92.37, 76.52, 70.19, 62.55, 59.50, 52.31, 48.40, 44.22, 43.92,
31.08, 29.66, 25.04, 24.21, 23.01, 22.93, 22.63, 9.84, 4.34 ppm; MS (ESI) m/z:
457.8 (MH+); Yield: 38.4%: (6d): 1H NMR 500 MHz CDCl3: d 7.70–7.65 (m, <1H),
7.58–7.53 (m, <1H), 7.50–7.44 (m, <1H), 6.70 (d, J = 10 Hz, 1H), 6.58 (d,
J = 10 Hz, 1H), 4.85–4.94 (m, 1H), 4.52 (d, J = 5 Hz, 1H), 3.67 (d, J = 5 Hz, 1H),
3.10 (d, J = 5 Hz, 1H), 3.02 (d, J = 15 Hz, 1H), 2.69–2.55 (m, 2H), 2.39 (d, J = 5 Hz,
2H), 2.31–2.21 (m, 1H), 2.2–2.12 (m, 1H), 2.12–2.02 (m, 1H), 1.96–1.85 (m, 1H),
1.70–1.57 (m, 2H), 1.53–1.44 (m, 2H), 1.44–1.36 (m, 1H), 1.30–1.19 (m, 2H),
0.95–0.89 (m, 7H), 0.89–0.79 (m, 2H), 0.58–0.50 (m, 2H), 0.19–0.09 (m, 2H)
ppm; 13C NMR (CDCl3) 300 MHz: d 175.58, 142.01, 140.98, 132.29, 130.83,
128.54, 123.11, 119.58, 118.66, 92.29, 70.12, 62.46, 59.43, 57.60, 48.33, 44.16,
39.24, 30.99, 29.67, 25.86, 24.34, 22.88, 15.56, 12.33, 9.81, 4.28 ppm; MS (ESI)
m/z: 457.9 (MH+); Yield: 40%: (6e): 1H NMR 500 MHz (DMSO-d6): d 6.59 (d,
J = 10 Hz, 1H), 6.53 (d, J = 10 Hz, 1H), 4.87 (s, broad), 4.45 (m, 2H), 3.64–3.57
(m, 1H), 3.03 (d, J = 5 Hz, 1H), 2.96 (d, J = 20 Hz 1H), 2.76 (t, J = 15 Hz, 2H),
2.62–2.53 (td, 2H), 2.40 (t, J = 15 Hz, 2H), 2.36–2.27 (m, 2H), 2.23–2.12 (m, 1H),
1.85–1.72 (m, 2H), 1.70–1.57 (m, 2H), 1.48 (d, J = 10 Hz, 1H), 1.33–1.19 (m, 3H),
0.87–0.79 (m, 1H), 0.42–0.51 (m, 2H), 0.16–0.06 (m, 2H) ppm; 13C NMR
(DMSO-d6) 300 MHz: d 171.49, 165.90, 141.62, 140.31, 131.11, 123.21, 118.63,
117.05, 90.43, 75.09, 69.33, 61.45, 58.33, 53.9, 47.30, 43.44, 37.93, 30.23, 29.28,
23.26, 22.15, 9.31, 3.78 ppm; MS (ESI) m/z: 415.9 (MH+); Yield: 32.2%.
Acknowledgment
This work was supported by NIH Grant R01 DA13425.
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