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E. Augustyn et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2616–2621
might lead to an increase in LAT-1 binding and an improvement in
substrate activity.
O
O
a
b
OH
O
To evaluate substrate activity, compounds were tested in two
different cell-based assays, using a LAT-1 overexpressing cell line
generated by transfection of HEK cells with human LAT-1 cDNA.24
HEK-LAT1 cells were used to identify ligands for LAT-1 using both
cis-inhibition and trans-stimulation assays.24 cis-Inhibition
involves competition of test compound with a radiolabeled amino
acid substrate (e.g., [3H]-gabapentin) for LAT-1 mediated cellular
uptake. Though the cis-inhibition assay can identify agents which
interact with LAT-1, it is exclusively used to identify inhibitors,
not substrates of the transporter. To determine whether a com-
pound was a substrate, a trans-stimulation assay27 was performed.
This assay exploits LAT-1’s alternating access mechanism28,29 by
pre-loading cells with [3H]-gabapentin followed by incubation
with test compound. We choose [3H]-gabapentin as a probe sub-
strate due to its selectivity against other transporters.29 The efflux
rate of radiolabeled amino acid in the presence of the test com-
pound is compared to the efflux rate in the absence of the test com-
pound and with both positive and negative control amino acids
(leucine and arginine, respectively) and parent amino acids
(phenylalanine and tyrosine) to assess whether a test compound
is a LAT-1 substrate. Substrates enhance the efflux rate of the radi-
olabeled amino acid compared with its efflux rate in the absence of
the substrate.
HN
N
R1
R1
Boc
H
: R = OBn
Boc
8
9
I
1
I
1
6
7
: R
=
: R = H
: R = OBn
1
1
O
O
c
OH
NH2 - HCl
O
N
R1
R1
Boc
R2
R2
1
1
H, R2 = Et
H, R2 = Bn
: R = H, R2 = Et
10
11
25
28
: R
: R
=
=
1
1
: R
=
H, R2 = Bn
12: R1 = OBn, R2 = Et
13: R1 = OBn, R2 = Et
35: R1 = OH, R2 = Et
x
d
Scheme 2. Synthesis of compounds 25 and 28. Reagents and conditions: (a) K2CO3,
CH2Br2, CH3CN, 100 °C, 8: 43%, 9: 32%; (b) Et2Zn, PdCl2(dppf)–DCM, THF, rt, 18 h, 10:
74%, 11: 60%, 12: 62%; (c) 1:1 6 N HCl (aq)/1,4-dioxane, 60 °C, 25: 60%, 28: 38%, 13:
29%; (d) H2 (balloon), Pd/C, no reaction.
robust approach for preparing several of the desired amino acid
analogs of Figure 1 (i.e., 26, 27, 34–36, and 40).
Though tyrosine analogs 37 and 38 (R2 = tert-butyl and benzyl,
respectively) could potentially be synthesized using the approach
of Scheme 3, we employed a different method43 for these two com-
pounds, taking advantage of the reactivity of tyrosine toward elec-
trophilic aromatic substitution. Use of tert-butyl alcohol as a source
of tert-butyl cation gave almost exclusively regioisomer 37,
whereas, the analogous reaction with benzyl alcohol gave a mix-
ture of products, presumably resulting from di- and tri-benzyla-
tion.44,45 Fortunately, the desired product 38 was easily
separated from the other isomers by preparative HPLC.
To prepare compounds 29 and 39, protected
L-phenylalanine 1
and
L
-tyrosine 2 were subjected to a Suzuki coupling30 with
phenylboronic acid followed by deprotection according to
Scheme 1. Attempts to introduce a methyl group at R2 using Suzuki
coupling with methyl boronic acid were unsuccessful,31 and NMR
analysis of the reaction mixture for conversion of 2 to 5 indicated
low yield (<25%) of desired product (not isolated).
