The Effects of Prodrug Size and a Carbonyl Linker on l-Type Amino Acid Transporter 1-Targeted Cellular and Brain Uptake

Article abstract of DOI:10.1002/cmdc.202000824

The l-type amino acid transporter 1 (LAT1, SLC7A5) imports dietary amino acids and amino acid drugs (e. g., l-DOPA) into the brain, and plays a role in cancer metabolism. Though there have been numerous reports of LAT1-targeted amino acid-drug conjugates (prodrugs), identifying the structural determinants to enhance substrate activity has been challenging. In this work, we investigated the position and orientation of a carbonyl group in linking hydrophobic moieties including the anti-inflammatory drug ketoprofen to l-tyrosine and l-phenylalanine. We found that esters of meta-carboxyl l-phenylalanine had better LAT1 transport rates than the corresponding acylated l-tyrosine analogues. However, as the size of the hydrophobic moiety increased, we observed a decrease in LAT1 transport rate with a concomitant increase in potency of inhibition. Our results have important implications for designing amino acid prodrugs that target LAT1 at the blood-brain barrier or on cancer cells.

Full text of DOI:10.1002/cmdc.202000824

Accepted Article
Title: The effects of prodrug size and a carbonyl linker on L-type amino
acid transporter 1-targeted cellular and brain uptake
Authors: Brooklynn Venteicher, Kasey Merklin, Huy X. Ngo, Huan-
Chieh Chien, Keino Hutchinson, Jerome Campbell, Hannah
Way, Joseph Griffith, Cesar Alvarado, Surabhi Chandra, Evan
Hill, Avner Schlessinger, and Allen Thomas
This manuscript has been accepted after peer review and appears as an
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the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000824
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01/2020

10.1002/cmdc.202000824
ChemMedChem
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The effects of prodrug size and a carbonyl linker on L-type amino
acid transporter 1-targeted cellular and brain uptake
Brooklynn Venteicher,[a] Kasey Merklin,[a] Huy X. Ngo,[b] Huan-Chieh Chien,[b] Keino Hutchinson,[c]
Jerome Campbell,[a] Hannah Way,[a] Joseph Griffith,[a] Cesar Alvarado,[a] Surabhi Chandra,[d] Evan Hill,[e]
Avner Schlessinger,[c] Allen A. Thomas*[a]
[a]
B. Venteicher,+ K. Merklin,+ J. Campbell, H. Way, J. Griffith, C. Alvarado, Prof. A. Thomas
Department of Chemistry
University of Nebraska at Kearney
2401 11th Ave, Bruner Hall of Science
Kearney, NE 68849 (USA)
E-mail: thomasaa@unk.edu
[b]
[c]
[d]
[e]
Dr. H. X. Ngo,+ Dr. H.-C. Chien+
Department of Bioengineering and Therapeutic Sciences
University of California, San Francisco
1550 4th St, Rm RH581
San Francisco, CA 94143 (USA)
K. Hutchinson,+ Prof. A. Schlessinger
Department of Pharmacological Sciences
Icahn School of Medicine at Mt. Sinai
1468 Madison Ave, Annenberg Building Floor 19
New York, NY 10029 (USA)
Prof. S. Chandra
Department of Biology
University of Nebraska at Kearney
2401 11th Ave, Bruner Hall of Science
Kearney, NE 68849 (USA)
Prof. E. Hill
Department of Psychology
University of Nebraska at Kearney
2507 11th Ave, Copeland Hall
Kearney, NE 68849 (USA)
[+]
These authors contributed equally to this work.
Supporting information for this article is given via a link at the end of the document.
Abstract: The L-type amino acid transporter 1 (LAT1, SLC7A5)
imports dietary amino acids and amino acid drugs (e.g. L-DOPA) into
the brain, and it plays a role in cancer metabolism. Though there have
been numerous reports of LAT1-targeted amino acid-drug conjugates
(prodrugs), identifying the structural determinants to enhance
substrate activity has been challenging. In this work, we investigated
the position and orientation of a carbonyl group in linking hydrophobic
moieties including the anti-inflammatory drug ketoprofen to L-tyrosine
and L-phenylalanine. We found that esters of meta-carboxyl L-
phenylalanine had better LAT1 transport rates than the corresponding
acylated L-tyrosine analogs. However, as the size of the hydrophobic
moiety increased, we observed a decrease in LAT1 transport rate with
a concomitant increase in potency of inhibition. Our results have
important implications for designing amino acid prodrugs that target
LAT1 at the blood-brain barrier (BBB) or on cancer cells.
DOPA,[5] which are used for treating brain disorders. Additionally,
drugs conjugated to phenylalanine (prodrugs) may also be LAT1
substrates.[6]
A major challenge of designing LAT1-targeted amino acid
prodrugs is that optimization of ligand-transporter interactions can
lead to inhibition rather than transport into the cell.[7] We have
shown that the structural differences between an inhibitor and
substrate are subtle.[8] For example, we[7a] and others[9] found that
substitution of phenylalanine’s aromatic ring at the meta position
generally resulted in improved uptake rate and/or binding potency
relative to the ortho and para positions. However, meta
substitution by larger, hydrophobic groups (e.g., phenyl or benzyl)
resulted in good LAT1 inhibition (IC50 5-10 M) but not
transport.[7a] Moreover, amino acids containing 4-5 aromatic rings
have been reported as highly potent LAT1 inhibitors rather than
substrates.[10]
We previously showed that LAT1 substrate SAR was
surprisingly tolerant of various functional groups at
phenylalanine’s meta position.[8] We also found that there was not
a direct correlation between the polarity of the meta substituent
and a ligand’s affinity. For instance, both tert-butyl- and
hydroxyethyl-substituted esters had comparable IC50 values and
transport rates. However, when a tert-butyl group was directly
attached at the meta position without an ester linkage, a
substantial decrease in transport rate was observed.[8]
Additionally, other carbonyl-containing functional groups (e.g.,
Introduction
The L-type amino acid transporter 1 (LAT1, SLC7A5) is a sodium-
independent heterodimeric transmembrane protein highly
expressed in both the blood-brain barrier[1] (BBB) and in
numerous cancer types.[2] LAT1 transports neutral amino acids
(e.g., Phe, Leu, Met) and L-histidine as well as amino acid-
containing drugs such as gabapentin,[3] melphalan,[4] and L-
1
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10.1002/cmdc.202000824
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amide, ketone, and carboxylic acid) resulted in substrate activity.
Given the carbonyl linker’s positive effect on substrate activity for
relatively small substituents, we hypothesized that larger moieties
might also benefit from its presence. Furthermore, since carbonyl
groups are a commonly used structural motif for attaching drugs
to amino acids in LAT1-targeted drug delivery,[6a,9b,11] it would be
advantageous to optimize their position and orientation within a
prodrug to increase its substrate activity. That combined with the
size and shape of the LAT1 binding site from recently solved
structures[12] and analysis of docked ligands[8] led to the idea of
studying the effects of changing the following variables for a
carbonyl linker:
1. position in relation to an aromatic “drug” (Fig. 1a, “drug” =
phenyl) compared with phenylalanine’s aromatic ring (Fig.
1b) and the amino acid moiety;
2. distance of the carbonyl-substituted aromatic “drug” from the
amino acid moiety (Fig. 1a: variable chain length, n);
3. mode of attachment (Fig. 1b vs. 1c) for larger, hydrophobic
moieties (“drug” = phenyl or ketoprofen) compared with a
methyl group.
also be noted that “reverse amide” analogs of 3a and 3b (i.e.
derived from aspartic and glutamic acid) have been described,
and they were shown to be relatively weak binders (0% and 57%
inhibition of [14C]-L-leucine uptake in rat brain perfusion
experiments, respectively).[17] Thus, we did not pursue this type of
carbonyl linkage in the current study.
To address variable 3, we prepared hydrophobic esters,
containing phenyl or ketoprofen moieties, employing two different
modes of attachment (Schemes 2-4). We selected ketoprofen, a
nonsteroidal anti-inflammatory drug, because the LAT1 activity of
ketoprofen-tyrosine conjugates (i.e., 21 and 22) had previously
been described.[6a,15] Moreover, the BBB permeability of prodrug
21 had been correlated with LAT1 uptake.[6a] Our models have
predicted the presence of polar side chains near the binding site
(e.g., Ser143, Ser338, Asn404) that are capable of hydrogen
bonding to a carbonyl substituent potentially leading to enhanced
potency.[8] Thus, from the results of our previous SAR study[8] (for
example, 10 vs. 11), we hypothesized that reversing the
orientation of the ester linkage as in 23 by employing a ketoprofen
derivative (alcohol 17) would lead to enhanced LAT1 potency
while maintaining uptake rate, resulting in greater BBB
permeability relative to 22. Likewise, we prepared and tested
phenoxycarbonyl analog 13 and benzoyloxy-substituted
phenylalanine derivatives 14 and 15 to determine the effect of the
ester’s mode of attachment for an intermediate-sized substituent
between the larger ketoprofen and a methyl group (i.e., 10 and
11).
