Discovery of indoximod prodrugs and characterization of clinical candidate NLG802

Article abstract of DOI:10.1016/j.ejmech.2020.112373

A series of different prodrugs of indoximod, including estesrs and peptide amides were synthesized with the aim of improving its oral bioavailability in humans. The pharmacokinetics of prodrugs that were stable in buffers, plasma and simulated gastric and intestinal fluids was first assessed in rats after oral dosing in solution or in capsule formulation. Two prodrugs that produced the highest exposure to indoximod in rats were further tested in Cynomolgus monkeys, a species in which indoximod has oral bioavailability of 6–10percent and an equivalent dose-dependent exposure profile as humans. NLG802 was selected as the clinical development candidate after increasing oral bioavailability (>5-fold), C_max (6.1–3.6 fold) and AUC (2.9–5.2 fold) in monkeys, compared to equivalent molar oral doses of indoximod. NLG802 is extensively absorbed and rapidly metabolized to indoximod in all species tested and shows a safe toxicological profile at the anticipated therapeutic doses. NLG802 markedly enhanced the anti-tumor responses of tumor-specific pmel-1 T cells in a melanoma tumor model. In conclusion, NLG802 is a prodrug of indoximod expected to increase clinical drug exposure to indoximod above the current achievable levels, thus increasing the possibility of therapeutic effects in a larger fraction of the target patient population.

Full text of DOI:10.1016/j.ejmech.2020.112373

European Journal of Medicinal Chemistry 198 (2020) 112373
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of indoximod prodrugs and characterization of clinical
candidate NLG802
Sanjeev Kumar a, Firoz A. Jaipuri a, Jesse P. Waldo a, Hima Potturi a,
Agnieszka Marcinowicz a, James Adams a, Clarissa Van Allen a, Hong Zhuang a,
, b,
Nicholas Vahanian a, Charles Link Jr. a, Erik L. Brincks a *, Mario R. Mautino a
a NewLink Genetics, Ames, IA, 50010, United States
b Lumos Pharma, Inc., Ames, IA, 50010, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of different prodrugs of indoximod, including estesrs and peptide amides were synthesized with
the aim of improving its oral bioavailability in humans. The pharmacokinetics of prodrugs that were
stable in buffers, plasma and simulated gastric and intestinal fluids was first assessed in rats after oral
dosing in solution or in capsule formulation. Two prodrugs that produced the highest exposure to
indoximod in rats were further tested in Cynomolgus monkeys, a species in which indoximod has oral
bioavailability of 6e10% and an equivalent dose-dependent exposure profile as humans. NLG802 was
selected as the clinical development candidate after increasing oral bioavailability (>5-fold), Cmax (6.1
e3.6 fold) and AUC (2.9e5.2 fold) in monkeys, compared to equivalent molar oral doses of indoximod.
NLG802 is extensively absorbed and rapidly metabolized to indoximod in all species tested and shows a
safe toxicological profile at the anticipated therapeutic doses. NLG802 markedly enhanced the anti-
tumor responses of tumor-specific pmel-1 T cells in a melanoma tumor model. In conclusion, NLG802
is a prodrug of indoximod expected to increase clinical drug exposure to indoximod above the current
achievable levels, thus increasing the possibility of therapeutic effects in a larger fraction of the target
patient population.
Received 2 March 2020
Received in revised form
20 April 2020
Accepted 21 April 2020
Available online 1 May 2020
Keywords:
Indoximod
Prodrugs
Drug exposure
Indoleamine 2,3-dioxygenase (IDO)
Amino acids
© 2020 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY
license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
maternal immune tolerance to the paternally-derived allogeneic
antigens expressed by the fetus [2e5]. The IDO pathway is induced
Indoximod (1-methyl-
the indoleamine 2,3-dioxygenase (IDO) pathway,
enzyme that catalyzes the oxidation of -tryptophan (Trp) into N-
D
-tryptophan, D1Mt, 1) is an inhibitor of
in tumor cells and by host immune cells and contributes to acquired
immunologic tolerance towards many types of tumors [6,7]. IDO
expression by tumor cells has been demonstrated in melanoma,
leukemia, pancreatic, ovarian, colorectal, endometrial and prostate
cancers, and its expression is associated with significantly worse
clinical outcomes [8e15]. IDO can also be expressed by cells of the
host immune system that are associated with tumors [16]. In
addition to IDO, the functionally analogous enzyme tryptophan 2,3-
dioxygenase (TDO), which is mainly expressed in the liver, also
participates in the degradation of Trp to Kyn and potentially pro-
vides a redundant or compensatory function to IDO in the creation
of local tolerance to tumors. In fact, TDO is highly expressed in
human glioblastoma tumors and other tumor cell lines [17], and its
inhibition in TDO-expressing tumor models enhances antitumor
activity [18]. Thus, the TDO pathway is also a potential player in the
creation of local tolerance to tumors by Trp degradation.
a
cytosolic
L
formyl kynurenine (Kyn). The IDO pathway is a counter-regulatory
mechanism that contributes to local control of inflammation and
participates in creation of acquired peripheral tolerance in normal
and pathological scenarios [1]. IDO-expressing cells are found
constitutively in many tissues, including epididymis, gut, lymph
nodes, spleen, thymus and lungs. In these tissues IDO regulates
local inflammation and immune response to foreign or uncommon
non-pathological antigens. IDO activity is also found at the
maternalefetal interface where it has a critical role in inducing
* Corresponding author. Lumos Pharma, Inc., 2503 S Loop Dr, Bldg 5, Suite 5100,
Ames, IA, 50010, USA.
The activities of the IDO and TDO enzymes result in reduction of
E-mail address: ebrincks@lumos-pharma.com (E.L. Brincks).
https://doi.org/10.1016/j.ejmech.2020.112373
0223-5234/© 2020 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

2
S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
Trp and increase in Kyn levels, Low levels of Trp activate the General
Control Nonrepressed-2 (GCN2) pathway [19,20], which leads to
the creation of a Trp insufficiency signal that inactivates MAP4K3/
GLK and mTOR [21]. Additionally, high Kyn levels (and its down-
stream metabolites) are sensed by the Aryl Hydrocarbon Receptor
(AhR), a ligand-activated transcription factor that controls the
expression of several genes involved in control of the immune
response, such as IDO1, FOXP3 and RORC [22e28]. The combination
of these signals has significant downstream effects on the pheno-
type and function of immune cells such as dendritic cells (DCs),
effector CD8þ T cells, CD4þ helper and regulatory T cells (Tregs),
leading to a generalized immunosuppressive response.
IND135600, IND016400). Available pharmacokinetic (PK) data ob-
tained after dosing indoximod FB capsules or indoximod HCl tab-
lets shows that indoximod has a linear and dose proportional PK
profile at doses of up to 1200 mg/dose, with average maximum
plasma concentration (Cmax) of 13.7
of 114 M h/dose, with inter dose exposure values AUC0e12h of
83 M h/dose, which under a 1200 mg bid dosing schedule result in
an average daily exposure value at steady state AUC0e24h,ss of
240 M h. Increasing doses above 1200 mg/dose up to 2400 mg/
dose results in a non-linear and less than dose proportional in-
crease in exposure, reaching Cmax values of 22 M and AUC∞ of
170 M h/dose at 2400 mg [30,31] (and unpublished results). The
mM and drug exposure (AUC∞)
m
m
m
m
m
Indoximod is an inhibitor of the IDO/TDO pathway, but it is
fundamentally different from other selective or dual IDO/TDO in-
hibitors in that it does not directly inhibit the enzymatic activity of
IDO or TDO, but instead opposes the effects elicited by these en-
zymes. Indoximod has a unique multi-faceted mechanism of action
based on reactivation of mTOR and on the modulation of AhR
function [21,29]. These effects are independent on the Trp metab-
olizing activity of IDO or TDO but oppose the effects of the enzy-
matic activity of both IDO and TDO. First, indoximod creates a Trp-
sufficiency signal which leads to reactivation of MAP4K3 and sub-
sequent activation of mTORC1 activity, thus opposing and bypass-
ing the effects of Trp deprivation that lead to GCN2 activation and
MAP4K3 and mTOR inactivation. This effect requires a relatively
pH-dependent solubility profile of indoximod (>20 mg/mL at pH 1
and < 2 mg/mL at pH 7.4), as well as the low permeability profile in
Caco-2 assays suggests that the non-linear PK profile of indoximod
is due to limiting intestinal permeability and absorption.
We hypothesized that increasing oral bioavailability and expo-
sure of indoximod in humans would increase the fraction of pa-
tients that respond favorably to the treatment by expanding the
pharmacodynamic effects to those mechanisms requiring higher
concentrations of indoximod as well as by minimizing the inter-
subject variability in exposure observed while dosing indoximod.
However, the non-linear pharmacokinetic profile of this drug
makes it unlikely that this could be solved by increasing the dose
given to patients. For the above-mentioned reasons, we investi-
gated whether indoximod prodrugs in different salt forms would
increase solubility and absorption rate or reduce blood clearance to
levels that increase the maximum concentration and exposure of
indoximod. Moreover, we looked for prodrugs and their salts that
could result in increased exposure when dosed orally and in pill
(capsule or tablet) dosage formulation. The results of these in-
vestigations showed that selected prodrugs resulted in exposure
increase upon oral administration.
high concentration of indoximod (approximately 20
mM under
normal Trp levels and 45 M under low Trp conditions), is observed
m
in both CD4þ and CD8þ T cells and leads to an increase in the
proliferative capacity of activated effector and helper T cells [29].
Second, indoximod skews the differentiation program of primary
CD4þ T cells, favoring a TH17 helper phenotype while inhibiting a
regulatory phenotype by modulating the activity of AhR, a tran-
scription factor which in the presence of Kyn or other ligands has
the opposite effects [29]. Indoximod induces the transcription of
RORC mRNA and represses the transcription of FOXP3 mRNA. This
pharmacodynamic effect takes place at clinically relevant concen-
2. Results and discussion
trations of indoximod (EC90 of 10
mM) and is independent of IDO/
2.1. Compound design
TDO activity or exogenous Kyn, though it happens to oppose the
Kyn/AhR effects on T cell differentiation [29]. Third, indoximod can
Incorporating amino acids as an ester or amide to the parent
drug is a known approach to improve the oral bioavailability of
poorly absorbed compounds [32e40]. Various transporters
expressed in the intestinal epithelial cells can be targeted via amino
acid derivatives to promote absorption of the amino acid based
prodrugs [41e46]. In the case of indoximod, which is an N-meth-
downregulate
IDO1
protein
expression
in
human
CD11cþCD123þCD83þ DCs (moDCs), and in murine IDOþ plasma-
cytoid DCs (pDCs) in tumor-draining lymph nodes (TDLN) [29].
These effects are observed with potency of approximately 30 mM,
via a mechanism that involves the function of AhR. This direct
modulation of IDO protein expression exerts inhibition on the IDO
pathway by blocking both the enzymatic and non-enzymatic
signaling functions of IDO1 that contribute to modulation of T cell
function. In fact, mixed-lymphocyte response (MLR) T cell prolif-
eration assays of both human or murine origin show that T cells
that are in an IDOþ environment restore proliferative activity at
ylated derivative of D-tryptophan, incorporating the amino acid is
possible via both the amino and carboxyl groups. Therefore, we
explored the formation of dipeptides with indoximod by conjuga-
tion of an amino acid to the amino or carboxyl group of indoximod,
with the purpose to facilitate absorption of amino acids through the
amino acid transporter LAT1 and LAT2 or peptide transporters
PEPT1 and PEPT2 present in the intestinal epithelial cells [47e51].
Given that indoximod is required at relatively high daily doses
(>2 g/day), we explored prodrugs of indoximod that were metab-
olized into molecules with a well-known and good safety profile,
concentrations of indoximod higher than 40 mM.
The therapeutic concentration requirements of indoximod have
been established using in murine tumor models. In this setting,
biological effects of indoximod are observed when mice are dosed
at 62 mg/kg bid, which results in a daily exposure for indoximod
such as other L-amino acids, or alcohols such as glycerol and
(AUC0e24h,ss) > 170
[29]. However, an even more profound antitumor benefit was
observed at higher doses (250 mg/kg dose bid), which resulted in
daily exposure of 700 mM h (Cav,ss of ~29 mM) and Cmax ~ 50 mM [29].
Indoximod, has been tested in human cancer clinical trials in
combination with different chemotherapeutic and immunothera-
peutic agents, such as docetaxel, paclitaxel, gemcitabine, nab-
paclitaxel, temozolomide, idaurubicin, daunorubicin, cytarabine,
ipilimumab, pembrolizumab, nivolumab, sipuleucel-T, and vaccines
in numerous clinical protocol (IND078189, IND120813, IND127155,
m
M h, (Cav,ss > 7
m
M) and/or Cmax above 16
m
M
ethanol, as well as other moieties. These molecules were conju-
gated with indoximod as esters or amide groups at the carboxyl
terminus to increase their LogP, thus potentially enhancing passive
absorption through the intestinal membrane [52e54]. Additionally,
we tested whether carbamates would improve absorption by
eliminating the ionization of amino group under physiological
conditions [53]. We synthesized the following classes of prodrugs:
1) esters of indoximod, derived from ethanol (2-Cl), isopropanol (3-
Cl), benzyl alcohol (6-Cl), glycerol (8-Cl), L-Ser (10-Cl), piperidin-4-
ylmethanol (12-Cl), 2-(piperidin-4-yl)ethan-1-ol (14-Cl), 2-

