L-Type amino acid transporter 1 activity of 1,2,3-triazolyl analogs of L-histidine and L-tryptophan

Article abstract of DOI:10.1016/j.bmcl.2019.06.033

A series of 1,2,3-triazole analogs of the amino acids L-histidine and L-tryptophan were modeled, synthesized and tested for L-type amino acid transporter 1 (LAT1; SLC7A5) activity to guide the design of amino acid-drug conjugates (prodrugs). These triazol

Full text of DOI:10.1016/j.bmcl.2019.06.033

Accepted Manuscript
L-type amino acid transporter 1 activity of 1,2,3-triazolyl analogs of L-histidine
and L-tryptophan
Colton Hall, Hannah Wolfe, Alyssa Wells, Huan-Chieh Chien, Claire Colas,
Avner Schlessinger, Kathleen M. Giacomini, Allen A. Thomas
PII:
S0960-894X(19)30414-7
https://doi.org/10.1016/j.bmcl.2019.06.033
BMCL 26511
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 April 2019
12 June 2019
19 June 2019
Please cite this article as: Hall, C., Wolfe, H., Wells, A., Chien, H-C., Colas, C., Schlessinger, A., Giacomini, K.M.,
Thomas, A.A., L-type amino acid transporter 1 activity of 1,2,3-triazolyl analogs of L-histidine and L-tryptophan,
Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.06.033
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

L-type amino acid transporter 1 activity of 1,2,3-triazolyl
analogs of L-histidine and L-tryptophan
Colton Hall,a Hannah Wolfe,a Alyssa Wells,a Huan-Chieh Chien,b Claire Colas,c§ Avner
Schlessinger,c Kathleen M. Giacomini,b Allen A. Thomasa*
aDepartment of Chemistry, University of Nebraska at Kearney, Kearney, NE, 69949, USA
bDepartment of Bioengineering and Therapeutic Sciences, University of California, San
Francisco, San Francisco, CA, 94158, USA
cDepartment of Pharmacological Sciences, Icahn School of Medicine at Mt. Sinai, New York,
NY, 10029, USA
*Corresponding author at: Department of Chemistry, University of Nebraska at Kearney,
Kearney, NE, 69949, USA. E-mail address: thomasaa@unk.edu (A. Thomas)
Keywords: membrane transporter; solute carrier family; triazole; click chemistry; amino acid;
cancer; blood-brain barrier
Abstract
A series of 1,2,3-triazole analogs of the amino acids L-histidine and L-tryptophan were modeled,
synthesized and tested for L-type amino acid transporter 1 (LAT1; SLC7A5) activity to guide the
design of amino acid-drug conjugates (prodrugs). These triazoles were conveniently prepared by
the highly convergent Huisgen 1,3-dipolar cycloaddition (Click Chemistry). Despite comparable
predicted binding modes, triazoles generally demonstrated reduced cell uptake and LAT1
binding potency relative to their natural amino acid counterparts. The structure-activity
relationship (SAR) data for these triazoles has important ramifications for treating cancer and
brain disorders using amino acid prodrugs or LAT1 inhibitors.

Conjugation of drugs with amino acids (“amino acid prodrugs”) has become an
increasingly common strategy for targeted drug delivery across the blood-brain barrier (BBB)
and into cancer cells by way of the L-type amino acid transporter 1 (LAT1; SLC7A5). LAT1 is a
sodium-independent membrane solute carrier protein1-3 found in several tissues throughout the
body, with high levels of expression in brain,4, 5 spleen, testis, placenta, thymus, skeletal muscle
and various cancer cells.6-10 LAT1 transports lipophilic amino acids such as phenylalanine,
leucine, isoleucine, methionine, tryptophan as well as thyroid hormones including thyroxine.
Additionally, LAT1 transports the hydrophilic amino acid histidine. We and others have shown
that LAT1 can also transport aryl-substituted phenylalanine analogs containing various
functional groups,2, 11-13 as well as amino acid bioisosteres such as hydroxamic acids and esters.14
Given these findings that LAT1 is relatively tolerant of structural changes to the amino acid
promoiety, we sought an amino acid scaffold that would be amenable to the rapid assembly of
prodrugs while maintaining substrate activity.
O
O
Drug
N
OH
N
X
OPG
NHPG
Drug
Drug
X
N
NH2
N
N
N
N-linked regioisomer
N
O
O
N
N
N
X
OH
OPG
NHPG
N
X
NH2
N
Drug
C-linked regioisomer
PG = protecting group, X = biodegradable linker
Figure 1. Click Chemistry design strategy for N- and C-linked -1,2,3-triazolyl amino acid
prodrugs.