Figure 1. Some general strategies for attaching drugs to amino acids involving
carbonyl groups (X = NH or O). (a) Carboxyl-containing drug conjugated to
lysine (X = NH, n = 4), serine (X = O, n = 1) or their homologs (variable n).[13] (b)
Alcohol- or amine-containing drug conjugated with meta-carboxyl
phenylalanine.[10b,11b,14] (c) Carboxyl-containing drug conjugated with meta-
tyrosine (X = O) or its anilino analog (X = NH).[9b,13b,15]
Results and Discussion
Synthesis
To address variables 1 and 2, we prepared a series of
benzoyl esters and amides derived from serine and lysine
homologs (Scheme 1). It had been previously shown that an
amide of ketoprofen and lysine (analogous to compound 3d)
resulted in 79% inhibition of the uptake of [14C]-L-leucine in a rat
brain perfusion experiment and was able to cross the BBB.[16]
However, it was not directly demonstrated whether this compound
was a LAT1 substrate or inhibitor or whether it might be gaining
access to the brain via a different transporter. Moreover, there has
not been a systematic study of the effect of chain length for
substituted serine and lysine homologs on LAT1 activity. It should
The syntheses of benzoyl amide and ester homologs of Table 1,
3a-d and 4a-b, respectively, were conveniently performed
according to Scheme 1. Benzoylation of commercially available
BOC-protected lysine homologs 1a-c (R = H) provided amides 2a-
c. Lysine derivative 2d (R = Me) was prepared from methyl ester
1d due to the convenience of availability at the time we initiated
the synthesis. Deprotection of 2a-d gave desired lysine homologs
3a-d. Esters 4a-b were easily prepared in one step by reaction of
L-serine or L-homoserine with benzoyl chloride in neat TFA to
avoid reaction at the alpha amino group.
Scheme 1. Synthesis of benzoyl amides 3a-d and esters 4a-b.
2
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Phenoxycarbonyl-
and
benzoyloxy-substituted
removed in one step using TFA to give desired phenoxycarbonyl
analog 13. Though benzoyloxy analog 15 was previously
synthesized in multiple steps using protecting groups,[17] we found
that in a manner analogous to 4a-b both benzoyloxy analogs 14
and 15 could be simply prepared by benzoylation of meta-L-
tyrosine and L-tyrosine in neat TFA, respectively.
phenylalanine derivatives of Table 2 (13-15) were synthesized
according to Scheme 2. The syntheses of all other phenylalanine
derivatives of Table 2 have previously been described.[7a,8]
Negishi coupling between aryl bromide 5, synthesized by
esterification of commercially available 3-bromobenzoic acid with
phenol, and organozinc 6[8] gave protected phenylalanine
derivative 7. BOC and tert-butyl groups were conveniently
Scheme 2. Synthesis of phenoxycarbonyl analog 13 and benzoyloxy-substituted phenylalanine derivatives 14 and 15.
The para-substituted ketoprofen-tyrosine conjugate 21 was
synthesized by acylation of L-tyrosine in TFA solvent as
previously described (Scheme 3).[6a] However, in our hands, only
a 3% yield of 21 was attained after preparative HPLC purification.
We found that a significantly higher yield (35%) could be achieved
for the synthesis of meta isomer 22 by acylation of BOC-protected
meta-L-tyrosine 8c, followed by deprotection using HCl in dioxane.
Scheme 3. Synthesis of para- and meta-substituted ketoprofen-tyrosine conjugates 21 and 22.
Compound 23, a “reverse ester” analog of the meta-
substituted ketoprofen-tyrosine conjugate 22, was prepared
according to Scheme 4. Alcohol 17 was obtained using a borane
reduction of ketoprofen.[18] DIC-mediated coupling of alcohol 17
and 9-BBN-protected 19, derived from meta-carboxyl
phenylalanine 18 (not depicted),[11b] gave ester 20, which was
subsequently deprotected using TBAF[19] to provide desired
analog 23.
3
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Scheme 4. Synthesis of ketoprofen-derived “reverse ester” analog 23.
SAR from HEK-hLAT1 Cell Assays
determined in the presence of negative control. The SAR data
from these two assays serve as a predictor of a compound’s LAT1
activity in vivo.
To determine LAT1 activity of our compounds, both cis-inhibition
and trans-stimulation assays were performed using HEK cells that
overexpress human LAT1, as previously described.[8,20] Both
positive and negative controls (i.e., phenylalanine and arginine,
respectively) were used in cell assays. A cis-inhibition assay
allows for evaluation of LAT1 potency. The %inhibition data were
determined by measuring the uptake of a radiolabeled substrate,
[3H]-gabapentin, in the presence of test compounds. IC50 values
were determined for selected compounds. Though a cis-inhibition
assay is vital for determining ligand potency, it is insufficient for
determining whether a compound is a LAT1 substrate. A trans-
stimulation assay was performed for determining whether a
compound is a LAT1 substrate. This assay takes advantage of
LAT1’s alternating-access mechanism.[21] In brief, HEK-hLAT1
cells were pre-loaded with [3H]-gabapentin and then incubated
with test compounds. Because there is a 1:1 stoichiometry for
exchange, the measured efflux rate of [3H]-gabapentin is directly
Benzoylated serine 4a and homoserine analog 4b (Table 1)
exhibited greater LAT1 transport rates than amides 3a and 3b
with the same chain length, as evidenced by larger %efflux of [3H]-
gabapentin in our trans-stimulation assay. However, both amides
(3a-b) and esters (4a-b) with shorter chain length (n = 1, 2) had
poor potency (%inhibition of LAT1 at 200 M: 4.6–16%). As the
chain length was increased to 3 carbon atoms for amide 3c, there
was an increase in binding potency. This agrees with its docking
pose (Fig. 2A), which suggests an additional pi-pi interaction
between its aromatic sidechain and Y259. Additionally, 3c had a
better docking score than all other compounds of Table 1
(supporting information, Table S1). In contrast, an interaction with
Y259 is not predicted for 3a or 3b (Fig. 2B), both of which are less
potent than 3c. While 3d, containing a chain of 4 carbon atoms,
is also predicted to make a pi-pi interaction with Y259, its
diminished potency may be due to an entropic penalty resulting
related to the rate of substrate uptake.[3,22] For ease of comparison, from the additional carbon atom. Transport rates for 3c and 3d
the compounds’ transport rates were normalized to the value for
L-phenylalanine (2.7 ± 0.3 fmol/min), which was set to 100%. For
a compound to be considered a substrate, its transport rate must
be greater than the background efflux rate of [3H]-gabapentin, as
were comparable to negative controls L-lysine and L-serine
indicating that they are more likely to be inhibitors than substrates.
Figure 2. Docking poses of compounds 3c (A) and 3b (B). Residues predicted to make hydrogen bonds (black dashes) are labeled. Oxygen, nitrogen, and carbon
atoms are represented in red, blue, and white, respectively.
4
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The only amide of Table 1 that was a LAT1 substrate was
enhancing LAT1 affinity.[7a,9b] Surprisingly, the bulkier phenyl ester
13 had both a lower transport rate and potency (higher IC50)
relative to its methyl ester counterpart 10, whereas 14 showed
significantly greater potency compared with its methyl analog 11.
Nonetheless, the substrate activity of phenyl ester 13 supports our
hypothesis that employing an ester linkage in which the carbonyl
is directly attached to the aromatic ring of phenylalanine allows for
transport of hydrophobic substituents (at least as large as a
phenyl group) that otherwise might lead to LAT1 inhibition (e.g. 9
vs. 13). Though our models have predicted that the ester carbonyl
of 13 may be able to form hydrogen bonds with residues Ser 143,
Ser 348, and Asn 404 in the binding site,[8] we are unable to
rationalize the dramatic differences in transport rate and potency
depending on the ester’s orientation (e.g. 13 vs. 14). Additionally,
while 13 and 14 have a similar pose to those of 3c and 3d, they
are predicted to make additional hydrophobic interactions in the
binding site (supporting information, Figure S1). Interestingly, 14
has a slightly better docking score than that of 13 (supporting
information, Table S1), in agreement with the experimental results
(Table 2).