S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
3
(dimethylamino)ethan-1-ol (16-Cl), 2-(tetrahydro-2H-pyran-4-yl)
ethan-1-ol (18-Cl); 2) Indoximod-C(O)NHR, where the C terminal
group forming an amide bond with indoximod is L-Val (20-Cl); 3)
carbamates 21 and 22; 4) N-AA-indoximod; dipeptides of indox-
and distribution through systemic circulation. For these reasons,
we tested the chemical stability of the prodrugs (5 M) after in-
m
cubation in buffered pH1 and SGF solutions for 1.5 h, an average
gastric residence time under fed conditions, and after a 6 h incu-
bation at pH 7.4 and SIF, to simulate stability through the small
intestine transit. Since these prodrugs were intended to be orally
dosed to animals in solution, we tested that they were chemically
stable in saline solution as a generic dosing vehicle. Additionally,
we tested the stability after a 2 h incubation in mouse and rat
plasma, and after a 6 h and 24 h incubation in human plasma,
where we expected to see moderate to high conversion of the
prodrugs into indoximod. Table 1 shows the percentage of prodrug
remaining after incubation at 37 ꢀC, in the corresponding solvent
for the indicated period, relative to a sample incubated in the same
vehicle for 0 min, immediately extracted with acetonitrile and
stored at ꢁ80 ꢀC until analysis. Each prodrug was quantified using a
specific LC-MS/MS analytical method developed for each test
compound.
At pH1 most of the prodrugs were stable (ꢂ50%) after 1.5 h, with
the exception of 8-Cl, 12-Cl and 58-Cl, which were moderately
stable (20e50%), and 10-Cl and 16-Cl which were found to be
highly unstable (<20%). Curiously, 10-Cl was more stable in SGF
than in buffer at pH 1, and 28-Cl was unstable despite being very
stable in buffer at pH 1 suggesting that it might be sensitive to
pepsin mediated cleavage.
At neutral pH7 most of the prodrugs were stable (ꢂ50%) except
for 10-Cl and 16-Cl, which were hydrolyzed completely after 6 h. In
SIF most prodrugs were stable except for 8-Cl, 12-Cl, and 28-Cl,
which were moderately stable and 10-Cl and 16-Cl which were
completely unstable. Compounds 12-Cl, 14-Cl, 16-Cl and 28-Cl
showed to be somewhat unstable after 24 h incubation in dosing
vehicle formulation. A higher stability for most of the tested pro-
drugs at pH 1 or 7 and in SGF and SIF indicates that the prodrugs
can easily pass through the stomach with minor degradation and
once absorbed can be easily hydrolyzed by liver and plasma to
release indoximod.
The results in plasma varied among plasma species and class of
compounds. In general, compounds of Class 4 and Class 5 (di-
peptides with natural or non-natural amino acids with no ester
protecting groups on the C-terminus) were stable in plasma of all
three species, with the exception of 31-Cl and 49-Cl which were
more unstable in rat plasma. Generally, compounds of Class 1 and
compounds of Class 6 (esters of indoximod) were very unstable in
rodent plasma and more stable human plasma even after longer
incubation periods suggesting differences in substrate preferences
for esterases present in rodent vs human plasma. Of note, 8-Cl, 10-
Cl, 12-Cl, 14-Cl, 16-Cl, 59-Cl and 62-Cl, showed moderate conver-
sion in human plasma, though only 14-Cl, 59-Cl, and 62-Cl satisfy
the criteria of being stable in gastric and intestinal simulated con-
ditions. These results suggested that these compounds can pass
through the stomach with slow degradation and the absorbed
molecules from the gastrointestinal tract can be rapidly converted
into the active form in plasma. Compounds of Class 2 (C-terminal
dipeptides) and Class 3 (carbamates), showed to be very stable in
plasma from all 3 species. Since prodrug conversion can take place
in the liver as well as in systemic circulation, the stability in plasma
was informative, but was not used a criterion to discard prodrugs
that were stable in plasma from further in vivo testing.
imod where the N-terminal amino acid is a natural L-amino acid
(AA) such as Gly (25-Cl), L-Ala (28-Cl), L-Val (31-Cl), L-Glu (34-Cl),
L-Gln (37-Cl), L-Ile (40-Cl), L-Leu (43-Cl), L-Lys (46-Cl), L-Phe (49-
Cl), L-Trp (52-Cl); 5) N-NNAA-indoximod; dipeptides where the
N-terminal amino acid is a non-natural amino acid (NNAA) such as
D-Trp (55-Cl) or indoximod (58-Cl); and 6) N-AA-indoximod-
COOEt; dipeptides of indoximod where the C-terminus of indox-
imod is an acetylated ester, and the N-terminal amino acid is a
natural -amino acid, such as L-Gln (59-Cl), L-Ile (60-Cl), L-Phe (61-
L
Cl), L-Leu (62-Cl), L-Met (64-Cl).
2.2. Chemistry
Ethyl 2-Cl and isopropyl 3-Cl esters of indoximod were syn-
thesized by the thionyl chloride assisted esterification with ethyl
and isopropyl alcohol, respectively.
Benzyl ester 6-Cl was synthesized via three step sequence
involving Boc protection of free amine, esterification with benzyl
bromide followed by Boc deprotection using hydrochloric acid in
ethyl acetate. Compounds 10-Cl, 12-Cl, 14-Cl, 16-Cl, 18-Cl and 20-Cl
were synthesized from Boc protected indoximod via a two-step
sequence involving HATU ester and amide coupling and Boc
deprotection using HCl or HCl/TFA. The deprotection of diol and Boc
deprotection in 7 was carried with a mixture of TFA/H2O/HCl in THF
to afford 8-Cl. Ethyl (21) and neopentyl (22) carbamates were
prepared by allowing indoximod to react with the corresponding
chloroformates in the presence of NaHCO3 (Scheme 1).
HATU coupling of ethyl (2-Cl) and benzyl (6-Cl) esters of
indoximod with several natural and non-natural amino acids
afforded the intermediates 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53,
56, and 63. The ester functionality was removed by hydrolysis to
generate the corresponding acid derivatives. Boc deprotection of
amides (24, 27, 30, 33, 35, 36, 38, 39, 41, 42, 45, 47, 48, 51, 54, 57 and
63) was achieved with HCl in dioxane to yield the hydrochloride
salts 25-Cl, 28-Cl, 31-Cl, 34-Cl, 59-Cl, 37-Cl, 60-Cl, 40-Cl, 62-Cl, 43-
Cl, 46-Cl, 61-Cl, 49-Cl, 52-Cl, 55-Cl, 58-Cl and 64-Cl, respectively
(Scheme 2).
The hydrochloric salt of prodrugs 8-Cl, 12-Cl, 59-Cl, 62-Cl, and
64-Cl were neutralized with aqueous sodium bicarbonate to yield
the corresponding free base forms; 25-Cl and 46-Cl were run
through an ion exchange chromatographic column to obtain the
corresponding free base form. The free bases were subsequently
converted to the monophosphospate (8-P, 12-P, 25-P, 46-P, 59-P,
62-P, and 64-P), mesylate (8-M, 59-M, and 62-M), besylate (62-B)
and bisulfate (8-S, 46-S, 59-S, and 62-S) salts with phosphoric,
methanesulfonic, benzene sulphonic and sulfuric acids in ethanol,
respectively (Scheme 3).
The selected prodrug, 62-Cl (also known as development com-
pound NLG802) was crystallized from ethanol/ethylacetate for
single crystal X-ray data (Fig. 1).
2.3. Biological evaluation
2.3.1. In vitro stability in biological matrices
The different prodrugs were evaluated by several in vitro and
in vivo criteria. First, we tested the stability of the prodrugs in
buffered solutions at pH1 and pH7.4, as well as in simulated gastric
fluid (SGF) and simulated intestinal fluid (SIF). Ideally, the prodrugs
should be sufficiently stable through the acidic conditions of the
stomach and neutral pH conditions of the small intestine and be
transformed to the desired metabolite after intestinal absorption
2.3.2. Pharmacokinetic testing of indoximod prodrugs in liquid
formulation in rats
A preliminary assessment of the pharmacokinetic profile of a
few prodrugs was carried out in rats dosed with the prodrug in
liquid formulation. Indoximod or its prodrugs were dosed to rats by
oral gavage at 50 mg/kg, blood samples were collected at 0, 0.5, 1, 2,