The Huisgen 1,3-dipolar cycloaddition, also known as “Click Chemistry”,15-20 provides a
synthetic strategy for connecting an amino acid moiety to a drug molecule via a 1,2,3-triazole
ring. Specifically, we envisioned using Click Chemistry to attach an alkyne- or azide-substituted
amino acid to a drug molecule possessing a biodegradable functional group that is linked to an
azide or alkyne moiety (Figure 1). These 1,2,3-triazoles (hereafter referred to as “triazoles”) are
structurally similar to the amino acids L-histidine and L-tryptophan. Thus, we hypothesized that
triazoles would be LAT1 substrates based on the similarity of their predicted binding modes to
these endogenous ligands (supplementary data). Moreover, L-histidine is one of the most potent
LAT1 substrates,12, 21 so we thought triazole analogs would also have good potency. Click
Chemistry offers the additional advantage of obtaining a single triazole regioisomer. Prior to
using this strategy for designing amino acid prodrugs, we wanted to test its feasibility by
determining the structure-activity relationship (SAR) for a series of triazole structural analogs
that we have designated as N-linked or C-linked based on which triazole atom is connected to the
amino acid beta carbon (Figure 1). We varied both the size and polarity of the triazole substituent
R to compare with the SAR of structurally-related histidine analogs and tryptophan. Compounds
were evaluated in trans-stimulation and cis-inhibition cell assays (as described below).
O
O
O
S
N
NH
Cl
O
O
O
O
O
Ph
S
O
O
O
HO
HO
OH
NHBoc
NHBoc
pyridine
Ph
NHBoc
1
2
3
O
NaN3
Bu4NBr
N
O
DMF
NHBoc
N
60% yield
N
4

Scheme 1. Synthesis of azide intermediate 4.
O
O
Cu(OAc)2
Na ascorbate
N
O
N
N
O
R
NHBoc
R
1:1 t-BuOH/H2O
N
NHBoc
N
N
35-100% yields
5a-e
4
6a-e
R group
O
TFA
Et3SiH
5a 6a
7a
)
TMS (
Ph
,
), H (
a
b
N
N
OH
R
DCM
(and TBAF for
NH2
7a-e
N
CH2OH
Me
7a
c
d
e
)
14-57% yields
Bn
Scheme 2. Synthesis of N-linked triazoles 7a-e.
O
O
Condition A
N
N
N
OH
OH
A
Cu(OAc)2
N
Bn
N
NH2
Na ascorbate
NH2
O
N
32% yield
1:1 t-BuOH/H2O
Bn
8e
11e
O
N
Condition B
N
N
O
O
B
Me
N
N
CuI, Et3N
CH3OH
NHBoc
NHBoc
N
60% yield
8d
Me
9
10d
O
TFA
Et3SiH
N
OH
N
NH2
11d
N
DCM
Me
33% yield
Scheme 3. Synthesis of C-linked triazoles 11d-e.