3a, which possessed the shortest chain length (n = 1). Although
esters 4a-b were substrates, they demonstrated significantly
lower potency (11-16% vs. 85% inhibition) and transport rates
(56-59% vs. 100% efflux) than positive control L-phenylalanine.
Due to the lack of commercial availability of synthetic precursors
and potentially difficult syntheses, longer chain homologs of
serine (i.e. X = O, n = 3 and 4) were not pursued.
Taken together with our previous SAR study[8] and the
results of Table 2, it is apparent that positioning the carbonyl linker
between the aromatic ring and amino acid backbone as in serine
and lysine homologs of Table 1 results in decreased potency
relative to carbonyl at the meta position (e.g., compounds 10 and
13). However, given that 3a and 4a-b are substrates, conjugation
of drugs with lysine and serine homologs may still be useful for
designing LAT1-targeted prodrugs. Also, it would probably be
more convenient to synthesize amino acid prodrugs from lysine
or serine and their homologs according to Scheme 1 than from
meta-substituted phenylalanine derivatives (Schemes 2-4).
Table 1. Relative exchange efflux rate and uptake inhibition of [3H]-gabapentin
in HEK-hLAT1 cells for benzoylated lysine (3a-3d) and serine (4a-b) homologs
with variable chain length.
Table 2. Relative exchange efflux rate, uptake inhibition of [3H]-gabapentin and
IC50 values in HEK-hLAT1 cells for meta- and para-substituted phenylalanine
derivatives 9-15 compared with positive and negative controls, L-phenylalanine
and L-arginine, respectively.
%L-Phe
Compound[a]
X
n
%Inhibition[c]
Efflux[b]
100
33
L-Phe
L-Lys
L-Ser
3a
-
1
4
1
1
2
3
4
1
2
85
-2.1
-1.0
4.6
8.6
54
%L-Phe
Efflux[b]
%Inhibi
tion[c]
IC50
(M)[d]
Compound[a]
R1
R2
-
-
35
L-Phe
L-Arg
9
H
-
H
100
28
29
89
37
67
52
30
36
85
49
98
81
54
37
85
100
49
69 ± 29
-
N
N
N
N
O
O
47
-
3b
37
PhCH2
MeO2C
MeCO2
H
H
7.3 ± 3
36 ± 23
-
3c
34
10
H
H
3d
26
38
11
4a
59
16
12
MeO2C
H
260 ± 90
70 ± 24
11 ± 3
-
4b
56
11
13
PhO2C
PhCO2
H
[a] Cell assay data was obtained at least in triplicate (wells). Amino acids were
purchased from commercial vendors or synthesized as depicted in Scheme 1.
All compounds above are single enantiomers of L configuration. [b] Compounds
were tested at 200 M for their ability to cause efflux (fmol/min) of [3H]-
gabapentin from pre-loaded HEK-hLAT1 cells. Efflux of [3H]-gabapentin was
calculated at 3 min after adding test compound. %Efflux was normalized relative
to L-Phe, which had an efflux rate of 2.7 ± 0.3 fmol/min, from an average of
seven experiments. [c] Compounds were tested at 200 M for their ability to
inhibit uptake of [3H]-gabapentin into HEK-hLAT1 cells. Data is presented as %
inhibition relative to background signal in the absence of a test compound.
14
H
15
PhCO2
[a] Cell assay data was obtained at least in triplicate (wells). L-Phenylalanine
and L-arginine were purchased from commercial vendors. Compounds 13-15
were synthesized as depicted in Scheme 2. We have previously published the
synthesis and LAT1 activity of all other amino acids of Table 2.[7a,8] [b,c]
Compounds were tested in trans-stimulation and cis-inhibition cell assays as
described for Table 1. [d] For IC50 determinations, varying concentrations of
each compound were added, from 0.1 µM to 500 M. IC50 and standard
deviation of each compound were calculated by Graphpad Prism version
5.0. %[3H]-Gabapentin uptake at each concentration was normalized relative
to %inhibition by BCH[23] at 2 mM, which was set to 100% inhibition.
Consistent with our previous SAR study (e.g. 10 vs. 11),[8]
having the carbonyl group directly attached to phenylalanine’s
aromatic ring in phenoxycarbonyl analog 13 resulted in a greater
transport rate than benzoylated meta-tyrosine 14. The latter is
likely a LAT1 inhibitor rather than a substrate due to its poor
transport rate. As with methyl ester 12, substitution at the para
position in 15 led to a decrease in potency, which is consistent
with previous reports that the meta position is preferred for
Given the differences in activity between regioisomers 13
and 14, we were curious as to whether these results would
transfer to a larger substituent than phenyl. As indicated above,
we selected ketoprofen and a ketoprofen derivative 17 as
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substituents, because ketoprofen prodrugs 21 and 22 had
previously been reported as LAT1 substrates.[6a,15] We
hypothesized that reversing the orientation of the ester linker (i.e.
22 vs. 23) would improve LAT1 transport rate leading to greater
brain uptake. Despite reports that both 21 and 22 are transported
by LAT1,[6a,15] based on the results of our trans-stimulation assay
in HEK-hLAT1 cells (Table 3), only compound 21 had a
significantly greater %efflux rate than negative control L-arginine.
Additionally, 21 and 22 were >50-fold more potent than L-
phenylalanine, which is consistent with previous observations that
potency is indirectly correlated with LAT1 substrate activity.[7]
Interestingly, reversing the orientation of the ester in 23 resulted
in an even greater increase in potency (IC50 = 0.31 ± 0.1 M) but
did not provide the desired substrate activity that had been
observed with phenyl ester 13. This result indicates that like 21
and 22, “reverse ester” 23 is most likely an inhibitor and not a
substrate, and it is among the more potent LAT1 inhibitors
reported in the literature.[10,24]
Table 3. Relative exchange efflux rate, uptake inhibition of [3H]-gabapentin and IC50 values in HEK-hLAT1 cells for meta- and para-substituted ketoprofen-tyrosine
conjugates 21, 22, and “reverse ester” analog 23.
IC50
Compound[a]
R1
R2
%L-Phe Efflux[b]
%Inhibition[c]
(M)[d]
69 ± 29
-
L-Phe
L-Arg
H
-
H
-
100
28
85
49
21
22
23
H
45
23
32
108
107
108
1.2 ± 0.2
1.2 ± 0.2
0.31 ± 0.1
H
H
[a] L-Phenylalanine and L-arginine were purchased from commercial vendors. Compounds 21 and 22 were synthesized as depicted in Scheme 3. Compound 23
was synthesized as shown in Scheme 4. [b-d] Cell assays were performed as described for Tables 1 and 2.
Uptake in HEK Uninduced and HEK-hLAT1 Cells
To corroborate the results of our trans-stimulation assay for 22
and 23, we measured their intracellular concentrations in HEK-
hLAT1 and uninduced HEK cells after a 30 min incubation time at
50 M using LC-MS analysis (Fig. 3). We used L-phenyl-d5-
alanine (Phe-d5) as
a positive control, which could be
distinguished from endogenous L-phenylalanine already present
within cells. Consistent with expectations, Phe-d5 displayed a 3.5-
fold greater uptake in HEK-hLAT1 cells relative to uninduced cells.
In contrast, the difference in uptake between induced and
uninduced cells for 22 and 23 was much smaller (1.2–1.5-fold).
The uptake of 22 in uninduced HEK cells was approximately one-
tenth the value reported in MCF-7 cells (0.013 ± 0.002 vs. 0.10 ±
0.01 nmol/mg/min),[15] which is likely due to a greater expression
of transporters on the latter’s surface. For example, 22 was also
reported to interact with organic-anion-transporting polypeptides
(OATP),[15] which are expressed in cancer cells.[25] Surprisingly,
compound 23 had about a 10-fold greater uptake into both
induced and uninduced HEK cells compared with 22. We currently
do not have an explanation for this discrepancy; however, it is
possible that compound 23 is gaining entry by a transporter that
is selective against 22 or maybe 23 has some passive diffusion.
Figure 3. Uptake of Phe-d5, 22, and 23 in HEK-hLAT1 cells and in HEK
uninduced cells (mean ± SD, n = 3) after a 30 min incubation time at 50 M,
normalized to milligrams of cell protein.