In order to prepare the desired alkyl-substituted amino acids of
Figure 1, we decided to examine the Negishi coupling32 as an alter-
native to the Suzuki reaction. Initial attempts to perform Negishi
coupling with diethyl zinc as a test case on 1 or 2 using conditions
described for a protected amino acid33 gave poor yields (<20%) of
the desired 3-ethyl substituted phenylalanine or tyrosine deriva-
tive. A literature search revealed no examples of Negishi coupling
reactions between iodo-substituted phenylalanine or tyrosine ana-
logs (e.g., 1 or 2) and alkyl zinc reagents. Apparently, Negishi cou-
pling of aromatic amino acids has been limited to substitution with
cyano34 or aromatic rings.35 We hypothesized that the desired
Negishi coupling might give a better yield if both the carbamate
‘N–H’ and carboxylic acid of 6 and 7 were masked, and this
prompted us to explore an oxazolidinone36,37 protecting group
(Scheme 2). To our knowledge, this synthetic method is a new
approach for obtaining meta-substituted phenylalanine deriva-
tives. In addition to serving the dual purpose of protecting both
the carbamate and carboxylic acid, the oxazolidinone was easily
removed under acidic conditions to simultaneously remove the
Boc group giving the meta substituted amino acids 13, 25 and 28.
Unfortunately the desired ethyl substituted tyrosine 35 was not
obtained using this route due to failure to deprotect the benzyl
ether of 13 presumably caused by poisoning of the palladium
catalyst used in hydrogenolysis.38
As deprotection of tyrosine intermediate 13 (Scheme 2) was
problematic, and moreover, we desired bulky alkyl groups at R2
(i.e., isopropyl and tert-butyl) that could be problematic using
the cross coupling strategy of Scheme 2 (e.g., conversion of 8 to
10), we explored the convergent approach depicted in Scheme 3
as an alternative. Conversion of commercially available alkyl iodide
14 to Jackson’s organozinc 1539,40 followed by an in situ Negishi
coupling41 to give 17a–f was successfully performed. Though this
route added additional synthetic steps to obtain noncommercial
meta substituted aryl iodides42 16c–f, we found Scheme 3 to be a
Tyrosine and phenylalanine analogs synthesized in Schemes 1–4,
were evaluated in cis-inhibition and trans-stimulation assays
(Fig. 1).24 Halogen size for phenylalanine derivatives 20–22 corre-
lated with an increase in substrate activity (blue bars, Fig. 1), but this
trend failed to hold at iodide 23, as a significant decrease was
observed (Fig. 1a). In contrast, a consistent trend of increasing %
inhibition (yellow bars, Fig. 1) with size was observed for the halo-
gens. A more noticeable trend in activity was observed for alkyl-
and aryl-substituted phenylalanines (24–29). Though methyl (24)
was found to be slightly better than hydrogen (i.e., parent Phe), lar-
ger, more lipophilic groups (benzyl and phenyl in 28 and 29, respec-
tively) lead to loss of substrate activity, but a rise in % inhibition.
cLogP values46 indicate that the lipophilicity of iodo (23: ꢀ0.43) lies
in between an ethyl (25: ꢀ0.53) and isopropyl group (26: ꢀ0.13).
This order roughly matches what is observed with activity. For com-
parison sake, compounds 28 and 29 have substantially higher cLogP
values (+0.51 and +0.33, respectively), and they both exhibited 98%
inhibition of [3H]-gabapentin in the cis assay. IC50 values for com-
pounds 28 and 29 were determined to be 7.3 and 6.6 lM, respec-
tively. This level of transporter inhibition is comparable to
lipophilic thyroid hormones (e.g., triiodothyronine), which also
demonstrated poor substrate activity that correlated with
lipophilicity.25 However, clearly other factors besides lipophilicity
impact activity as methyl analog 24’s cLogP value (ꢀ1.1) lies in
between fluoro (20: ꢀ1.4) and chloro (21: ꢀ0.84), yet 24 had the
greatest substrate activity of all the tested compounds (efflux
rate = 3.8 fmol/min).
Surprisingly, a different trend was apparent for halogenated
tyrosines (30–33, Fig. 1b), where the best substrate contained a
meta fluoro substituent (30). And there was a decrease in activity
going from chloro (31) to bromo (32), with a slight, but signifi-
cant increase in substrate activity from bromo (32) to iodo (33)