TMS
OTf
O
O
O
TFA
Et3SiH
CsF
N
O
N
N
O
N
N
OH
NHBoc
NHBoc
DCM
N
NH2
N
CH3CN
N
N
39% yield
4
12
13
72% yield
Scheme 4. Synthesis of benzotriazole 13.
Scheme 1 shows the synthesis of azide intermediate 4 used to prepare N-linked triazoles.
Starting with commercially available Boc-protected serine 1, we prepared tert-butyl ester 2 using
tert-butyl (Z)-N,N'-diisopropylcarbamimidate.22 We were unable to prepare a tosylate of alcohol
2 by reaction with tosyl chloride presumably due to steric crowding of the Boc and tert-butyl
ester groups. Instead we prepared benzyl sulfonate 3 as previously described.23 Subsequent
nucleophilic substitution with sodium azide using tetrabutylammonium bromide as a phase-
transfer agent provided the desired azide 423 in moderate yield (60%).
Using methodology similar to what was previously published for preparing triazoles 7a-
c,24 cycloaddition of azide 4 with various substituted alkynes (5a-e) was catalyzed by copper(I)
ion generated in situ from sodium ascorbate reduction of copper(II) acetate to give protected
triazole analogs 6a-e in 35-100% yields (Scheme 2). Both the tert-butyl ester and Boc protecting
groups were conveniently removed in one step using trifluoroacetic acid (TFA) in the presence
of carbocation scavenger triethylsilane (Et3SiH). Additionally, Et3SiH has been shown to
increase the rate of Boc deprotection.25 These deprotection conditions were also used in the
synthesis of C-linked triazole 11d (Scheme 3) and benzotriazole 13 (Scheme 4).

Scheme 3 illustrates the synthesis of the two C-linked triazoles 11d and 11e. Benzyl
triazole 11e was prepared from benzyl azide 8e and unprotected, commercially available (S)-2-
aminopent-4-ynoic acid using Click Chemistry conditions “A” as had been applied in the
synthesis of N-linked triazoles of Scheme 2. As 11e was chronologically the first triazole analog
we synthesized in this project, we had hoped to avoid using protecting groups. However, we
found that separation of the amino acid 11e from the other reactants (i.e. diacetoxycopper and
sodium ascorbate) proved challenging; thus, we modified our synthesis to use protected amino
acids in the Click Chemistry reactions for all other analogs of Schemes 2-4. Hence, alkyne 926
was prepared by Boc and tert-butyl ester protection of (S)-2-aminopent-4-ynoic acid. For
unknown reasons, we found that methyl azide 8d (prepared in situ from sodium azide and
iodomethane) did not react with alkyne 9 using the same conditions as for N-linked triazoles of
Scheme 2. Alternative conditions27 “B” employing CuI and triethylamine (Et3N) were found to
give desired 10d in 60% yield. Methyl triazole 11d was obtained following deprotection of 10d
using TFA and Et3SiH.
Benzotriazole 12 was prepared by a [3+2] cycloaddition of azide 4 and benzyne
generated in situ by a cesium fluoride mediated elimination of 2-(trimethylsilyl)phenyl triflate
(Scheme 4; 72% yield).28 Desired amino acid 13 was obtained by deprotection of 12 with TFA
and Et3SiH, as performed for triazoles of Scheme 2.
Triazoles of Schemes 2-4 were tested for LAT1 activity as previously described (24-well
poly-D plate configuration)12, 29 using human embryonic kidney cells containing inducible LAT1
(HEK-hLAT1)30 in two different assays: cis-inhibition and trans-stimulation, to determine ligand
potency and relative transport rates, respectively. In the cis-inhibition assay, test compounds
were incubated with cells in the presence of a known substrate, [3H]-gabapentin (6 nM).

Compounds were screened at 200 M to determine %inhibition of [3H]-gabapentin uptake
relative to a LAT1 inhibitor BCH (2 mM),31 set to 100% inhibition. Select compounds were then
tested at various concentrations to determine the half maximal inhibitory concentration (IC50).
cis-Inhibition indicates ligand potency, however, it does not distinguish between LAT1 inhibitors
and substrates. To identify whether a compound was a substrate, we performed a trans-
stimulation assay. This experiment is possible due to the alternating access mechanism of
LAT1,32 in which an exchange occurs between intra- and extra-cellular amino acids in a 1:1 mole
ratio. The assay is performed by loading cells with [3H]-gabapentin prior to exposure to test
compounds. The efflux rate of [3H]-gabapentin (fmol/min) was calculated at 3 minutes after
adding test compound. % Efflux was normalized relative to L-phenylalanine 18, which had an
efflux rate of 2.7 ± 0.3 fmol/min, from an average of seven experiments.
Positive controls included natural amino acid substrates L-histidine 15, L-phenylalanine
18 and L-tryptophan 22. Non-substrate L-arginine 14 was used as a negative control, which had a
relative efflux rate of 28% in our trans-stimulation assay. Non-substrates generally show
20−35% relative efflux due to background levels of exported [3H]-gabapentin. Low levels of
endogenous amino acids present in the cell assay may cause efflux via LAT1 or an alternate
transporter.
Unfortunately, triazole analogs of Table 1 (7a-e, 11d-e) were considerably less potent
(lower %inhibition and greater IC50 values) in our cis-inhibition assay than related histidine
analogs (15-17) of Table 2. Unsubstituted N-linked triazole 7a (R = H) showed the highest
relative efflux rate and greatest potency of the triazole series. However, both the potency (160
M vs. 110 M) and relative %efflux (82% vs. 140%) for 7a were inferior to L-histidine 15. In
contrast to histidine analogs 16-17, substitution in the triazole series was less well tolerated. Only