Taken together, these results indicate that if LAT1 is
contributing to cell uptake for 22 or 23, it is small in comparison
with Phe-d5. Perhaps, 22 and 23 are too large to be
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accommodated in the binding site as LAT1 changes
conformations from outward-facing to inward-facing. Alternatively,
they may bind a different conformation (e.g. outward-facing
conformation) so strongly that the barrier to conformational
change is prohibitive.
ketoprofen brain levels, the amount of 22 transported into the rat
brain was approximately one-third the value of Phe-d5.
Unfortunately, neither 23 nor its expected metabolite 17,
were detected in rat brain (<0.3 nmol/g/min). This result contrasts
with the higher uptake of 23 into HEK uninduced cells relative to
Phe-d5 and 22. It may be that 23 is effluxed by an ABC transporter
faster than uptake at the BBB. Alternatively, 23 may be entering
HEK cells by transporters that are less abundant at the BBB,
which could also help explain its dramatically greater cell uptake
compared with 22. Nonetheless, these findings support our
interpretation of data from HEK-hLAT1 cells that 23 is not a LAT1
substrate. Though we cannot rule out the possibility of LAT1
playing a role in BBB transport of 22, its brain levels were
significantly lower than Phe-d5. Moreover, as has been
reported,[15] 22 is a substrate for the low affinity-high capacity
transporter OATP2 (SLC21A5) and thus could be crossing the
BBB primarily by this mechanism,[29] particularly in light of the high
concentration (500 M) we used in our perfusion experiments.
Rat Brain Perfusion Studies
Given the surprisingly good uptake of 23 into HEK cells relative to
Phe-d5 and 22, we wondered whether it might be BBB permeable,
even if LAT1’s contribution toward transport was small. Thus, 23
was tested alongside Phe-d5 and 22 in a modified in situ rat brain
perfusion experiment.[26] Though many techniques exist to study
transport across the BBB,[27] in situ brain perfusion effectively
measures brain uptake in live mammalian models. Also, due to
direct injection into the rat carotid artery, which is flushed with
perfusion buffer prior to adding compound, we would expect
prodrugs such as 22 or 23 to be the major species being
presented at the BBB rather than their metabolites (i.e. ketoprofen
or alcohol 17, respectively). For this experiment, test compounds
were perfused at 500 M for 1 min, which we found to be needed
to reliably determine brain levels using our single quad LC-MS
system. It is likely that at this high concentration most active
transport mechanisms would be saturated, including LAT1.
Conclusion
We have shown that both the position and orientation of a
carbonyl group used to link hydrophobic substituents to an amino
acid can have a considerable impact on LAT1 transport rate and
potency. Additionally, we found that serine and homoserine are
likely better amino acid promoieties than lysine and its homologs
for use in LAT1-targeted drug delivery, as amides of the latter had
poorer transport rates and were generally not substrates.
However, benzoylated serine and homoserine derivatives were
considerably less potent compared with meta-substituted
phenylalanine analogs, providing further evidence that the
carbonyl’s position relative to the phenyl ring and the amino acid
group are important for recognition by LAT1. Consistent with our
previous findings for a methyl ester,[8] having a carbonyl group
directly attached at the meta position of phenylalanine gave a
better transport rate for
a phenyl-substituted ester than
attachment via the ester oxygen.
Despite the benefits of having a carbonyl directly attached
at the meta position, as the size of the substituent was increased
to include two aromatic rings (i.e. ketoprofen analog 17), the
transport rate decreased along with a dramatic increase in
potency for “reverse ester” 23. Despite previous reports that
ketoprofen-tyrosine prodrugs (i.e., 21 and 22) are LAT1
substrates,[6a,15] based on our experiments using an inducible cell
line, HEK-hLAT1, we found them to be potent LAT1 inhibitors and
not substrates. Moreover, rat brain perfusion experiments
corroborated our interpretation of cell assay data, as prodrug 22
and its analog 23, in which the orientation of the ester group was
reversed, had poor to no brain uptake, relative to L-phenyl-d5-
alanine. Taken together, we conclude that though LAT1 can
transport phenylalanine derivatives with relatively small
substituents, at least up to the size of a phenyl ring, larger
substituents are more likely to lead to LAT1 inhibition and not
substrate activity. These findings are highly relevant to LAT1-
targeted treatment of both neurological diseases and cancer.
Figure 4. Rat brain uptake after perfusion of Phe-d5, 22, and 23 at 500 M for
1 min, normalized to brain hemisphere mass (mean ± SD, n = 3). Brain levels
of respective metabolites ketoprofen and alcohol 17 are plotted on the same
graph for comparison sake. Both 23 and 17 were below our limit of detection of
0.3 nmol/g/min.
Phe-d5 showed considerably greater rat brain uptake (26 ±
3 nmol/g/min) compared with ketoprofen-derived prodrugs 22 and
23 (Fig. 4). Uptake of 22 was at the limit of detection for our LC-
MS (0.3 nmol/g/min). Consistent with what had been reported for
rat brain perfusion of the para isomer 21,[6a] we also observed
extensive intrabrain metabolism of 22 to give parent drug
ketoprofen (7.1 ± 2.3 nmol/g/min).[28] Compound 22 has been
shown to be cleaved by esterases in various species and tissue
types, including rat brain S9 fraction to give ketoprofen as the sole
metabolite besides the phenylalanine-derived promoiety.[15]
Assuming a direct relationship between uptake of 22 and
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000824
ChemMedChem
FULL PAPER
was stirred overnight at rt. Acidified the cooled reaction mixture with 3M
aq. HCl. Extracted products with diethyl ether, dried (MgSO4), and
concentrated in vacuo. Crude products were taken forward to the next step
without purification. Yields: 95-100%.
Experimental Section
This experimental section includes full details for synthesis of final
products, cell assay conditions, modeling methods, and procedures for rat
brain and cellular uptake studies. The following can be found in supporting
information: docking poses of compounds 13 and 14 (Figure S1); docking
scores of compounds from Tables 1-3 (Table S1); synthesis and summary
of deuterated standards for test compounds used in LC-MS analysis of rat
brain tissue samples (Table S2); 1H and 13C NMR spectra of newly
synthesized, final products (3b-d, 4b, 13, 14, 23); and relative uptake of
[3H]-gabapentin into un-transfected, un-induced and induced HEK-hLAT1
cells in the presence and absence of LAT1 inhibitor BCH at 2 mM (Figure
S2).
General procedure for BOC deprotection to give benzoylated lysine
homologs (3a-c): BOC-protected homologs 2a-c (2.0 mmol, 1.0 equiv)
were stirred overnight at rt with triethylsilane (3.0 equiv) and 1:1 TFA (10
equiv)/DCM. Reaction mixture was concentrated in vacuo and then
purified by preparative HPLC (Method B). 3a: Yield: 56 mg (14%); >98%
purity by LC-MS (254 nm, Method B), 1H NMR ((CD3)2SO)  8.72 (s, 1H),
7.84 (d, J = 7Hz, 2H), 7.58-7.43 (m, 3H), 4.09 (m, 1H), 3.58 (m, 2H); in
accordance with reference.[32] m/z (ESI-pos) M+1 = 209.1. 3b: Yield: 54
mg (12%), >98% purity by LC-MS (254 nm, Method C), mp 210-
213 °C, [훼]2퐷4 +14° (c 0.45, 1M aq. HCl), H NMR (D2O + 5%DCl)  7.01
1
Ligand Docking. Molecular docking was performed using Glide from the
Schrödinger suites. All ligands were docked to the recently solved cryo-
EM structure of LAT1 (PDB: 6IRT).[12b] The 6IRT structure was prepared
for docking with the Maestro Protein Preparation Wizard[30] under default
parameters. The ligand binding site was defined based on the coordinates
of LAT1 inhibitor BCH[23] from the 6IRT structure. The receptor grid for
docking was generated via Maestro Receptor Grid Generation panel.[30]
The small molecules used in molecular docking were prepared for docking
using LigPrep of the Schrödinger suite.[30] The docking results were
visualized via PyMOL.[31]
(m, 2H), 6.88 (m, 1H), 6.78 (m, 2H), 3.48 (t, J = 6Hz, 1H), 2.89 (t, J = 6Hz,
2H), 1.59 (m, 2H); 13C NMR ((CD3)2SO + 5% DCl)  170.9, 168.9, 134.1,
131.6, 128.5, 127.5, 50.1 (m), 35.5 (m), 30.0. m/z (ESI-pos) M+1=223.1.