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S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
Scheme 1. Synthesis of 2-Cl, 3-Cl, 6-Cl, 8-Cl, 10-Cl, 12-Cl, 14-Cl, 16-Cl, 18-Cl, 20-Cl, 21 and 22. Reagents and conditions: a) ROH, SOCl2, 80 ꢀC, 17 h; b) NaOH, Boc2O, dioxane, 0 - rt,
16 h, 98%; c) BnBr, Cs2CO3, DMF, rt, 2 h, 91%; d) HCl, EtOAc, 0 ꢀC - rt, 2.75 h, 88%; e) R1OH or R2NH2, HATU, DIPEA, CH3CN, 0 ꢀC to rt, 17 h, 38e92%; f) HCl (4 M in dioxane), dioxane, rt,
2.5 h, 42e94%; g) i) ClCO2R, NaHCO3, THF, 0.5 h; ii) HCl, 0 ꢀC. *a mixture of TFA and HCl was used.
4, 10, 24, 48 and 72 h, plasma concentration of indoximod was
determined by LC-MS/MS, and pharmacokinetic parameters for
indoximod calculated using non-compartmental analysis. Since all
rats were orally dosed at 50 mg/kg, but each prodrug has different
molecular weight, in order to compare the values of maximum
peak concentration (Cmax) and total area under the curve (AUC∞)
obtained after dosing each prodrug vs. those obtained after dosing
indoximod, the measured Cmax and AUC∞ values were normalized
by multiplying them by the ratio of MWProdrug/MWindoximod, thus
assuming linear pharmacokinetics within an approximately 2-fold
dose range. Table 2 shows that the ethyl ester of indoximod (2-Cl)
produced a relative increase in both Cmax and AUC∞ compared to
dosing indoximod, which is consistent with this prodrug being
stable in simulated gastric and intestinal fluids and very labile in rat
plasma. However, the similar prodrugs isopropyl ester 3-Cl and
benzyl ester 6-Cl showed a moderate decrease in exposure, while
carbamate prodrugs 21 and 22 showed a marked decrease in
exposure parameters and were not characterized any further. No
apparent correlation was observed between changes in exposure
parameters and increase in cLogP in this reduced set of prodrugs.
A caveat of the comparison between dosing prodrugs vs
indoximod as a free base was that prodrugs were fully soluble in the
dosing formulation, while indoximod was insoluble at doses of
50 mg/kg. This may result in a time-dependent controlled release
effect for indoximod which could result in lower Cmax but higher
AUC∞ than when dosed in fully soluble form. Therefore, for further
comparisons and to ensure full dissolution of indoximod and each
prodrug appropriate vehicles were used (saline solution, Crema-
phor®: EtOH: saline (10:10:80), or Chremaphor: EtOH: saline: HCl
(10:10:80:0.1 N)) and the dose was reduced to 10 mg/kg. The use of
solution formulation ensured that only the differences in intestinal
permeability and in vivo conversion of prodrugs to indoximod
would be assessed by characterization of the PK parameters,
excluding the effects of differences in solubility or solubilization
rate of different prodrugs, different salt forms and polymorphs,
which could indirectly impact the observed absorption rate.
Table 3 shows the relative change in indoximod Cmax and AUC∞
and their normalized values after orally dosing each one of the test
prodrugs in solution at 10 mg/kg. In general, there did not seem to
be a correlation between the class or prodrug or their cLogP and the
change in the normalized values of Cmax and/or AUC∞. Generally,
prodrugs from classes 1, 4, or 6 produced an increase in AUC∞,
though some prodrugs from those classes also resulted in relative
decreases in AUC∞, suggesting that a combination of factors might
be affecting the performance of each prodrug.
Table 3 shows that some prodrugs resulted in an effective in-
crease in either Cmax, AUC∞ or both pharmacokinetic parameters. In
particular, exposure (AUC∞) to indoximod seems to be enhanced
when dosing 12-Cl, 62-Cl, 59-Cl, 46-Cl, 18-Cl, 16-Cl, 43-Cl, 60-Cl,
25-Cl, 8-Cl, 10-Cl, and 34-Cl, while indoximod Cmax seems to be
enhanced when dosing 16-Cl, 8-Cl, 25-Cl, 59-Cl, 43-Cl, 2-Cl and 12-
Cl. 2-Cl resulted in a 11% reduction in AUC when dosed at 10 mg/kg
but in a 126% increase in AUC at 50 mg/kg, possibly due to satu-
ration of elimination mechanisms after absorption. Since the pro-
drugs were administered in completely soluble form, this suggests
that those prodrugs that show enhanced exposure of indoximod in
plasma do so by a mechanism that involves a combination of factors
including enhanced permeability of the prodrug through the

S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
5
Scheme 2. HATU coupling and ester hydrolysis of indoximod prodrug intermediates. Reagents and conditions: a) R1CO2H, HATU, DIPEA, CH3CN, 0 ꢀC-rt, 17 h, 71e97%; b) i)
LiOH.H2O, THF, H2O, rt, 2 h; *for 33 NaOH, MeOH, THF, 0 ꢀC, 1 h; ii) 1 M HCl, 40e99%; c) HCl (4 M dioxane), 0 ꢀC - rt, 2.5e18 h.
Scheme 3. Synthesis of different salts from the free base form of 8-Cl, 12-Cl, 25-Cl, 46-
Cl, 59-Cl, 62-Cl and 64-Cl. Reagents and conditions: a) X ¼ H3PO4, or CH3SO3H, or
PhSO3H, or H2SO4, EtOH, 0 ꢀC to rt, 5e18 h.
intestinal cell wall, reduced clearance of the prodrug with respect
to indoximod and good rate of hydrolysis of the prodrug to
indoximod in vivo.
2.3.3. Pharmacokinetic testing of indoximod prodrug salts in solid
capsule formulation in rats
To test which prodrugs have the best combined set of phar-
macological properties (solubilization rate, solubility, intestinal
Fig. 1. Single crystal X-ray of NLG802.
permeability, clearance rate and rate of metabolization to indox-
imod) that would result in increased exposure to indoximod after
oral dosing in a capsule formulation, the prodrugs that showed
enhanced indoximod Cmax or AUC∞ when dosed in solution were
prepared in different salt forms and mixed with excipients to form a
powder blend. These blends were formulated in capsules contain-
ing the same molar dose of each prodrug. Gelatin capsules were
prepared containing 11, 28 or 50 mmol of indoximod or each pro-
drug per capsule (Supplementary Information, Table 1). One to
three capsules were administered to rats by intra stomach delivery
at target doses of 37, 185 or 500 mmol/kg, equivalent to indoximod