a methyl substituent in compound 7d displayed comparable %efflux to its related histidine
analog 16, but showed a 2-fold decrease in potency (440 M vs. 190 M). Moreover, benzyl
substitution in triazoles 7e and 11e resulted in loss of potency relative to analog 17. Though one
could interpret the negative %inhibition for 7e and 11e as due to LAT1 activation for [3H]-
gabapentin uptake, we think it is possible that a change in plasma membrane integrity could
cause an uptake signal increase compared with control in the absence of any test compounds.
Nonetheless, these negative %inhibition values do not change the interpretation of our data, that
triazoles 7e and 11e have poor potency. However, triazoles of Table 1 (7a-e, 11d-e)
demonstrated relative %efflux greater than our negative control L-arginine 14 (37-82% vs. 28%),
suggesting that they are being transported into cells by LAT1.
It should be noted that we were unable to prepare the C-linked analog of triazole 7a by
reaction of TMS-azide with alkyne 9 using either conditions A or B of Scheme 3. An alternate,
multi-step synthesis (not involving Click Chemistry) of this unsubstituted C-linked triazole in
racemic form has previously been described;33 however, given the lack of potency for C-linked
triazoles 11d and 11e, we decided not to pursue this compound.
Relative to triazoles of Table 1, benzotriazole 13 (Table 2) demonstrated improved
%efflux and potency. Additionally, it appeared to be a better substrate in terms of %efflux
compared with the natural substrate tryptophan 22 (100% vs. 59%), albeit with a 2-fold decrease
in potency. Thus, all of the triazole analogs of Tables 1 and 2 had diminished potency relative to
their corresponding histidine and tryptophan analogs.
Based on our models, both triazoles and the corresponding histidine analogs dock within
a model of the LAT1 binding site in a comparable manner (supplementary data). Therefore, we
cannot explain the difference in activity based on their predicted binding modes. Furthermore,

comparing cLogP values for triazoles with their histidine congeners did not provide any
additional insight into why triazoles have decreased potency. For example, N-linked triazoles
(7a-7e) exhibit an almost order of magnitude higher cLogP34 than their histidine counterparts
(e.g. cLogP: 7a = -2.9 vs. 15 = -3.7). Whereas, C-linked triazoles, which were even less potent,
had more negative cLogP values (e.g. 11d = -4.2 vs. 16 = -3.7). Moreover, pyridyl analogs 19-
21, which we previously reported as potent LAT1 substrates,12 had a comparable cLogP to
triazole 7a (-3.1 vs. -2.9, respectively). The only apparent difference between these triazoles and
their related histidine and pyridyl analogs is heterocycle basicity. A 1,2,3-triazole ring is non-
basic with a reported pKa of 1.15 for its conjugate acid.35 However, our models, representing
snapshots with the highly dynamic transport cycle do not indicate the presence of acidic side
chains in the vicinity of the ligand binding site. Thus, we cannot determine the potential
functional role of a basic heterocycle (i.e. imidazole and pyridine). Furthermore, imidazole and
pyridine rings would be primarily neutral under the conditions of our cell assay (pH 7.4), even
though their protonation states inside the binding site is unknown. Given the fact that most
natural LAT1 substrates do not contain a basic heterocycle (e.g. phenylalanine 18, tryptophan 22,
leucine, methionine, tyrosine, etc.), it is unclear to us why triazoles have decreased potency
compared with structurally-related histidine and pyridyl analogs.