3c: Yield: 280 mg, (60%), >98% purity LC-MS (254 nm, Method C), mp
229-231°C dec, [훼]2퐷4 +19° (c 0.51, 1M aq. HCl), 1H NMR (D2O)  7.72 (m,
2H), 7.58 (m, 1H), 7.49 (m, 2H), 3.76 (m, 1H), 3.41 (m, 2H), 1.93 (m, 2H),
1.70 (m, 2H); 13C NMR (D2O, CH3CN added as internal standard)  172.6,
171.6, 134.1, 132.6, 129.3, 127.5, 53.3, 39.5, 27.7, 24.7. m/z (ESI-pos)
M+1=237.1.
Methyl
Commercially
N6-benzoyl-N2-(tert-butoxycarbonyl)-L-lysinate
(2d):
Synthesis General. Flash Column Chromatography was performed either
using silica gel (porosity 60 Å, particle size: 40-63 m, 230 x 400 mesh)
from Sorbent Technologies in Chemglass columns or using a Teledyne
ISCO NextGen300+ Flash Chromatography System (RediSepRf High
Performance GOLD silica cartridges). Preparative HPLC performed on a
Gilson PLC 2020. Column: Synergi 4 Fusion-RP by Phenomenex, 150 x
21.2 mm, protected with a SecurityGuard PREP Cartridge, C12, 15 x 21.2
mm. Preparative HPLC methods: Each of the following methods employed
isocratic elution at 20 mL/min flow rate with the indicated percentage (%)
of CH3CN in non-buffered Milli-Q deionized water (Integral 5 Water
Purification System). Method A: 0% (i.e. water only); B: 2%; C: 5%; D:
10%; E: 30%. Compounds purified by HPLC were concentrated by
lyophilization using a Labconco Freezone 2.5 Plus. LC-MS analysis was
performed using an Agilent G6125 single quad ESI source and a 1260
available methyl (tert-butoxycarbonyl)-L-lysinate
hydrochloride 1d (0.35 g, 1.2 mmol) and DMAP (14 mg, 0.12 mmol) were
dissolved in DCM (4 mL) and stirred at 0 °C. N-Ethyl-N-isopropylpropan-
2-amine (0.52 mL, 2.5 mmol) was added and the reaction was left to stir
for 10 min, followed by addition of benzoyl chloride (0.21 mL, 1.8 mmol)
all-at-once. Reaction mixture was stirred for 1 h at rt. Then, the reaction
was acidified with 1M aq. HCl (10 mL), extracted with diethyl ether (2 x 10
mL), dried (MgSO4) and filtered, and concentrated in vacuo. Crude product
was carried forward to the next step without purification. Yield: 0.475 g
(100%).
N6-Benzoyl-L-lysine (3d): Saponification of the methyl ester functional
group in 2d was achieved using LiOH in a manner similar to that described
previously.[33] Crude product was carried directly forward into BOC
deprotection by stirring with 1:1 TFA/DCM (5 mL) overnight at rt. Removed
solvents in vacuo. Desired product 3d was purified by preparative HPLC
(Method D). Yield: 0.12 g (41%), >98% purity LC-MS (254 nm, Method A),
mp 234-240 °C dec, [훼]2퐷4 +19.2° (c 0.89, 1M aq. HCl), 1H NMR (D2O) 
7.59 (m, 2H), 7.46 (m, 1H), 7.37 (m, 2H), 3.98 (m, 1H), 3.29 (m, 2H), 1.88
(m, 2H), 1.56 (m, 2H), 1.39 (m, 2H). 13C NMR ((CD3)2SO + 5% DCl)  173.4,
173.0, 135.4, 133.9, 131.5, 129.9, 54.9, 42.5, 31.6, 30.0, 24.2. m/z (ESI-
pos) M+1=251.1.
Infinity HPLC system (G7112B Binary Pump and G7114A Dual

Absorbance Detector). Column: Synergi 4 Fusion-RP by Phenomenex,
150 x 4.6 mm. The following LC-MS methods (A-F) were performed using
1.0 mL/min flow rates: Method A: gradient elution, starting with 10%
CH3CN in 0.1% formic acid and ramping to 80% over 10 min. For LC-MS
methods B-F, isocratic elution was performed using a mobile phase
containing the following percentages (%) of CH3CN in 0.1% formic acid.
LC-MS Method: B, 5%; C, 10%; D, 20%; E, 30%; F, 50%.
1H and 13C NMR were recorded on an Avance III HD Bruker instrument
operating at 400 and 100 MHz, respectively. Unless indicated otherwise,
NMR spectra were obtained as CDCl3, CD3OD, D2O and (CD3)2SO
General procedure for benzoylation of serine and homoserine (4a-b):
L-Serine or L-homoserine (1 equiv) were stirred with TFA (1.7M) at 0 ºC
for 15 min. Then, benzoyl chloride (1.5 equiv) was added all-at-once and
the mixture was stirred vigorously overnight at rt. The reaction mixture was
diluted with cold diethyl ether (50 mL), and the suspension was filtered and
the resulting solids were dried under high vacuum. The crude product was
purified by preparative HPLC (Method C). 4a: Yield: 65 mg (24%), >95%
purity by LC-MS (254 nm, Method C), 1H NMR ((CD3)2SO + 5%DCl)  8.13-
7.36 (m, 5H), 4.68 (m, 2H), 4.48 (m, 1H). Our 1H NMR was consistent with
the reported spectrum.[34] m/z (ESI-pos) M+1=210.1. 4b: Yield: 50 mg
(16%), >98% purity by LC-MS (254 nm, Method B), mp 198-200°C,
solutions (reported in ppm), using residual solvent peaks in the 1H and 13
C
NMR spectra (CDCl3: 7.27, 77.23 ppm; CD3OD: 3.31, 49.15 ppm; D2O,
4.79; and (CD3)2SO: 2.50, 39.51 ppm) as the reference standard,
respectively. All J values are given in units of Hz. Optical rotations were
measured on a Rudolph Research Autopol III polarimeter (using sodium D
line, 589 nm) and []D given in units of (degrees-mL)/(dm-g), and
concentration (c) is reported in units of g/100 mL. All water used in analysis
and for preparative HPLC was purified by a Milli-Q® Integral 5 Water
Purification System. Melting points were obtained using a Mel-Temp
apparatus and are uncorrected. Sonication was performed using a VWR
Aquasonic Model 75T.
[훼]2퐷4 +37° (c 0.66, 1M aq. HCl), H NMR (D2O + 5%DCl)  7.72 (m, 2H),
1
7.43 (m, 1H), 7.27 (m, 2H), 4.26 (m, 2H), 4.08 (m, 1H), 2.25 (m, 2H); 13C
NMR (D2O, acetone added as internal standard)  172.0, 168.9, 134.6,
130.0, 129.5, 129.4, 61.4, 51.0, 29.3. m/z (ESI-pos) M+1=224.1.
General procedure for benzoylation of BOC protected lysine
homologs (2a-c): Commercially available BOC-protected lysine
homologs 1a-c (1.0 equiv) and NaOH (2.1 equiv) were dissolved with
stirring in 1:1 dioxane/water (0.5 M). The solution was cooled in an ice bath,
and benzoyl chloride (1.2 equiv) was added dropwise. Reaction mixture
Phenyl 3-bromobenzoate (5): To a stirred mixture of DMAP (0.12 g , 1.0
mmol), 3-bromobenzoic acid (2.0 g, 10 mmol), phenol (0.94 g, 10 mmol),
and THF (3 mL) was added N,N'-diisopropylcarbodiimide (1.3 g, 10 mmol)
8
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10.1002/cmdc.202000824
ChemMedChem
FULL PAPER
at 0 °C. Reaction mixture was left to stir overnight at rt. Then, the mixture
was suspended in 1:1 DCM/hexanes (10 mL). The suspension was filtered
to remove insoluble diisopropyl urea and the filtrate was concentrated in
vacuo. Mixture was purified by flash chromatography (10%
EtOAc/hexanes). Yield: 2.1 g (74%). 1H NMR (CDCl3)  8.37 (s, 1 H), 8.16
(d, J = 8Hz, 1H), 7.79 (d, J = 8Hz, 1H), 7.44 (m, 3H), 7.31 (m, 1H), 7.23
(m, 2H).
(S)-3-(2-Amino-2-carboxyethyl)benzoic acid hydrochloride (18): The
title
compound
was
prepared
from
(S)-2-amino-3-(3-
cyanophenyl)propanoic acid (5.0 g, 26 mmol) using a procedure described
by Peura[11b]. Yield: 5.1 g (79%).
