A. A. Zur et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
O
R
NH
-HCl
3e: Leucinamide
2
NH
2
c
1
R
e: (CH
f: (S)-CH
g: CH
2 2
S(CH ) -
3
)
2
CHCH
2
-
O
O
d (and e for
O
3
CH (CH )CH-
2
3
R
a
R
12g only)
R
3
OH
OMe
NHOH
2
-HCl
NH
2
NH
2
-HCl
NH
12e:
1
e-g (Leu, Ile, Met)
9e-f
HA-Leu
12f: HA-Ile
2g: HA-Met
9g
b
1
Boc-9g
Scheme 1. Synthesis of compounds 13e and 12e–12g. Reagents and conditions: (a) SOCl
MeOH, 50 °C, sealed tube, 13e: 60%; (d) 50% NH
2
, MeOH, 9e: 56%, 9f: 86%, 9g: 67%; (b) Boc
OH in water, MeOH or 1,4-dioxane, 12e: 23%, 12f: 3%, 12g: 20% (2 steps); (e) 4 N HCl in 1,4-dioxane. 13e and 12e–g were
2
O, DCM, Boc-9g: 85%; (c) 7 N NH
3
in
2
purified by conversion to their HCl salts and recrystallization to >99% purity by HPLC.
better (60% recrystallized yield) than for the corresponding HA
analogs. Generally HA’s demonstrated poor solubility in both water
and organic solvents; however, we found that solubility was dra-
matically improved by conversion to the hydrochloride salt.
Of these three bioisosteres, only the hydroxamic acid 12a had
significant activity in our trans-stimulation assay relative to non-
substrates Gly and Arg (Table 1). We were surprised by this result.
We had expected the tetrazole 11 and acylsulfonamides 10a to
have been better surrogates for the acidic carboxylic acid than
to be a LAT1 substrate,32 to our knowledge this was the first time
that 13e or any amino acid primary amide has been tested for
LAT1 activity. Interestingly, 13e did not demonstrate significant
substrate activity (efflux rate: 0.69 fmol/min). This result also indi-
cated that 13e was sufficiently stable to the assay conditions so as
not to generate adequate parent Leu 1e to cause trans-stimulation.
Furthermore, it is apparent that the hydroxamic acid ‘–OH’ group
plays an important role in the observed LAT1 activity; whether that
be due to its effect on acidity, hydrogen bonding, or some other
factor is currently unclear.
1
(
2a given that the measured pKa1 values61 for the former
pKa1 = 2.5 and 1.8, respectively) were much closer to that of par-
A different story unfolded for ester 9e. In a trans-stimulation
3
2
ent amino acid Phe 1a (pKa1 = 1.8) than HA-Phe 12a (pKa1 = 6.9)
was. It is worth noting that our pKa1 values were considerably
lower than those reported for these bioisosteres when they were
present as isolated functional groups,34 which demonstrates as
experiment performed by Nagamori, both ester 9e and its parent
Leu appeared to have almost identical activity. In our hands, 9e
exhibited significantly less activity than parent Leu in both trans-
stimulation (efflux rate: 2.1 vs 3.2 fmol/min) and cis-inhibition
might be expected that the
a-amino group depresses their pK
a
as
assays (IC50: >200 lM vs 87 lM). One possible explanation for this
it would for an adjacent carboxylic acid.
disparity may be due to the fact that the cells used by Nagamori were
different from the cells we used. Nagamori et al. used non-trans-
fected HeLa S3 cells (a cervical cancer cell line) whereas, we used
To determine whether HA’s of other LAT1 amino acid substrates
e.g., Leu, Ile, Met) were LAT1 ligands, compounds of Table 2 were
(
5
2
prepared and tested. All of the HA’s, with the exception of 12h, had
diminished activity in both our trans-stimulation and cis-inhibition
assays relative to the parent amino acids. Based on their % inhibi-
HEK-hLAT1 cells that demonstrated 8-fold higher uptake of
3
[ H]-gabapentin relative to the control cell line HEK-EV (Supple-
mentary material). LAT1 expression may have been higher in our
transfected cells and the contribution of other transporters could
differ between the two cell lines. We selected HEK cells due to their
3
tion of [ H]-gabapentin cell uptake or IC50 values, it is clear that
HA’s are weaker ligands of LAT1 than the parent amino acids.