Table1. LAT1 trans-stimulation and cis-inhibition cell assay activity for N- and C-linked
triazoles 7a-e and 11d-e.
O
O
N
OH
OH
R
R
N
N
N
N
NH2
NH2
N
N-linked
C-linked
Relative
%
Compounda
R
H
Regioisomer
IC50 (μM)d
160 ± 27
>1000
%Effluxb
Inhibitionc
7a
7b
N
N
N
N
C
N
C
82
58
Ph
37
57
70
63
49
43
13
14
38
20
-6
7c
CH2OH
Me
>1000
7d
440 ± 190
>1000
11d
7e
Me
Bn
NDe
11e
Bn
-19
NDe
aCell assay data was obtained at least in triplicate (24-well poly-D plate configuration) in each
condition. All compounds above are single enantiomers of L configuration. bCompounds 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 (18), which had an efflux rate of 2.7 ± 0.3
fmol/min, from an average of seven experiments. cCompounds were tested at 200 M for their
ability to inhibit uptake of [3H]-gabapentin into HEK-hLAT1 cells. Data are presented as %
inhibition relative to background signal in the absence of a test compound. dFor IC50
determinations, varying concentrations of each compound were added, from 0.1 µM to 500 M.
IC50 and standard deviation of each compound was calculated by GraphPad Prism version 5.0.
%[3H]-Gabapentin uptake at each concentration was normalized relative to %inhibition by
BCH31 at 2 mM, which was set to 100% inhibition. eNot determined.

Table 2. LAT1 trans-stimulation and cis-inhibition cell assay activity for some naturally
occurring amino acids and structural analogs including benzotriazole 13.a
Relative
%Efflux Inhibition
%
IC50
(μM)
Compound
R
14
(L-Arg)
NH2(NH)CNH(CH2)2-
28
140
85
49
92
70
74
85
84
98
99
-
15
(L-His)
110 ± 93
190 ± 25
170 ± 53
69 ± 29
160 ± 56
69 ± 17
100 ± 53
16
17
81
18
(L-Phe)
100
140
130
89
19
20
21
22
(L-Trp)
59
79
160 ± 87

13
100
59
340 ± 104
aCell assay conditions were as described for Table 1.
A series of substituted 1,2,3-triazolyl amino acids were synthesized and tested for LAT1
substrate activity. The synthesis of these triazole analogs is convenient, potentially serving as a
template for conjugation of drug molecules with amino acids (i.e. hypothetical prodrugs of
Figure 1) via the highly convergent Huisgen 1,3-dipolar cycloaddition. However, only a
benzotriazole analog 13 exhibited a better uptake rate than the corresponding natural amino acid
analog L-tryptophan 22, and all of the triazoles of the current work were less potent than their
corresponding histidine and tryptophan equivalents. Despite having poor potency for inhibiting
LAT1 uptake of [3H]-gabapentin, triazoles may be weak binding substrates, as evidenced by
their activity in a trans-stimulation assay relative to non-substrate L-arginine 14. Though we
have no evidence to explain why a basic heterocycle (e.g. imidazole and pyridine) provides
greater potency than these non-basic triazoles, our results suggest that the binding site is
sensitive to relatively subtle changes in ligand properties. Moreover, it was previously noted that
LAT1 is selective for aromatic amino acids relative to aliphatic ones, and this preference does
not correlate well with lipophilicity.36, 37 Our data expand upon that observation by showing that
aromaticity does not guarantee good ligand binding. Thus, for reasons that are not obvious to us,
amino acids containing a 1,2,3-triazole ring appear to be inferior LAT1 substrates compared with
histidine and tryptophan structural analogs. These findings could influence the design of amino
acid containing prodrugs as well as LAT1 inhibitors for treating brain disorders and cancer.