3-(((1R,4'S,5S)-5'-Oxo-94-boraspiro[bicyclo[3.3.1]nonane-9,2'-
[1,3,2]oxazaborolidin]-4'-yl)methyl)benzoic acid (19): The following
synthesis is a modification of a procedure described by Peura.[11b] Charged
a dry round bottom flask and stir bar with meta-carboxyl phenylalanine
hydrochloride 18 (4.0 g, 16 mmol), anhyd. DMF (40 mL), and anhyd.
pyridine (2.6 mL, 33 mmol). Cooled flask in an ice bath and added 9-
methoxy-9-borabicyclo[3.3.1]nonane (1M in THF, 16 mL, 16 mmol) to the
stirred solution, dropwise. Allowed reaction to slowly warm to rt and stirred
overnight. Transferred mixture to a separatory funnel using EtOAc (150
mL). Washed organic phase with 1M aq. KHSO4 (50 mL). Re-extracted
aqueous phase with EtOAc (50 mL). Combined organic phases were
washed with water (2 x 50 mL), brine (50 mL), dried (MgSO4), filtered and
concentrated in vacuo. Heated the crude product with EtOAc (50 mL) to
near boiling, using a spatula to break up the larger pieces of solid. Allowed
the suspension to cool to rt before filtering. Dried resulting white solid
under high vacuum overnight. Yield: 3.7 g (69%). 1H NMR ((CD3)2SO) 
12.9 (br s, 1H), 7.97 (s, 1H), 7.82 (d, J = 6Hz, 1H), 7.60 (d, J = 6Hz, 1H),
7.44 (m, 1H), 6.44 (m, 1H), 5.92 (m, 1H), 3.87 (m, 1H), 3.24 (m, 1H), 2.99
(m, 1H), 1.27-1.84 (m, 12H), 0.45 (m, 2H).
Phenyl
(S)-3-(3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-
oxopropyl)benzoate (7): The title compound was prepared from tert-butyl
(R)-2-((tert-butoxycarbonyl)amino)-3-iodopropanoate[8] (1.9 g, 5.0 mmol)
and aryl bromide 5 (1.4 g, 5.0 mmol) using Negishi coupling conditions that
we previously described.[8] Crude was partially purified via flash
chromatography (10% EtOAc/hexanes), and then carried forward without
further purification. Yield: 1.0 g (45%).
(S)-2-((tert-Butoxycarbonyl)amino)-3-(3-hydroxyphenyl)propanoic
acid (8c): BOC protection of meta-L-tyrosine 8b (1.0 g, 5.5 mmol) was
performed in a manner similar to that previously described for L-DOPA.[35]
Yield: 1.2 g (80%). Product was carried to the next step without purification.
(S)-2-Amino-3-(3-(phenoxycarbonyl)phenyl)propanoic
acid
hydrochloride (13): BOC deprotection of phenyl (S)-3-(3-(tert-butoxy)-2-
((tert-butoxycarbonyl)amino)-3-oxopropyl)benzoate 7 (1.0 g, 2.3 mmol)
was performed using TFA/Et3SiH in
a manner as we previously
2-(3-Benzoylphenyl)propyl
3-(((1R,4'S,5S)-5'-oxo-94-
described.[36] Product was purified via preparative HPLC (Method E) and
converted to an HCl salt by freeze drying in the presence of aqueous HCl.
Yield: 20 mg (3%), >98% purity through analysis of LC-MS (254 nm,
Method E), mp 208-211 °C dec, [훼]2퐷4 +2.7° (c 0.38, 1M aq. HCl), 1H NMR
(D2O + 5% DCl)  8.30-8.37 (m, 2H), 7.79-7.91 (m, 2H), 7.73 (m, 2H), 7.59
(m, 1H), 7.47 (m, 2H), 4.56 (t, J = 7Hz, 1H), 3.57 (m, 2H); 13C NMR (D2O
+ 5% DCl, and 5% CD3CN as internal standard)  171.5, 167.4, 151.6,
136.3, 136.2, 131.8, 130.9, 130.7, 130.4, 127.5, 122.7, 119.9, 54.7, 36.4.
m/z (ESI-pos) M + 1 = 286.1.
boraspiro[bicyclo[3.3.1]nonane-9,2'-[1,3,2]oxazaborolidin]-4'-
yl)methyl)benzoate (20): Charged a dry round bottom flask and stir bar
with boroxazolidone 19 (569 mg, 1.73 mmol), alcohol 17 (415 mg, 1.73
mmol), DMAP (21 mg, 0.17 mmol) and anhydrous THF (5 mL). The stirred
mixture was cooled in an ice bath under argon. N,N′-
Diisopropylcarbodiimide (0.54 mL, 3.5 mmol) was added dropwise. The
mixture was allowed to warm to rt, and stirring was continued overnight.
The reaction mixture was concentrated in vacuo. The resulting residue was
suspended in DCM (2-3 mL), filtered to remove diisopropylurea, and then
purified by an ISCO flash chromatography system (40 g silica cartridge),
eluting with a gradient of 20%-100% EtOAc/hexanes. Yield: 605 mg (64%).
1H NMR (CDCl3)  7.93 (m, 1H), 7.80-7.90 (m, 2H), 7.76-7.80 (m, 2H),
7.59-7.68 (m, 1H), 7.36-7.59 (m, 7H), 5.45 (m, 1H), 4.28-4.58 (m, 2H),
4.02-4.28 (m, 2H), 3.14-3.52 (m, 3H), 1.06-1.99 (m, 15H), 0.63 (s, 1H),
0.25 (d, J = 17 Hz, 1H).
(S)-2-Amino-3-(3-(benzoyloxy)phenyl)propanoic acid hydrochloride
(14): The title compound was prepared from meta-L-tyrosine (0.15 g, 0.82
mmol) using reaction conditions similar to those of 4a-b. After removal of
volatiles by concentration in vacuo, the product was converted to its HCl
salt by dissolution in p-dioxane (2 mL) and 4M HCl in dioxane (1.2 mL).
Concentrated in vacuo and then triturated resulting solids with diethyl ether.
The solids were filtered, rinsing with diethyl ether and dried under high
vacuum. Yield: 71 mg (30%), >98% purity by LC-MS (254 nm, Method D),
mp 183-187 °C dec, [훼]2퐷4 0° (c 0.091, DMSO), 1H NMR (D2O)  8.35 (m,
2H), 7.94 (m, 1H), 7.79 (m, 2H), 7.70 (m, 1H), 7.49 (m, 1H), 7.38 (m, 2H),
(2S)-3-(3-((2-(3-Benzoylphenyl)propanoyl)oxy)phenyl)-2-((tert-
butoxycarbonyl)amino)propanoic acid (22-BOC):
A solution of
±ketoprofen acid chloride 16 [prepared by refluxing thionyl chloride with
±ketoprofen as described by Napoleon[37] (444 mg 1.63 mmol)] dissolved
in DCM (1 mL) was added dropwise to a stirred mixture of BOC-protected
meta-L-tyrosine 8c (458 mg, 1.63 mmol), Et3N (0.69 mL, 4.9 mmol), and
DMAP (20 mg, 0.16 mmol) in DCM (1 mL) at 0 °C under argon. The
reaction was allowed to warm to rt slowly and stirring was continued
overnight. The reaction mixture was then cooled in an ice bath, and 1M aq.
HCl (10 mL) was carefully added. Extracted product with DCM (2 x 10 mL).
Dried combined organic phases (MgSO4), filtered and concentrated.
Purified using an ISCO flash chromatography system (12 g silica cartridge)
eluting with a gradient of 10%-50% EtOAc/hexanes (both mobile phases
containing 1% HOAc). Dried product under high vacuum overnight. Yield:
382 mg (45%). 1H NMR (CDCl3)  7.86 (m, 1H), 7.82 (m, 2H), 7.70 (m, 1H),
7.58-7.65 (m, 2H), 7.46-7.52 (m, 3H), 7.28 (m, 1H), 7.04 (m, 1H), 6.92 (m,
1H), 6.84 (m, 1H), 4.99 (m, 1H), 4.58 (m, 1H), 4.03 (q, J = 7Hz, 1H), 3.13
(m, 2H), 1.64 (d, J = 7Hz, 3H), 1.39 (br s, 9H).
4.51 (m, 1H), 3.56 (dd, J = 6, 15Hz, 1H), 3.43 (dd, J = 8, 15Hz, 1H); 13
C
NMR (D2O + 5% DCl, CD3CN added as internal standard)  171.8, 168.2,
151.9, 137.3, 135.6, 131.7, 131.0, 130.0, 129.6, 128.7, 123.7, 122.4, 120.1,
54.9, 36.3. m/z (ESI-pos) M+1=286.1.