6
S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
Table 1
Chemical stability of indoximod prodrugs.a.
Compound
Class
Saline
Buffer
Simulated Fluids
SGF
Plasma
pH 1
pH 7.4
24 h
SIF
Mouse
2 h
Rat
2 h
Human
24 h
1.5 h
24 h
6 h
1.5 h
24 h
3 h
24 h
6 h
24 h
2-Cl
3-Cl
8-Cl
1
1
1
1
1
1
1
1
1
2
3
3
4
4
4
4
4
4
4
4
4
4
5
5
6
6
6
6
100
ND
99
ND
70
23
46
47
76
98
ND
ND
99
40
99
91
78
98
99
100
92
61
87
91
85
97
90
87
59
ND
37
ND
10
36
74
3
100
ND
99
ND
70
23
46
47
76
98
ND
ND
99
40
99
91
78
98
99
100
92
61
87
91
85
97
90
87
94
ND
50
ND
0
92
91
0
52
ND
29
ND
0
12
22
0
83
ND
62
ND
56
75
90
2
85
ND
58
ND
56
73
90
2
64
ND
34
ND
0
45
62
0
0
ND
10
ND
0
3
8
0
0
64
ND
ND
38
12
76
85
0
0
0
0
0
80
0
0
0
0
89
97
100
100
94
57
100
49
80
88
77
66
90
82
65
0
0
0
0
93
ND
33
ND
0
35
ND
11
ND
0
2
4
2
54
39
ND
ND
68
0
47
86
83
86
82
6-Cl
0
10-Cl
12-Cl
14-Cl
16-Cl
18-Cl
20-Cl
21
84
19
0
0
0
44
27
10
85
68
ND
ND
78
65
79
90
93
84
87
52
51
ND
ND
64
100
98
99
65
92
92
89
96
52
53
45
73
95
99
90
85
99
ND
ND
83
58
91
81
100
100
95
93
89
55
81
100
67
97
96
92
0
85
54
ND
ND
80
20
55
90
83
81
95
97
79
81
51
63
108
98
89
92
60
49
ND
ND
78
25
50
88
83
77
97
92
80
73
48
49
88
97
86
88
67
62
ND
ND
64
36
78
88
53
94
66
96
91
88
53
81
70
91
65
91
37
ND
ND
65
34
44
60
74
100
95
57
92
33
45
34
17
63
96
48
100
100
100
56
92
9
100
100
54
100
95
19
76
79
80
0
22
25-Cl
28-Cl
31-Cl
34-Cl
37-Cl
40-Cl
43-Cl
46-Cl
49-Cl
52-Cl
55-Cl
58-Cl
59-Cl
60-Cl
61-Cl
62-Cl
0
0
96
0
69
51
77
1
38
0
30
96
87
97
77
92
23
75
68
53
78
75
94
32
82
0
16
11
6
0
0
0
0
0
0
a: % remaining reported. ND: not determined. Mur: murine.
Table 2
Change in Cmax and AUC for Indoximod after Orally Dosing Rats with Solutions of Indoximod or its Prodrugs at a dose of 50 mg/kg.
Drug/Prodrug
Salt
MW
Dose
n
Cmax
Dose Norm. Cmax
% Change in Norm. Cmax
%
AUC∞
Mh
Dose Norm. AUC∞
Mh
% Change in Norm AUC∞
%
cLogP
g/mol
mg/kg
m
M
m
M
m
m
e
Indoximod
2-Cl
3-Cl
6-Cl
21
HCl
HCl
FB
HCl
FB
218
246
261
345
290
333
50
50
50
50
50
50
1
1
1
1
1
1
27
58
23.4
17.8
4.5
27
66
28
28
6.0
0.15
0%
143%
4%
1323
2645
877
650
172
3.6
1323
2988
1051
1028
229
0%
ꢁ1.10
1.96
2.27
3.14
2.34
3.66
126%
ꢁ21%
ꢁ22%
ꢁ83%
ꢁ100%
4%
ꢁ78%
22
FB
0.10
ꢁ99%
5.5
n: number of rats; Cmax
Norm. Cmax M): Cmax normalized to the same molar dose (
in Norm. Cmax: Calculated as [Cmax (indoximod from Prodrug)/Cmax(indoximod from indoximod) - 1] x 100; AUC∞
in plasma. Value is the average of n values; Norm. AUC∞ M.h): Average AUC∞ normalized to the same molar dose (
AUC∞ of indoximod in plasma by MWprodug/MWindoximod; % Change in AUC∞: calculated as [AUC∞ (indoximod from prodrug)/AUC∞ (indoximod from indoximod) - 1] x 100.
(m
M): maximum concentration of indoximod observed in plasma. Value is the average of n values.
mol/kg) obtained by multiplying the observed Cmax of indoximod in plasma by MWprodug/MWindoximod % Change
M.h): Area under the curve [indoximod] vs Time observed
mol/kg); obtained by multiplying the observed average
(
m
m
(
m
(
m
m
dosing at 8, 40 and 110 mg/kg, respectively (See methods).
Table 4 shows that 12-Cl or 12-P resulted in a significant
decrease in Cmax (69e79%, p < 0.004) and AUC∞ (54e64%,
p < 0.014) for indoximod. Since this compound showed an increase
in Cmax (24%) and AUC∞ (75%) when administered via oral solution,
the difference in solubilization rate or final solubility may account
for the observed differences when administered in powder form.
Other prodrugs such as 59, 59-Cl, 59-P, 59-M, 25-Cl, 25-P, 46, 46-Cl,
46-S or 46-P produced minor and non-statistically significant var-
iations in the Cmax or AUC∞ for indoximod compared to an equiv-
alent molar dose of indoximod.
(59e152%) and AUC∞ (49e76%) at doses ꢂ 185
mmol/kg in its hy-
drochloride or phosphate salt forms.
The L-leucyl prodrug 62 in its hydrochloride (62-Cl), phosphate
(62-P), mesylate (62-M) or besylate (62-B) salt forms dosed at
37e185
mmol/kg was able to significantly increase AUC∞ of
indoximod by 33e127%, while its sulfate salt (62-S) did not result in
a significant increase in Cmax or AUC∞ at equivalent doses. Similarly,
significant increases in Cmax were observed for 62-Cl, 62-P or 62-M
salts. At doses of 500 mmol/kg, 62-Cl, showed a minor increase in
Cmax and AUC∞ compared to indoximod indicating a possible
saturation of absorption or prodrug conversion at these high dose
levels.
The glycerol ester 8-P produced a significant increase in Cmax
(37e153%) and AUC∞ (21e75%) at doses of up to 185 mmol/kg, while
8-Cl, 8-S and 8-M resulted in no significant changes in indoximod
exposure.
2.3.4. Pharmacokinetic testing of indoximod prodrug salts in solid
capsule formulation in Cynomolgus monkeys
A new methionyl derivative prodrug 64-Cl introduced at this
stage of development showed a significant increase in Cmax
Since the rat shows a non-saturable linear increase in exposure
with doses of indoximod of up to 100 mg/kg, while humans show a

S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
7
Table 3
Change in Cmax or AUC∞ for Indoximod after Orally Dosing Rats with Solutions of Indoximod or its Prodrugs.
Prodrug ID
Class
Salt form
MW
Dose
n
Cmax
Norm. Cmax % Change in Norm. Cmax AUC∞
Norm. AUC∞ % Change in Norm AUC∞
cLogP
g/mol
mg/kg
m
M
m
M
%
m
M,h
m
M,h
%
Indoximod
12-Cl
62-Cl
59-Cl
46-Cl
18-Cl
16-Cl
43-Cl
60-Cl
25-Cl
8-Cl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
HCl
218
389
396
411
419
367
362
368
396
312
329
378
384
283
402
368
383
354
430
368
326
455
354
441
402
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
5
5
3
5
5
3
3
3
3
3
5
3
3
3
3
3
3
3
3
3
3
3
3
3
3
17.3
12.1
9.3
13
8.7
8.9
23.8
14.5
7.1
19.6
22.1
7.7
10
17.3
21.5
16.2
24.4
16.7
15
39.5
24.4
12.8
28
33.3
13.3
17.6
22
11.9
12
8.5
14.2
7.9
12.5
13.4
3
1.6
3.2
2.4
0
24
508
500
490
428
414
460
440
366
334
419
395
339
326
350
231
246
212
209
167
187
207
126
125
90
508
889
888
806
795
774
731
617
606
599
595
588
574
454
425
415
372
338
329
316
310
262
202
182
110
0
75
75
58
56
52
44
21
19
18
17
16
ꢁ1.10
1.66
3.16
ꢁ0.11
ꢁ0.04
2.21
1.75
0.24
3.16
ꢁ1.52
ꢁ0.04
ꢁ1.16
ꢁ2.10
1.96
1
6
6
4
1
1
4
6
4
1
1
4
1
4
4
4
4
6
4
4
5
2
5
1
ꢁ6
41
ꢁ3
ꢁ14
128
41
ꢁ26
62
92
10-Cl
34-Cl
2-Cl
ꢁ23
2
13
17
27
ꢁ11
ꢁ16
ꢁ18
ꢁ27
ꢁ33
ꢁ35
ꢁ38
ꢁ39
ꢁ48
ꢁ60
ꢁ64
ꢁ78
49-Cl
52-Cl
37-Cl
31-Cl
61-Cl
40-Cl
28-Cl
58-Cl
20-Cl
55-Cl
14-Cl
6.4
7.1
4.8
8.8
4
7.4
9
1.5
1
ꢁ31
ꢁ31
ꢁ51
ꢁ18
ꢁ54
ꢁ28
ꢁ22
ꢁ83
ꢁ91
ꢁ82
ꢁ86
0.20
0.19
ꢁ3.02
0.28
3.12
0.24
ꢁ1.21
0.66
ꢁ0.28
2.19
2.19
1.6
1.3
59.9
n: number of rats; Cmax
Norm. Cmax M): Cmax normalized to the same molar dose (
in Norm. Cmax: Calculated as [Cmax (indoximod from Prodrug)/Cmax(indoximod from indoximod) - 1] x 100; AUC∞
observed in plasma. Value is the average of n values; Norm. AUC∞ M.h): Average AUC∞ normalized to the same molar dose (m
(m
M): maximum concentration of indoximod observed in plasma. Value is the average of n values.
mol/kg) obtained by multiplying the observed Cmax of indoximod in plasma by MWprodug/MWindoximod % Change
M,h): Area under the curve [indoximod] vs Time
mol/kg); obtained by multiplying the observed
(
m
m
(m
(
m
average AUC∞ of indoximod in plasma by MWprodug/MWindoximod; % Change in AUC∞: calculated as [AUC∞ (indoximod from prodrug)/AUC∞ (indoximod from indoximod) - 1]
x 100.
saturable exposure above doses of 10 mg/kg [30,31], we tested in
primates two of the prodrugs that showed the largest increase in
AUC∞ to investigate whether the monkey could be a better model
to predict human pharmacokinetics for indoximod than rats. Cyn-
omolgus monkeys were dosed with indoximod, 62-Cl or 64-Cl at
2.3.5. Selection of clinical candidate
Of all the tested prodrugs, 62-Cl was selected as the develop-
ment candidate. First, it was stable in simulated gastric and intes-
tinal fluids, and demonstrated a good conversion in human plasma.
Second, it produced increased exposure when dosed to rats in so-
lution and in capsule form, and most importantly, it resulted in
increased exposure of indoximod when dosed in capsule form to
non-human primates. Among other salts of 62-Cl, only the mesy-
late salt showed better increase in exposure than the hydrochloride
salt when dosed to rats in capsule form. However, the mesylate had
disadvantages for pharmaceutical development, such as higher
molecular weight, hygroscopicity, and the need to implement strict
controls to avoid the formation of genotoxic impurity ethyl
methane sulfonate. Since the estimated daily dose of these pro-
drugs in humans is in the range of 1.2e2.4 g/dose, trying to mini-
mize the molecular weight is important to reduce the drug load per
tablet capsule form. Additionally, a daily dose of more than 2 g
requires strict controls in the levels of genotoxic impurities.
Compared to 62-Cl, 64-Cl produced slightly smaller exposure
doses of 92, 275 or 875 mmol/kg in a crossover study design where
each animal received the same single molar dose of either indox-
imod, 62-Cl or 64-Cl every 7 days. These prodrugs were mixed with
excipients and formulated into capsules of different dose strengths
(Suppl. Materials, Table 2). Animals were orally dosed with 1 or 3
capsules A (458 mmol/capsule) or 4 capsules B (1032 mmol/capsule).
Blood samples were collected at 0, 5 min, 15 min, 30 min, 1, 2, 4, 8,
12, 24, 26 and 48 h post-dose, the concentrations of prodrug and
indoximod were analyzed by validated LC-MS/MS methods and PK
parameters were calculated by non-parametric methods.
Table 5 shows that both 62-Cl and 64-Cl significantly increase
parameters of exposure for indoximod, with 62-Cl showing slightly
higher increases in AUC∞ than equivalent molar doses of 64-Cl and
64-Cl showing higher Cmax values at the highest dose level. A
comparison of the dose dependent exposure parameters for
indoximod (Cmax and AUClast) after orally dosing indoximod cap-
sules with similar excipient compositions at allometrically com-
parable doses to either monkeys or humans shows that monkeys
are a good model to predict the dose-dependent exposure of
indoximod (Fig. 2), both in the values of Cmax and AUC and with
comparable dose-dependent saturation profile. Administration of
these prodrugs to monkeys produced a larger and significant in-
crease in indoximod exposure in monkeys (~4-5-fold) compared to
rats, suggesting that a significant improvement in indoximod
exposure cold be expected in humans after administration of 62-Cl
or 64-Cl.
values for Cmax and AUC∞ at doses of up to 3300
m
mol/m2 in
monkeys, has higher molecular weight, potential long-term sta-
bility concerns of the pharmaceutical preparation, and possible
formation of undesired oxidation metabolites due to the presence
of the methionine residue. For these reasons, 62-Cl (NLG802) was
selected as the preclinical development candidate.
2.4. Pharmacological characterization of NLG802
2.4.1. Characterization of NLG802 drug substance
NLG802 (62-Cl, MW 395.93 g/mol) is a crystalline white powder,
with LogP 2.6, LogD7.4 of 2.3 and pKa of 7.76. It is not hygroscopic