And none of the HA’s had IC50 values below 200
6
5
l
M in our assay.
having relatively low levels of transporters, so we would expect
the observed activity in our assays to be due solely to LAT1. Our
IC50 value for 9e was consistent with earlier SAR presented by
Conversely, all of the HA’s demonstrated greater efflux rates of
3
[
H]-gabapentin from pre-loaded HEK-hLAT1 cells than did the
4
negative controls Arg and Gly. The notable exception to this trend
was HA-Gly 12h, which we did not expect to be a LAT1 substrate
by analogy to its non-substrate, parent amino acid Gly. Of the
HA’s tested, 12a and 12e–g (HA’s of Phe, Leu, Ile, Met) demon-
strated significant activity relative to the negative controls. The lar-
ger efflux rates measured for HA-Leu 12e and HA-Ile 12f relative to
HA-Phe 12a (1.5 vs 1.3 fmol/min) were juxtaposed with the activ-
ity of the parent amino acids, in which Phe 1a demonstrated a
superior efflux rate (3.6 fmol/min). It has previously been shown
Uchino that the methyl ester of phenylalanine poorly inhibited
1
4
uptake of L-[ C]-Phe into oocytes expressing LAT1. But the ostensi-
ble interpretation of the results from our trans-stimulation assay is
the same as Nagamori’s—that methyl ester 9e does appear to be a
LAT1 substrate, despite lacking an acidic carboxylic acid functional
group.
Because of our concerns and those raised by others about the
potential for a false positive result in LAT1 cell assays,37 we evalu-
ated how much parent Leu 1e would need to be present as an impu-
rity in test compounds (i.e., 9e, 12e, or 13e), either from the
synthesis or formed under the conditions of the cell assay, to result
in a significant efflux rate (>1 fmol/min) in our trans-stimulation
assay. We tested the efflux rate at concentrations ranging from
that both Leu and Ile have slightly greater LAT1 transport capacity
3,64
(V
max) values than Phe.6
Since the trans-stimulation assay relies
3
upon the kinetics of exchange between intracellular [ H]-gabapen-
tin and extracellular test compound, it is conceivable that a similar
trend for Vmax applies to the HA’s as it does to the parent amino
acids. However, considering the bounce in our assay relative to
the observed efflux rates, we cannot confidently distinguish the
4 lM up to 200 lM covering a range of Leu 1e impurity from 2%
to 100%, respectively (in relation to previous studies). The back-
ground efflux rate (0.7 ± 0.05 fmol/min) was subtracted from total
3
substrate activity of the HA’s from each other.
efflux and the net contribution to [ H]-gabapentin efflux rate is
Given the disparity in the literature4
,32,37
as to whether esters
depicted in Figure 1. The effect of increasing concentrations of Leu
are LAT1 substrates, we also tested the Leu methyl ester 9e, which
was an intermediate in the preparation of HA-Leu 12e (Scheme 1).
And to further probe the SAR for close-in derivatives of the HA’s
1e on the net LAT1 efflux rate was fitted to the Michaelis–Menten
equation (K
The K and Vmax were similar to previously reported values.
A Leu 1e concentration of 4 M did not increase the LAT1
exchange rate significantly in comparison to background signal
m
of 36.8 ± 9.8 lM and Vmax of 1.99 ± 0.19 fmol/min).
64
m
(Table 2), we also decided to test the structurally-related primary
l
amide Leucinamide 13e. Though ester 9e was recently reported