(S)-2-Amino-3-(4-(benzoyloxy)phenyl)propanoic acid hydrochloride
(15): The title compound was prepared from L-tyrosine (1.0 g, 5.5 mmol)
according to the procedure for compound 14, and its synthesis has
previously been described.[17] Yield: 430 mg (27%), >98% purity by LC-MS
(254 nm, Method D). 1H NMR was consistent with the previously reported
spectrum.[17] 1H NMR (CD3OD)  8.19 (d, J = 8Hz, 2H), 7.72 (m, 1H), 7.59
(m, 2H), 7.43 (m, 2H), 7.28 (m, 2H), 4.32 (m, 1H), 3.14-3.34 (m, 2H,
overlaps with CHD2OD). m/z (ESI-pos) M+1=286.1.
(3-(1-Hydroxypropan-2-yl)phenyl)(phenyl)methanone (17): The title
compound was prepared as previously described,[18] however in our hands
the yield was considerably lower than what had been reported. Purified
product using an ISCO flash chromatography system (40 g silica cartridge),
eluting with a gradient of 10-50% EtOAc/hexanes. Yield: 0.49 g (26%). 1H
NMR (CD3OD)  8.16 (m, 2H), 7.68 (m, 1H), 7.55 (m, 2H), 7.39 (d, J = 8Hz,
2H), 7.24 (d, J = 9Hz, 2H), 4.29 (m, 1H), 3.37 (dd, J = 6, 16Hz, 1H), 3.28
(m, 3H), 3.17 (dd, J = 8, 15Hz, 1H).
(2S)-2-Amino-3-(3-((2-(3-
benzoylphenyl)propanoyl)oxy)phenyl)propanoic acid hydrochloride
(22): The title compound has also been synthesized by an alternate
route.[15] To 22-BOC (380 mg, 0.74 mmol) was added HCl (4M in dioxane,
2.8 mL, 11 mmol), and the mixture was stirred for 30 min at rt. Solvent was
removed in vacuo, and the resulting powder was triturated with diethyl
ether and filtered, rinsing several times with diethyl ether. Product was
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000824
ChemMedChem
FULL PAPER
dried overnight under high vacuum. Yield: 260 mg (79%), >98% purity by
LC-MS (254 nm, Method F). H NMR was consistent with the previously
reported spectrum.[15] 1H NMR ((CD3)2SO)  13.7 (br s, 1H), 8.42 (br s, 3H),
7.50-7.90 (m, 9H), 7.38 (m, 1H), 7.20 (m, 1H), 7.03 (m, 2H), 4.26 (m, 1H),
4.15 (m, 1H), 3.15 (m, 2H), 1.55 (d, J = 7Hz, 3H). m/z (ESI-pos) M + 1 =
418.2.
trans-Stimulation assay. HEK-hLAT1 cells were seeded at 200,000
cells/well and grown on poly-D-lysine coated 24-well plates in DMEM
medium to achieve at least 90% confluence after 48 h. As previously
described,[8] cells were treated with 1 µg/mL doxycycline and 2 mM sodium
butyrate for 24 h before the uptake assay. During uptake assays, cells
were first washed with sodium-free choline buffer (140 mM choline chloride,
2 mM potassium chloride, 1 mM magnesium chloride, 1 mM calcium
chloride, and 1 M Tris) and incubated for 10 min in the same buffer. After
that, the buffer was removed and replaced with preincubation buffer
(sodium-free choline with 6 nM of [3H]-gabapentin) and pre-incubated for
30 min. The pre-incubation buffer was removed, and cells were washed
twice with sodium-free choline buffer. The trans-stimulation buffer
containing sodium-free choline buffer and tested compounds at 200 µM
was then added. An aliquot of the trans-stimulation buffer was collected at
3 min for extracellular radioactivity measurement. The extracellular
radioactivity was determined by scintillation counting on a LS6500
Scintillation Counter (Beckman Coulter). Efflux of [3H]-gabapentin was
calculated at 3 min after adding test compound. %Efflux was normalized
relative to L-Phe, which had an efflux rate of 2.7 ± 0.3 fmol/min, from an
average of seven experiments.
1
(2S)-2-Amino-3-(3-((2-(3-
benzoylphenyl)propoxy)carbonyl)phenyl)propanoic
acid
hydrochloride (23): To a solution of boroxazolidone 20 (555 mg, 1.01
mmol) in THF (5.5 mL) was added TBAF (1M in THF, 4.0 mL, 4.0 mmol),
and the mixture was stirred overnight at rt. Consistent with previously
described deprotection conditions for amino acid-containing
boroxazolidones,[19] water (10 mL) was added and the mixture was stirred
for 5 min. Acidified the mixture to pH 3-4 using neat formic acid (1-2 mL).
Concentrated mixture in vacuo, and then diluted the resulting residue to
approximately 30 mL total volume using 1:1 CH3CN/water. Additional
formic acid (1-2 mL) was added and the mixture was sonicated to aid
dissolution. Undissolved solids were removed by a 0.45 m syringe filter
prior to purification by preparative HPLC (gradient elution of 10% CH3CN
in water ramping to 80% over 15 min). Product-containing fractions were
lyophilized. Desired product was converted to its HCl salt by suspending
the lyophilized solids in dioxane (3 mL) and adding 4M HCl in dioxane
(0.25 mL). After sufficient mixing and sonication, the suspension was
diluted with diethyl ether (10 mL) and the solids were filtered, rinsing
several times with diethyl ether. Dried solids under high vacuum overnight.
Yield: 179 mg (38%). >98% purity by LC-MS (254 nm, Method E). mp 166-
172 °C dec, [훼]2퐷4 +8.7° (c 1.1, DMSO). 1H NMR (CD3OD)  7.92 (s, 1H),
7.87 (d, J = 8Hz, 1H), 7.70-7.80 (m, 3H), 7.58-7.68 (m, 3H), 7.41-7.58 (m,
5H), 4.62 (d, J = 6 Hz, 2H), 4.27 (m, 1H), 3.36 (m, 2H), 3.21 (dd, J = 8, 15
Hz, 1H), 1.44 (d, J = 7 Hz, 3H). 13C NMR ((CD3)2SO + 5% DCl)  196.0,
170.0, 165.6, 143.8, 137.3, 137.1, 135.9, 134.6, 132.8, 131.9, 130.4, 130.0,
129.7, 129.1, 128.8, 128.7, 128.5, 128.2, 127.9, 69.3, 52.9, 38.4, 35.3,
17.7. m/z (ESI-pos) M + 1 = 432.2.
HEK-hLAT1 Cell Uptake Studies. HEK-hLAT1 cells were seeded at
300,000 cells/well and grown on poly-D-lysine coated 6-well plates in
DMEM medium to achieve at least 90% confluence after 48 h. As
previously described,[8] cells were treated with 1 µg/mL doxycycline and 2
mM sodium butyrate for 24 h before the uptake assay. During uptake
assays, cells were first washed with sodium-free choline buffer (140 mM
choline chloride, 2 mM potassium chloride, 1 mM magnesium chloride, 1
mM calcium chloride, and 1 M Tris) and incubated for 10 min in the same
buffer. After that, the buffer was removed and replaced with uptake buffer
(2.5 mL per well) (sodium-free choline with tested compounds at 50 µM).
Uptake was performed at 37 °C for 30 min, and then terminated by
washing the cells twice with ice-cold choline buffer. Acetonitrile (500 µL)
was added to each well and pipetted up and down ~ 20 times. The
acetonitrile cell extracts were transferred to 1.5 mL microcentrifuge tubes
and evaporated by blowing nitrogen. Once fully evaporated, the cell
extracts were frozen at -20 °C. Remaining cells in each well were lysed via
lysis buffer (800 µL) (0.1 N NaOH and 0.1% SDS) and allowed to sit at rt
for 3 h. An aliquot of cell lysates was collected for protein quantification via
a Bradford protein assay (Bio-Rad Laboratories, Hercules, CA).
Cell culture and characterization. TREx HEK-hLAT1 (XenoPort, Inc.,
Santa Clara, CA)[20b] is a tetracycline inducible cell line encoding both
LAT1 and 4F2hc (SLC3A2). The cell line was previously validated for
uptake of known LAT1 substrates.[8] Cells were grown and maintained in
Dulbecco's Modified Eagle Medium (DMEM) H-21 media supplemented
with 10% fetal bovine serum, 100 units/mL penicillin, 100 units/mL
streptomycin, 1 µg/mL Fungizone, 2 mM L-glutamine and 3 µg/mL
blasticidin. The incubation conditions were 37 °C and 5% CO2. The
maximum cell passage used in this study was 15. Uptake of [3H]-
gabapentin into induced HEK-hLAT1 cells (supporting information: Figure
S2) was verified prior to each cell assay.