Conflicts of interest
The authors confirm no conflict of interest.
Acknowledgements
This work was supported by the National Institutes of Health’s National Institute of Neurological
Disorders and Stroke grant R15 NS099981 (to A.A.T), the National Institutes of Health's
National Institute of General Medical Sciences grant R01 GM117163 (to H.C.C. and K.M.G.)
and R01 GM108911 (to A.S. and C.C.). We appreciate OpenEye Scientific Software Inc. for
granting us access to its high-performance molecular modeling applications through its academic
license program.
Appendix A. Supplementary data
Full experimental details, compound characterization data, including 1H and 13C NMR spectra
for newly synthesized compounds 7d and 11d, and ligand docking description along with models
of compounds 7e, 11e and 17 docked in the LAT1 active site can be found in the online version.
Current Author Addresses
§(C.C.) Pharmacoinformatics Research Group, Department of Pharmaceutical Chemistry,
University of Vienna, Althanstraße 14 (UZA II), 1090 Vienna, Austria.
References and notes
1. Kanai, Y.; Segawa, H.; Miyamoto, K.; Uchino, H.; Takeda, E.; Endou, H. J. Biol. Chem. 1998,
273, 23629.

2. Uchino, H.; Kanai, Y.; Kim, D. K.; Wempe, M. F.; Chairoungdua, A.; Morimoto, E.; Anders,
M. W.; Endou, H. Mol. Pharmacol. 2002, 61, 729.
3. del Amo, E. M.; Urtti, A.; Yliperttula, M. Eur. J. Pharm. Sci. 2008, 35, 161.
4. Boado, R. J.; Li, J. Y.; Nagaya, M.; Zhang, C.; Pardridge, W. M. Proc. Natl. Acad. Sci. USA
1999, 96, 12079.
5. Kido, Y.; Tamai, I.; Uchino, H.; Suzuki, F.; Sai, Y.; Tsuji, A. J. Pharm. Pharmacol. 2001, 53,
497.
6. Yanagisawa, N.; Satoh, T.; Hana, K.; Ichinoe, M.; Nakada, N.; Endou, H.; Okayasu, I.;
Murakumo, Y. Cancer Biomark. 2015, 15, 365.
7. Kobayashi, H.; Ishii, Y.; Takayama, T. J. Surg. Oncol. 2005, 90, 233.
8. Ebara, T.; Kaira, K.; Saito, J.; Shioya, M.; Asao, T.; Takahashi, T.; Sakurai, H.; Kanai, Y.;
Kuwano, H.; Nakano, T. Anticancer Res. 2010, 30, 4223.
9. Ichinoe, M.; Mikami, T.; Yoshida, T.; Igawa, I.; Tsuruta, T.; Nakada, N.; Anzai, N.; Suzuki,
Y.; Endou, H.; Okayasu, I. Pathol. Int. 2011, 61, 281.
10. Takeuchi, K.; Ogata, S.; Nakanishi, K.; Ozeki, Y.; Hiroi, S.; Tominaga, S.; Aida, S.; Matsuo,
H.; Sakata, T.; Kawai, T. Lung Cancer 2010, 68, 58.
11. Ylikangas, H.; Malmioja, K.; Peura, L.; Gynther, M.; Nwachukwu, E. O.; Leppanen, J.;
Laine, K.; Rautio, J.; Lahtela-Kakkonen, M.; Huttunen, K. M.; Poso, A. ChemMedChem 2014, 9,
2699.
12. Chien, H. C.; Colas, C.; Finke, K.; Springer, S.; Stoner, L.; Zur, A. A.; Venteicher, B.;
Campbell, J.; Hall, C.; Flint, A.; Augustyn, E.; Hernandez, C.; Heeren, N.; Hansen, L.; Anthony,
A.; Bauer, J.; Fotiadis, D.; Schlessinger, A.; Giacomini, K. M.; Thomas, A. A. J. Med. Chem.
2018, 61, 7358.