8
S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
Table 4
Comparison of Cmax and total exposure (AUC∞) between indoximod free base vs. its prodrugs in different salt forms after oral dosing of rats with capsules.
Drug/Prodrug
Salt form
Dose
n
Cmax
M)
% Change in Cmax
p
AUC∞
M,h)
% Change in AUC∞
p
m
mol/kg
(
m
(
m
Indoximod
Free base
37
185
500
37
37
37
37
37
37
37
37
37
37
37
37
37
37
37
185
37
37
37
185
37
185
500
37
185
500
37
185
37
37
11
8
6
4
4
4
4
4
4
4
4
4
4
4
4
4
4
8
7
4
4
8
8
8
8
6
4
8
6
7
10
4
4
4
15.9
20.8
76.2 25
4.9 0.4
8
4
0
0
0
ꢁ69
ꢁ79
ꢁ17
8
390 166
1080 478
2871 1379
180 18
141 45
340 57
350 83
446 101
325 61
327 12
386 89
280 21
314 105
342 97
394 103
403 153
472 58
571 95
1896 765
285 39
472 120
400 136
1236 498
439 114
1898 852
4269 1255
664 134
1860 609
3300 391
628 145
1433 858
886 273
565 82
0
0
0
12-Cl
12-P
46
46-Cl
46-S
46-P
59
59-Cl
59-P
59-S
59-M
25-Cl
25-P
8-Cl
8-P
HCl
H3PO4
free base
HCl
H2SO4
H3PO4
free base
HCl
H3PO4
H2SO4
CH3SO3H
HCl
H3PO4
HCl
H3PO4
H3PO4
CH3SO3H
H2SO4
H3PO4
H3PO4
HCl
0.008
0.004
0.26
0.39
0.44
0.15
0.43
0.33
0.12
0.25
0.45
0.35
0.45
ꢁ54
ꢁ64
ꢁ13
ꢁ10
10
ꢁ17
ꢁ16
ꢁ1
ꢁ28
ꢁ20
ꢁ12
1
0.014
0.006
0.28
0.33
0.27
0.23
0.24
0.48
0.11
0.21
0.3
0.48
0.45
3.3
1
2
13.3
17.2
15.3
11.5
16.7
17.8
10.9
19
16.5
17.5
15.4
20.2
21.7
52.8 23
11.6
17.6
21.0 11
31.1
19.2
52.4 15
121 46
30.4 10
44.2 10
80.0 22
29.2 13
35.3
33.6
20.5
12.2
9
5
4
9
4
3
ꢁ4
4
5
12
ꢁ32
20
4
10
ꢁ3
28
37
153
ꢁ27
2
32
49
21
152
59
92
112
5
84
69
111
29
ꢁ23
8
6
2
5
5
3
3
21
46
75
0.16
0.18
0.032
0.0002
0.16
0.34
0.13
0.0067
0.014
0.12
0.19
0.45
8-M
8-S
64-P
5
2
ꢁ27
21
2
14
13
76
49
70
87
15
61
33
127
45
ꢁ5
8
6
0.003
0.16
0.27
0.24
64-Cl
62-Cl
62-P
HCl
HCl
HCl
HCl
<0.0001
0.031
0.005
<0.0001
0.39
0.008
0.0001
0.0004
0.14
0.017
0.048
0.006
<0.0001
0.26
0.003
0.024
0.0004
0.034
0.41
HCl
H3PO4
H3PO4
CH3SO3H
Besylate
H2SO4
7
3
2
4
62-M
62-B
62-S
37
0.19
369 145
Table 5
Comparison of Cmax and total exposure (AUC0->∞) between indoximod free base vs. NLG802 in different salt forms after oral dosing of Cynomolgus monkeys with capsules.
Drug/Prodrug ID
Indoximod
62-Cl
Salt form
free base
HCl
Dose
n
Cmax
M)
8.2 0.4
% Change in Cmax
p
AUC∞
M,h)
% Change in AUC∞
p
(
m
mol/kg)
(
m
(m
92
275
825
92
275
825
92
3
3
3
3
3
2
3
3
3
0
0
0
518
476
230
305
402
411
e
38.5
74.9
4
5
0
0
0
195
518
416
136
393
369
e
e
17.5
27.8
50.6
3
8
8
e
e
165 52
114
e
0.0004
0.003
0.005
0.0005
0.009
0.013
2
<0.0001
<0.0001
0.017
0.0007
0.005
0.059
101 28
92 17
463 36
853 349
90.7 11
370 113
761 516
64-Cl
HCl
33
5
275
825
88 32
142 57
(less than 0.08% weight gain in dynamic vapor sorption cycle from
0 to 80%RH) and soluble in water (19.8 mg/mL at 25 ꢀC), SGF
(17.1 mg/mL at 37 ꢀC), FaSSIF (19.1 mg/mL, at 37 ꢀC) as well as in
buffered solutions ranging from pH 1.58e6.7 (16.3e21 mg/mL).
hydrolysis to M9 which is further metabolized by hydrolysis to
indoximod. Most species except human show a significant dehy-
drogenation to indoximod (M1). Since this metabolite is not formed
after incubation of indoximod in primary hepatocytes from any
species, it is likely that the M1 is formed by hydrolysis of M6/M7. In
a carboxylesterase metabolism phenotyping study using recombi-
nant CES enzymes expressed in insect cell Supersomes (CES1b,
CES1c and CES2), formation of metabolite M9 was observed with
CES1b and CES1c and not with CES2. Based on the sum of differ-
ences in percent of abundance for each metabolite with respect to
that in humans, rat and monkey hepatocytes were the two species
that showed a metabolite profile most similar to humans.
2.4.2. Metabolization of NLG802
Metabolite identification of NLG802 was carried out after in-
cubation for 2 h with primary hepatocytes from CD-1 mouse,
Sprague Dawley rat, Beagle dogs, Cynomolgus monkey and human.
A total of fifteen metabolites were detected by LC-UV-MSn. Table 6
and Fig. 3 show the main metabolites, quantified by their %UV
response. Metabolization profiles were very similar among rat, dog,
monkey and human, with a few differences in the relative amounts
of each metabolite formed. Mouse showed two additional metab-
olites. Overall, NLG802 is extensively metabolized to indoximod via
NLG802 showed moderate direct (IC50
dependent (7-fold shift) inhibition of CYP3A4, weak inhibition to
CYP2D6 (IC50 19 M) and CYP2C19 (IC50 47 M), no inhibition of
5 mM) and time-
m
m