Cell extract was diluted to 1.0 mL with either 0.1% formic acid (for Phe-d5)
or with 1:1 CH3CN/water (for compounds 22 and 23). Samples were then
passed through a syringe filter (0.45 m) and analyzed directly by single
quad LC-MS in selected ion monitoring (SIM) mode (see instrument and
column details provided in “Synthesis General” section, above) eluting with
either isocratic 0.1% formic acid for 8 min, then ramping to 80% CH3CN +
20% 0.1% formic acid over 4 min, and holding for 2 min for analysis of
Phe-d5; or eluting with isocratic 40% CH3CN + 60% 0.1% formic acid for 7
min, then ramping to 80% CH3CN + 20% 0.1% formic acid over 1 min, and
holding for 2 min for analysis of 22 and 23. Peak areas for parent ions
(M+1) were converted to amount (nmol) using standard curves generated
over a 20-fold concentration range for analyte in the presence of cell
extract. Cell uptake rate (nmol/min/mg) was calculated by dividing amount
of test compound (nmol) by cell uptake time (30 min) and normalized to
amount of protein (mg), as determined above. All cell uptake experiments
were performed in triplicate for each compound and analysis by LC-MS
was also in triplicate.
cis-Inhibition assay and IC50 determinations. HEK-hLAT1 cells were
seeded at 200,000 cells/well and grown on poly-D-lysine coated 24-well
plates in DMEM medium to achieve at least 90% confluence after 48 h. As
previously described,[8] cells were treated with 1 µg/mL doxycycline and 2
mM sodium butyrate for 24 h before the uptake assay. During uptake
assays, cells were first washed with sodium-free choline buffer (140 mM
choline chloride, 2 mM potassium chloride, 1 mM magnesium chloride, 1
mM calcium chloride, and 1 M Tris) and incubated for 10 min in the same
buffer. After that, the buffer was removed and replaced with uptake buffer
(sodium-free choline with 6 nM of [3H]-gabapentin and inhibitors at
appropriate concentrations). The primary screen was done at 200 µM. The
IC50 determination experiments were performed with concentrations from
0.1 to 500 µM. Uptake was performed at 37 °C for 3 min, and then
terminated by washing the cells twice with ice-cold choline buffer. Cells
were lysed via lysis buffer (800 µL) (0.1 N NaOH and 0.1% SDS) and
allowed to sit at rt for 3 h. Intracellular radioactivity was determined by
scintillation counting on a LS6500 Scintillation Counter (Beckman Coulter).
IC50 and standard deviation of each compound were analyzed using
GraphPad Prism version 5.0. %[3H]-Gabapentin uptake at each
concentration was normalized relative to %inhibition by BCH[23] at 2 mM,
which was set to 100% inhibition.
In Situ Rat Brain Perfusion Technique. Laboratory rats (Rattus
norvegicus) of the Long-Evans strain, ranging in age from 6-96 weeks
(average was about 72 weeks) and of both sexes, were used in all brain
perfusion experiments, similar to a previously described method.[26] Rats
were anesthetized via i.p. injections using a Ketamine:Xylazine aqueous
solution (88 mg/kg : 30 mg/kg). During the procedure, rats showed slowed,
but normal respiratory activity. A cannula with an internal diameter of 0.159
mm was custom-made from 30-gauge PTFE tubing. The cannula was pre-
filled with a heparin solution (1 mg/mL, IU≥100/mg) before it was inserted
10
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10.1002/cmdc.202000824
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FULL PAPER
into the left carotid artery and secured with surgical silk. The chest cavity
was opened, and the ventricles were severed to stop systemic circulation.
Immediately, the brain was perfused at 10.5 mL/min for 1 min 20 sec with
a standard perfusion buffer[26] at 37 °C: 128 mM NaCl, 24 mM NaHCO3,
4.2 mM KCl, 2.4 mM NaH2PO4, 1.5 mM CaCl2, 0.9 mM MgCl2. Perfusion
buffer was oxygenated using 95% O2 + 5% CO2 for 10 min prior to use. D-
Glucose was added immediately before perfusion to give a concentration
of 9 mM. Then, test compound dissolved in perfusion buffer (500 M) was
perfused for 1 min 15 sec (15 sec was the time required for compound to
reach the carotid artery via a peristaltic pump (Control Company, model
3386), as determined by observing the movement of colored solutions).
Lastly, capillaries were flushed with perfusion buffer for 1 min 20 sec. The
brain was then extracted, hemispheres were separated, and the left brain
hemisphere was frozen using liquid nitrogen and stored in a -30 °C freezer
prior to analysis.
approved by the University of Nebraska at Kearney Animal Care
and Use Committee.
Acknowledgements
This work was supported by the National Institutes of Health’s
National Institute of Neurological Disorders and Stroke grant R15
NS099981 (A.A.T), the National Institutes of Health's National
Institute of General Medical Sciences grant R01 GM117163 (H.N.
and H.C.C.) and R01 GM108911 (A.S.) and T32 GM062754
(K.H.). We appreciate OpenEye Scientific Software Inc. for
granting us access to its high-performance molecular modeling
applications through its academic license program. We are
grateful to Prof. Kathleen Giacomini (UCSF) for assistance with
manuscript editing and cell biology consultation. Additionally,
A.A.T. and E.H. thank Prof. Janet Steele (UNK Department of
Biology), Dr. Arik A. Zur (BioLineRx), and Prof. Howard
Gendelman (UNMC Department of Pharmacology and
Experimental Neuroscience) for assistance and consultation
regarding rat brain perfusion experiments.
Rat Brain Homogenization. Test compounds were extracted from brain
hemisphere (after recording its mass) using a homogenization technique
modified from the literature.[9b] Left brain hemisphere was added to a 50
mL centrifuge tube on ice. The brain hemisphere was spiked with a
deuterated isotopomer (synthesis of deuterated analogs of test
compounds are provided in supporting information) or structurally similar
analog of the test compound (Table S2, supporting information; 2.5 mL, 2
M in 20% CH3CN/H2O). In 10 sec pulses, each hemisphere was
pulverized using a Fisher 850 homogenizer (speed 16,000). Then, a 10%
aqueous trichloroacetic acid solution (2 mL) was added to precipitate
proteins. The mixture was vortexed for 2 min and placed on ice for 10 min.
The homogenate was centrifuged at 7,500g for 10 min at 7 °C using a
Beckman Avanti J-25 high speed centrifuge. The supernatant was
removed for LC-MS analysis using a Pasteur pipette.
Conflict of Interest
The authors declare no competing financial interest.
Keywords: amino acids • blood-brain barrier • drug delivery •
membrane proteins • prodrugs
Rat Brain LC-MS Analysis. The supernatant from tissue homogenization
was freeze dried to concentrate and then the residue was diluted with 1:1
CH3CN/H2O (1 mL). The mixture was then filtered through a 0.2 m syringe
filter and analyzed by single quad LC-MS in selected ion monitoring (SIM)
mode (instrument and column details provided in “Synthesis General”
section, above; masses of analyte ions are summarized in Table S2,
supporting information). Brain uptake (nmol/g/min) was calculated by
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Abbreviations Used
LAT1, L-type amino acid transporter 1; BCH, 2-
aminobicyclo[2.2.1]heptane-2-carboxylic acid; DIC, N, N’-
diisopropylcarbodiimide; P(o-tolyl)3, tri(o-tolyl)phosphine; Pd2dba3,
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tris(dibenzylideneacetone)dipalladium(0);
9-BBN,
9-
borabicyclo[3.3.1]nonane; DMEM, Dulbecco's Modified Eagle
Medium; HEK-hLAT1, human embryonic kidney cell line inducible
for human L-type amino acid transporter 1; MCF-7, Michigan
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microscopy; ABC, ATP-binding cassette.
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All animals were maintained following the Guide for the Care and
Use of Laboratory Animals. All procedures were conducted in
accordance with Animal Care Protocol #190226, which was
11
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Entry for the Table of Contents
Larger amino acids were inhibitors of the L-type amino acid
transporter 1 (LAT1) and had poor rat brain uptake. Small- to
medium-sized esters of meta-carboxyl L-phenylalanine were
substrates. However, larger ester substituents caused a
decrease in LAT1 transport rate and an increase in potency of
inhibition (23, IC50 = 0.31 ± 0.1 M).
Institute and/or researcher Twitter usernames:
@Alzchemist1970
@SchlessingerLab
@cas_unk
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