13. Augustyn, E.; Finke, K.; Zur, A. A.; Hansen, L.; Heeren, N.; Chien, H.-C.; Lin, L.;
Giacomini, K. M.; Colas, C.; Schlessinger, A.; Thomas, A. A. Bioorg. Med. Chem. Lett. 2016,
26, 2616.
14. Zur, A. A.; Chien, H.-C.; Augustyn, E.; Flint, A.; Heeren, N.; Finke, K.; Hernandez, C.;
Hansen, L.; Miller, S.; Lin, L.; Giacomini, K. M.; Colas, C.; Schlessinger, A.; Thomas, A. A.
Bioorg. Med. Chem. Lett. 2016, 26, 5000.
15. Zhao, Z.; Yao, Z.; Xu, X. Curr. Org. Chem. 2017, 21, 2240.
16. Singh, M. S.; Chowdhury, S.; Koley, S. Tetrahedron 2016, 72, 5257.
17. Wang, C.; Ikhlef, D.; Kahlal, S.; Saillard, J.-Y.; Astruc, D. Coord. Chem. Rev. 2016, 316, 1.
18. Schoffelen, S.; Meldal, M. Alkyne-azide reactions, 1st ed.; Wiley-VCH Verlag GmbH & Co.
KGaA: Weinheim, Germany, 2014.
19. Heravi, M. M.; Tamimi, M.; Yahyavi, H.; Hosseinnejad, T. Curr. Org. Chem. 2016, 20,
1591.
20. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. Int. Ed. Engl. 2001, 40, 2004.
21. Yanagida, O.; Kanai, Y.; Chairoungdua, A.; Kim, D. K.; Segawa, H.; Nii, T.; Cha, S. H.;
Matsuo, H.; Fukushima, J.; Fukasawa, Y.; Tani, Y.; Taketani, Y.; Uchino, H.; Kim, J. Y.;
Inatomi, J.; Okayasu, I.; Miyamoto, K.; Takeda, E.; Goya, T.; Endou, H. Biochim. Biophys. Acta
2001, 1514, 291.
22. West, K. R.; Bake, K. D.; Otto, S. Org. Lett. 2005, 7, 2615.
23. Zou, Y.; Fahmi, N. E.; Vialas, C.; Miller, G. M.; Hecht, S. M. J. Am. Chem. Soc. 2002, 124,
9476.
24. Gajewski, M.; Seaver, B.; Esslinger, C. S. Bioorg. Med. Chem. Lett. 2007, 17, 4163.
25. Mehta, A.; Jaouhari, R.; Benson, T. J.; Douglas, K. T. Tetrahedron Lett. 1992, 33, 5441.

26. McConathy, J.; Zhou, D.; Shockley, S. E.; Jones, L. A.; Griffin, E. A.; Lee, H.; Adams, S. J.;
Mach, R. H. Mol. Imaging 2010, 9, 329.
27. Kaur, J.; Bhardwaj, A.; Sharma, S. K.; Wuest, F. Bioorg. Med. Chem. 2013, 21, 4288.
28. Shi, F.; Waldo, J. P.; Chen, Y.; Larock, R. C. Org. Lett. 2008, 10, 2409.
29. Geier, E. G.; Schlessinger, A.; Fan, H.; Gable, J. E.; Irwin, J. J.; Sali, A.; Giacomini, K. M.
Proc. Natl. Acad. Sci. USA 2013, 110, 5480.
30. Zerangue, N. U.S. Patent 7,462,459 B2, 2008.
31. Segawa, H.; Fukasawa, Y.; Miyamoto, K.; Takeda, E.; Endou, H.; Kanai, Y. J. Biol. Chem.
1999, 274, 19745.
32. Fotiadis, D.; Kanai, Y.; Palacin, M. Mol. Aspects Med. 2013, 34, 139.
33. Ikeda, Y.; Kawahara, S.-i.; Taki, M.; Kuno, A.; Hasegawa, T.; Taira, K. Protein Eng. 2003,
16, 699.
34. cLogP was calculated using ChemDraw v.17.1.
35. Catalan, J.; Abboud, J. L. M.; Elguero, J. Adv. Heterocycl. Chem. 1987, 41, 187.
36. Ylikangas, H.; Peura, L.; Malmioja, K.; Leppanen, J.; Laine, K.; Poso, A.; Lahtela-
Kakkonen, M.; Rautio, J. Eur. J. Pharm. Sci. 2013, 48, 523.
37. Puris, E.; Gynther, M.; Huttunen, J.; Petsalo, A.; Huttunen, K. M. J. Controlled Release
2017, 261, 93.

Graphical Abstract
O
O
LAT1 substrates
N
OH
OH
R
R
N
O
N
O
N
NH2
NH2
O
N
N
OH
OH
HN
NH2
N
NH2
OH
N
N
HN
NH2
N
L-histidine
L-tryptophan
1,2,3-triazolyl analogs