S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
9
Fig. 2. Indoximod prodrugs 62-Cl and 64-Cl increase exposure to indoximod in monkeys.
Fig. 2. Comparison of Cmax and AUClast for indoximod after oral dosing of indoximod capsules of similar compositions to humans or monkeys, and after dosing capsules with
indoximod prodrugs 62-Cl and 64-Cl. For better inter-species and inter-drug comparisons doses were allometrically scaled by surface area by multiplying the dose in mg/kg by 12
for monkeys or 37 for humans and then converted to mmoles. Human PK data was aggregated from several human clinical trials (NCI8045 [30,31], NCI8784, NLG2100 and NLG2102)
after single oral dosing of indoximod free base capsules in the range of 200 mge2000 mg, using 82 kg as the average patient weight for dose conversion.
Table 6
Metabolite identification in primary hepatocytes.
Metabolite Relative Abundance (%UV area)
NLG802
M1
M2
M3
M4
M5
M6
M7
M8
M9
M10
M11
M12
Mouse
Rat
Dog
Monkey
Human
0.3
0.4
1.1
1.3
0.9
12.2
16.5
10.5
11.9
1.9
57.8
74.4
62.1
70.7
89
0.3
1.0
e
0.5
0.1
0.8
1.3
0.1
0.2
0.1
0.6
1.3
0.1
e
0.2
0.6
1.9
0.4
1.6
e
e
2.2
3.8
2.6
7.6
1.2
e
3.1
e
16.5
e
1.8
5.0
2.6
1.3
0.4
4.8
1.0
3.8
e
e
e
0.5
e
1.3
e
e
e
e
e
Fig. 3. Metabolic transformations for NLG802 in rat, dog, monkey and human hepatocytes. a) hydrolysis, b) dehydrogenation, c) hydrolysis and cyclization, d) oxygenation, e) di-
oxygenation.

10
S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
ꢂ573
mmol/kg suggesting saturation pharmacokinetics above a
certain dose level.
Further characterization of pharmacokinetics of NLG802 was
carried out in male and female cynomolgus monkeys, after a single
intravenous dose (15 mg/kg) and after single (75, 225, 675 mg/kg)
and repeated (75, 150, 375 mg/kg bid) oral doses of NLG802. In
these studies, quantification of the parent prodrug NLG802, as well
as the intermediate metabolite M9 and the final drug indoximod
were quantified in plasma (Table 8). A similar characterization was
carried out in male and female rats dosed orally with NLG802 at
100e900 mg/kg bid (not shown).
In general, there were no marked sex differences in rats and
monkeys. In all species, NLG802 was rapidly absorbed and exten-
sively metabolized to the intermediate metabolite M9, which is
subsequently metabolized to indoximod with Tmax for indoximod
of 1e8 h. Not all samples yielded measurable levels of NLG802 or
M9 at every time point to allow calculation of all PK parameters.
Metabolite to parent exposure ratios (indoximod: NLG802) were
>39000 in rats and 690 to 8700 in monkeys, reflecting an extensive
conversion of the NLG802 to the active drug indoximod. No drug
accumulation was observed for NLG802, M9 or indoximod in rats or
monkeys after repeat dose administration.
In general, half-life or clearance could not be determined for
NLG802 and M9 in mice, rats or monkeys. Apparent clearance (CL/
F) for NLG802 and M9 after oral dosing in monkeys was much
higher than liver blood flow (>10,000 mL/min/kg) indicating fast
conversion of NLG802 to M9 and subsequent metabolization to
indoximod. In those few cases where half-life could be measured,
NLG802 showed a half-life between 3 and 9.7 h and M9 showed a
half-life of 3.7e8.2 h in monkeys. In general, indoximod half-life
was 2.2e3.4 h in mice, 23e80 h in rats and 1.8e4.3 h in mon-
keys. After intravenous dosing of NLG802, clearance for indoximod
in monkeys was 10 mL/min/kg and the volume of distribution (Vd)
was 1 L/kg (Table 8).
Fig. 4. Dose dependent antitumor effect of NLG802 in a murine melanoma tumor
model
Fig. 4. Graphical summary of tumor measurements taken on day 11. Significance was
determined using ANOVA (overall significance p ¼ 0.002025) with a follow up Dun-
nett’s multiple comparisons test comparing each dosing group against the
vehicle þ vaccine group (*p ¼ 0.0219; **p ¼ 0.0167; ***p ¼ 0.00977).
CYP450 1A2, 2B6, 2C8 and 2C9 (IC50 > 50 mM) and no induction of
CYP1A2, 2B6 or 3A4 in human hepatocytes. NLG802 showed
moderate inhibition of P-gp transporter activity in MDR1-MDCKII
cells (IC50 27
porters OATP1B1, OATP1B3, OAT1, OAT3, OCT1 and OCT2 in
HEK293 cells expressing these transporters (IC50 > 50 M) or the
mM), does not inhibit the anion and cation trans-
m
BRCP transporter in Caco-2 cells. Given the almost total conversion
of NLG802 to downstream metabolites after absorption (Fig. 3),
these weak inhibitory activities do not raise any safety concerns for
potential drug interactions.
Oral bioavailability could not be determined for NLG802 due to
its rapid conversion to indoximod. Therefore, oral bioavailability in
monkeys was measured via its conversion to indoximod after
dosing of NLG802 to Cynomolgus monkeys orally at 75 mg/kg
2.4.3. Pharmacokinetics of NLG802
The pharmacokinetics of NLG802 was investigated in mice, rats
and monkeys, in single and repeated doses administered orally
(and IV in monkeys), at doses ranging from 20 mg/kg to 900 mg/kg,
in suspension or capsule formulation.
A comparative pharmacokinetic analysis was carried out in mice
to bridge the dose-dependent indoximod exposure obtained after
dosing indoximod or NLG802 with the known pharmacodynamic
effects of indoximod in mouse models. Table 7 shows that indox-
imod Cmax increases 4 to 2-fold when dosing NLG802 compared to
equivalent molar doses of indoximod in the dose range of
(210 mmol/kg) or intravenously at 15 mg/kg (42 mmol/kg) (Table 8).
Oral bioavailability in monkeys was 61.8% and 50.1% for males and
females, respectively. This compares favorably with the oral
bioavailability of indoximod in male monkeys, which was deter-
mined to be 10% and 6% after oral dosing of indoximod at 275 and
825 mmol/kg, respectively (not shown). This indicates that NLG802
increases oral bioavailability of indoximod in monkeys approxi-
mately 5 to 6-fold at comparable molar dose levels.
NLG802 exhibited moderate binding (ꢂ50.0% and <95.0%) to
plasma proteins from Beagle dog, Cynomolgus monkey and human.
The bound values were non-calculable in CD-1 mouse and Sprague-
Dawley rat plasma due to rapid metabolism. Metabolite M9 also
exhibited a medium binding to plasma proteins from mouse, rat
and monkey and was low (<50.0%) in dog and human plasma.
After a single dose of NLG802 to rats at 100 mg/kg/dose,
approximately 40% of the administered dose was eliminated pre-
dominantly in the urine as indoximod. After a single dose of
NLG802 to monkeys at 75 mg/kg/dose, approximately 15% of the
administered dose was eliminated in the urine and feces within
12 h post dose, predominantly in the urine as indoximod.
143e1147
observed after dosing NLG802 increases 3 and 2-fold compared
with dosing indoximod at 143 and 287 mol/kg, respectively.
mmol/kg. Daily exposure (AUC0-24) of indoximod
m
However, exposure values become comparable at dose levels
Table 7
Comparison of indoximod pharmacokinetics after single dose of indoximod or
NLG802 in mice.
Test Article Dose Level
Cmax
AUC0-24
M$h
36.4
77.7 21.6
153.6 79.2
Tmax
h
T1/2
h
mmol/kg
m
M
m
Indoximod
NLG802
143
287
573
1147
143
9.3 2.2
17.2 3.7
26 11.1
51.7 25.4
35 25.3
68.9 3.9
65.7 34.1
9
0.6 (0.5e1)
0.6 (0.5e1)
1.0 (0.5e2)
3
2.8
3.1
3.4
2.9
2.2
3.1
3.2
2.4.4. Toxicology of NLG802
Toxicology studies were conducted in rats (40 males and 40
females) dosed orally at 0, 20, 60, 180 (F) or 150 (M) mg/kg bid for
28 days. The severely toxic dose in 10% of the animals (STD10) was
concluded to be 150 mg NLG802 free base/kg/dose bid. The Cmax
348.2 115.2 1.8 (0.5e6)
105.4 68.5
142.3 64.6
150.8 76.1
1.4 (0.5e2)
0.5
0.8 (0.5e1)
287
573
1147
101.4 38.9 374.3 180.7 0.6 (0.5e1)
and AUC0e12h for indoximod on Day 28 were 169
mM and

S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
11
Table 8
Single and repeated dose pharmacokinetics of NLG802 in monkeys.
Route
Dose
mg/kg
15
Dose
mol/kg
42
Analyte
Study Day
Sex
n
Cmax
nM
Tmax
h
AUC0-24
T1/2
h
CL or CL/F
mL/min/kg
Vd
m
nM∙h
L/kg
IV
NLG802
Indoximod
1
1
M
M
F
F
F
M
F
M
F
M
F
M
M
F
M
M
M
F
M
F
F
F
F
M
F
1
3
3
1
3
3
3
3
3
3
3
3
2
3
3
2
2
3
3
1
1
2
2
2
2
2
3
3
135
39500 2300
43100 3410
8.46
9.71 0.874
7.74 2.53
55000 12900
74400 23000
59300 7140
47300 12600
10.5 2.25
17.1 7.63
192
63.7
71800 9100
70600 14900
28.7
35.6 6.15
39.7 12.4
177000 93800
222000 42100
179000 15600
223000 49500
57.0 14.3
80.6 4.88
305
NC
2
10918
9.8 1.3
10.2 2.2
121000
99400 16000
92700 24500
25.6 17.5
16.0 3.41
19.5 1.64
16.2 4.05
128000 37400
86500 5250
29500
309
1.06
0.97
1.5
NA
NA
NA
1.8
3.9
2.7
2.7
3
8.5
NA
2.7
2.8
NA
NA
3.8
2.9
3.4
NA
3.8
5
PO
75
209
NLG802
1
8
2
2
1
2
2
1
1
1
2
1
2
2
3
2
1
2
2
Indoximod
1
8
8
PO
PO
150
225
417
626
NLG802
M9
Indoximod
8
8
79000 8520
93400 19300
12.6
403000 67500
509000 58800
60.2
17.6 2.83
13.8 1.70
227000
NLG802
M9
Indoximod
1
1
1
353
858
28000
68500 36500
97700 29600
403
181000 102000
525000 193000
2400
82.2 66.6
22.1 9.37
7240
24700
24.5
86800
137000
20100
38200
PO
PO
375
675
1043
1878
NLG802
M9
Indoximod
NLG802
8
8
8
1
308
203000
75.9
148
2
2.5
1
2
1.5
1
703
1660000
777
966
3610
2880
9.7
8.2
3.7
10.2
4.3
M9
1
1
1580
1760
387000 506000
311000 438000
M
F
M
Indoximod
2
4
6450000 9510000
4470000 7170000
111 176
134 184
T
max: time of Cmax; T1/2: half-life; CL: clearance; CL/F: apparent clearance; Vd: volume of distribution. Average values SD are calculated when 3 values were available. Study
Day 1 reflects PK after a single dose; Study Day 8 reflects PK after bid dosing at the stated dose level.
1410
and 170
m
M h, respectively, in the males dosed at 150 mg/kg/dose bid
Correlating this dose with dose-dependent exposure parameters
for indoximod after administration of NLG802 (Table 7) suggests
that antitumor effect mediated by NLG802 takes place at daily ex-
m
M and 1380 M h, respectively, in the females dosed at
m
180 mg/kg bid. The no adverse effect level (NOAEL) for this study
was determined to be 60 mg NLG802 free base/kg/dose bid which
posures for indoximod of AUC0-24 ꢂ 290
m
M h (twice the measured
was associated with a Cmax and AUC0e12h of 110
mM and 953
mM h,
exposure of 145
m
M h/dose due to bid dosing) and Cmax ꢂ 50 M.
m
respectively, on Day 28 for the active metabolite indoximod.
Among adverse events noticed in less than 10% of animals in the
highest dose group were mortality (2 due gavage errors, 2 of un-
known causes), abnormal respiratory sounds, labored breathing,
distended abdomen and decreased food consumption.
Additionally, a toxicology study was conducted in monkeys (12
males and 12 females) dosed orally at 0, 60, 120, 180 or 150 mg/kg
bid for 28 days. The NOAEL was 120 mg/kg/dose bid, which was
3. Conclusions
Indoximod is a clinical investigational drug candidate for the
treatment of cancer intended to be administered in combination
with other cancer therapies that involve stimulation of an immune
response to the tumor, such as cancer immunotherapy, chemo-
therapy or radiotherapy. Indoximod, has been tested in human
cancer clinical trials in combination with different chemothera-
peutic and immunotherapeutic agents, such as docetaxel, pacli-
taxel, gemcitabine, nab-paclitaxel, temozolomide, idaurubicin,
daunorubicin, cytarabine, ipilimumab, pembrolizumab, nivolumab,
sipuleucel-T, and vaccines. Available PK data for indoximod shows a
linear PK profile at doses of up to 1200 mg/dose, associated with an
associated with sex averaged Cmax of 103
mM and AUC0e12h of
320 M h for the active metabolite indoximod.
m
NLG802 is not genotoxic or clastogenic, does not alter behavioral
parameters in rats or monkeys and does not affect cardiovascular or
respiratory parameters in monkeys.
2.4.5. Pharmacodynamic activity
average daily exposure at steady state of approximately 240
mM h
In order to verify that NLG802 preserves the pharmacodynamic
effects of indoximod, we evaluated its antitumor effect in a mouse
melanoma tumor model which involved: 1) inoculation of B16F10
tumor cells on Day 1, 2) dosing NLG802 twice a day by oral gavage
from Day 6 to Day 11 at different dose levels, 3) vaccination with a
gp100 vaccine on Day 7, 4) adoptive transfer of pmel-1 CD8þ T cells
on Day 7, and 5) evaluation of tumor size on day 11. All groups of
mice received adoptive transfer of pmel-1 CD8þ T cells. Control
groups of mice were those that did not receive vaccination or did
not receive NLG802 treatment. Mice were orally dosed at 143, 287,
(i.e. Cav,ss 10 M). In vitro assays and animal models suggest that
m
different pharmacodynamics effects take place at different con-
centration levels, with effects on human CD4þ TH17 and Tregs taking
place at lower concentrations (EC90 10 mM) while other effects such
as stimulation of CD8þ T cell proliferation or downregulation of IDO
protein expression in moDCS require higher concentrations of
indoximod (EC50 ꢂ 30
mM). Mouse animal models suggest that
increasing indoximod exposure above the currently achievable
levels would improve the observed antitumor effects. The saturable
dose-dependent exposure of indoximod suggested that its
bioavailability is limited by absorption rather than by clearance
mechanisms. Therefore, here we investigated prodrugs of indox-
imod designed to increase oral bioavailability of indoximod by
573 and 1147
in tumor size was obtained when mice were dosed at the doses
ꢂ287 mol/kg/dose (equivalent to 62 mg/kg of indoximod).
mmol/kg/dose BID. A statistically significant reduction
m

12
S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
enhancing its intestinal absorption.
liquid chromatography with electrospray ionization in positive ion
We investigated derivatization of the amino and carboxyl
groups of indoximod by formation of amides, esters, carbamates or
a combination of them, with protecting groups that are metabo-
lized into molecules with a good safety profile. Since the prodrug is
intended to be stable through the gastric and intestinal transit and
be metabolized once entering systemic circulation, we discarded
those prodrug candidates that were unstable in simulated gastric
and intestinal conditions. As expected, some of the ester prodrugs
showed to be unstable under these conditions. A preliminary
assessment of indoximod exposure was determined by evaluation
of pharmacokinetics in rats after oral dosing in solution vehicles. In
general, indoximod has high bioavailability in rats (~50% depending
on dose), and a long half-life (~30 h), so improvements above 2-fold
in exposure parameters were not expected in this model. Since
solubility and solubilization rate affects oral bioavailability, it was
considered useful to test the selected candidate prodrugs in
different salt forms after dosing them orally in a capsule formula-
tion, at different dose levels. This test indicated that the best pro-
drugs were dipeptides, bearing a non-polar amino acid such as Leu
or Met in the amino terminus, and an ethyl ester in the carboxyl
terminus (62-Cl and 64-Cl). These prodrugs were evaluated in
monkeys, a species which has low oral bioavailability for indox-
imod and a similar dose-exposure profile as humans, which
showed that both prodrugs increased the Cmax and AUC, >4-fold
compared to equivalent molar doses of indoximod. The selected
clinical development candidate NLG802 increased oral bioavail-
ability to indoximod in monkeys >5-fold.
mode time of flight mass spectrometry (HPLC-ESI-TOF/MS).
4.1.1. Synthesis of ethyl 1-methyl-D-tryptophanate hydrochloride
(2-Cl)
SOCl2 (1.34 mL, 18.3 mmol) was added to a suspension of
indoximod (4.00 g, 18.3 mmol) in ethanol (50 mL) at 0 ꢀC and the
mixture was stirred at 80 ꢀC overnight. After cooling to rt, the
solvent was distilled-off and the crude was diluted with diethyl
ether (100 mL), the white solid was filtered-off and washed with
dry ether (20 mL) to afford 2-Cl (5.1 g, 98%). LCMS (ESI, m/z): 247.31
[M þ H]þ. 1H NMR (DMSO‑d6):
d
1.08 (t, 3H, J ¼ 7.1 Hz), 3.20e3.35
(m, 2H), 3.75 (3H, s), 4.08 (q, 2H, J ¼ 7.1 Hz), 4.15 (dd, 1H, J ¼ 7.0,
5.7 Hz), 7.05 (ddd, 1H, J ¼ 7.9, 7.0, 1.0 Hz), 7.16 (ddd, 1H, J ¼ 8.2, 7.0,
1.2 Hz), 7.22 (1H, s), 7.41 (dt, 1H, J ¼ 8.2, 0.9 Hz), 7.54 (dt, 1H, J ¼ 7.9,
1.0 Hz), 8.63 (br s, 3H).
4.1.2. Synthesis of isopropyl 1-methyl-D-tryptophanate
hydrochloride (3-Cl)
SOCl2 (0.167 mL, 2.29 mmol) was added to a suspension of
indoximod (0.500 g, 2.29 mmol) in isopropanol (15 mL) at 0 ꢀC rt,
and the mixture was stirred at 80 ꢀC overnight. After cooling to rt,
the solvent was distilled off and the crude was basified with 25% aq
NaHCO3 (20 mL); the product was extracted with CH2Cl2, the
combined organic extract was dried over Na2SO4 and the solvent
was distilled-off under reduced pressure. The free base was con-
verted to its hydrochloride salt by adding dry HCl in dioxane, the
solvent was removed under reduced pressure to afford 3-Cl as
white solid (0.252 g, 37%). LCMS (ESI, m/z): 261.31 [M þ H]þ. 1H
NLG802 is stable under gastric and intestinal conditions, is
extensively absorbed and rapidly metabolized by carboxyl esterases
(CES1b and CES1c) to a dipeptide Leu-indoximod (M9), and sub-
sequently hydrolyzed by unidentified peptidases to individual
amino acids Leu and indoximod.
The safety dose levels established in monkeys (NOAEL) were
120 mg/kg bid, which is equivalent to an allometrically equivalent
dose of 3200 mg bid in humans. This dose resulted in sex averaged
NMR (DMSO‑d6):
d
0.95 (d, 3H, J ¼ 6.3 Hz), 1.10 (d, 3H, J ¼ 6.2 Hz),
3.14e3.31 (m, 2H), 3.72 (s, 3H), 4.08 (t, 1H, J ¼ 6.6 Hz), 4.79e4.85
(m, 1H), 7.00e7.04 (m, 1H), 7.12e7.18 (m, 2H), 7.39 (dd, 1H, J ¼ 8.5,
2.2 Hz), 7.52 (d, 1H, J ¼ 7.8 Hz), 8.51 (br s, 3H).
4.1.3. Synthesis of Na-(tert-butoxycarbonyl)-1-methyl-
D-
tryptophan (4)
exposure parameters of Cmax of 103
m
M and AUC0e12h of 320
m
M h
To a mixture of indoximod (3.0 g, 13.75 mmol) in dioxane
(70 mL) at 0 ꢀC, NaOH (550 mg dissolved in 30 mL DI water) was
added, followed by the addition of Boc2O. The reaction was stirred
at 0 ꢀC for 4 h and stirred overnight at rt. The solution was
concentrated under reduced pressure to approx. one third the
original volume. The reaction was acidified with 1 N HCl at 0 ꢀC and
the product was extracted with EtOAc (3 ꢃ 50 mL). The organic
extract was washed with brine (30 mL) and dried over Na2SO4, the
solvent was evaporated under reduced pressure to afford the
product that was used directly in the next step without further
(i.e. Cav,ss of 27 M) for the active metabolite indoximod. We predict
m
that these exposure levels would bring additional therapeutic
benefits for indoximod in humans.
4. Experimental section
4.1. Chemistry
All reagents and solvents were purchased from commercial
sources. All commercial reagents and solvents were used as
received without further purification. The reactions were moni-
tored using analytical thin layer chromatography (TLC) with
0.25 mm EM Science silica gel plates (60F-254). The developed TLC
plates were visualized by short wave UV light (254 nm) or im-
mersion in potassium permanganate solution followed by heating
on a hot plate. Flash chromatography was performed with Selecto
purification (4.3 g, 98%). 1H NMR:
d 1.24 and 1.42 (2 s, 9H),
3.25e3.37 (m, 2H), 3.71 (s, 3H), 4.62e4.67 (m, 1H), 5.01e5.04 (m,
1H), 6.89 (s, 1H), 7.07e7.12 (m, 1H), 7.19e7.21 (m, 1H), 7.26e7.72 (m,
1H), 7.59 (d, 1H, J ¼ 7.9 Hz).
4.1.4. Synthesis of benzyl Na-(tert-butoxycarbonyl)-1-methyl-D-
tryptophanate (5)
Scientific silica gel, 32e63
m
m particle sizes. All reactions were
Na-(tert-butoxycarbonyl)-1-methyl-
D
-tryptophan
(3.00
g,
performed in flame or oven-dried glassware under a nitrogen at-
mosphere. All reactions were stirred magnetically at ambient
temperature unless otherwise indicated. 1H NMR spectra were
obtained with a Bruker DRX400, Varian VXR400 or VXR300.1H
9.42 mmol) was dissolved in 60 mL of DMF, followed by the addi-
tion of Cs2CO3 (1.78 g, 5.47 mmol) and benzyl bromide (1.61 mL,
9.42 mmol). The resulting suspension was stirred at room tem-
perature for 2 h. After the end of reaction (TLC), the DMF was
removed under reduced pressure followed by suspending the res-
idue in ethyl acetate (100 mL) before washing with distilled water
(3 ꢃ 50 mL) and brine. The organic layer was dried over anhydrous
sodium sulfate and concentrated under vacuum. The residue was
purified by column chromatography on silica gel (3.5 g, 91%). 1H
NMR spectra were reported in parts per million (d) relative to TMS
(0.0), DMSO‑d6 (2.50) or MeOH-d4 (4.80) as an internal reference.
All 1H NMR spectra were taken in CDCl3 unless otherwise indicated.
MS was conducted on Waters ACQUITY UPLC system with a QDa
detector. Purity of all final target compounds was 95% or higher.
High resolution mass spectrometric analysis for NLG802 was per-
formed on the Agilent G6224A TOF LC/MS using high performance
NMR:
d 1.28 (s, 9H), 3.25e3.27 (m, 2H), 3.62 (s, 3H), 4.65e4 0.70 (m,
1H), 5.02e5.17 (m, 3H), 7.07e7.12 (m, 1H), 7.19e7.28 (m, 5H),

S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
13
7.30e7.36 (m, 3H), 7.53 (d, 1H, J ¼ 8.0 Hz).
92% yield. 1H NMR:
d 0.95e1.05 (m, 2H), 1.47 (s, 18H), 1.32e1.40 (m,
3H),1.55 (d, 2H, J ¼ 2.4 Hz), 2.59 (dt, 2H, J ¼ 2.7,12.8 Hz), 3.25 (d, 2H,
J ¼ 5.6 Hz), 3.74 (s, 3H), 3.99e4.05 (m, 2H), 4.94e5.00 (m, 2H), 5.08
(d, 1H, J ¼ 8.0 Hz), 6.52 (br s, 1H), 6.86 (s, 1H), 7.09 (t, 1H, J ¼ 7.4 Hz),
7.21 (t, 1H, J ¼ 7.6 Hz), 7.28 (d, 1H, J ¼ 8.0 Hz), 7.53 (d, 1H, J ¼ 8.0 Hz).
4.1.5. Synthesis of benzyl 1-methyl-D-tryptophanate hydrochloride
(6-Cl)
Ethyl acetate (26.9 mL) and MeOH (8.9 mL) in an RB flask
equipped with a septum and a needle vent were cooled in an ice
bath with stirring. Acetyl chloride (14.22 mL) was added slowly. The
resulting solution was stirred at 0 ꢀC for 20 min and MeOH (0.5 mL)
was added. A flask containing benzyl Na-(tert-butoxycarbonyl)-1-
4.1.6.5. 2-(Dimethylamino)ethyl Na-(tert-butoxycarbonyl)-1-methyl-
D-tryptophanate (15). It was obtained in 38% yield. 1H NMR:
d 1.33
(s, 1H), 1.43 (s, 8H), 2.23 (s, 5H), 2.29 (s, 1H), 2.43e2.60 (m, 4H), 3.27
(d, 2H, J ¼ 5.6 Hz), 3.74 (s, 3H), 4.1e4.23 (m, 2H), 4.63 (m, 1H), 5.10
(m, 1H), 6.91 (s, 1H), 7.10 (ddd, 1H, J ¼ 8.0, 6.8, 1.2 Hz), 7.21 (ddd, 1H,
J ¼ 8.0, 6.8, 1.2 Hz), 7.28 (d, 1H, J ¼ 8.0 Hz), 7.54 (d, 1H, J ¼ 8.0 Hz).
methyl-D-tryptophanate (3.5 g, 8.6 mmol) was placed in an ice
bath and the cold, freshly prepared HCl (4 M in EtOAc) was slowly
poured into the flask containing benzyl Na-(tert-butoxycarbonyl)-
1-methyl-
0
D-tryptophanate. The solution was stirred vigorously at
ꢀC for 15 min where the formation of a white suspension was
4.1.6.6. 2-(Tetrahydro-2H-pyran-4-yl)ethyl
ycarbonyl)-1-methyl-D-tryptophanate (17). It was obtained in 60%
yield. 1H NMR:
1.29e1.35 (m, 2H), 1.42 (s, 9H), 1.60e1.67 (m, 5H),
Na-(tert-butox-
observed, and the flask was removed from the ice bath. The sus-
pension was stirred vigorously for 2.5 h. The solution was cooled in
an ice bath diluted with ether (50 mL) and the suspension was
filtered, and the solid cake washed with cold ether. The solid dried
under high vacuum and 6-Cl was isolated as a colorless solid
(6.45 g, 88%). LCMS (ESI, m/z): 309.31 [M þ H]þ. 1H NMR (DMSO‑d6):
d
3.17e3.35 (m, 4H), 3.74 (s, 3H), 3.84e3.93 (m, 2H), 4.10 (dq, 2H,
J ¼ 10.4, 6.4 Hz), 4.55e4.65 (m, 1H), 5.06 (d, 1H, J ¼ 8.2 Hz), 6.86 (s,
1H), 7.09 (ddd, 1H, J ¼ 8.0, 7.0, 1.1 Hz), 7.21 (ddd, 1H, J ¼ 8.2, 6.9,
1.1 Hz), 7.28 (d, 1H, J ¼ 7.4 Hz), 7.48e7.59 (m, 1H).
d
3.28 (dd, 2H, J ¼ 5.6, 15.2 Hz), 3.70 (s, 3H), 4.26e4.29 (m, 1H), 5.08
(d, 1H, J ¼ 12.4 Hz), 5.13 (d, 1H, J ¼ 12.4 Hz), 7.04 (t, 1H, J ¼ 7.6 Hz),
7.06 (s, 1H), 7.10e7.18 (m, 3H), 7.30e7.35 (m, 3H), 7.42 (d, 1H,
J ¼ 8.0 Hz), 7.53 (d, 1H, J ¼ 8.0 Hz).
4.1.6.7. tert-Butyl Na-(tert-butoxycarbonyl)-1-methyl-D-tryptophyl-
L-valinate (19). It was obtained in 91% yield. 1H NMR:
d 0.69 (d, 3H,
J ¼ 6.8 Hz), 0.75 (d, 3H, J ¼ 6.8 Hz), 1.42 (s, 18H), 1.98e2.03 (m, 1H),
3.18 (dd, 1H, J ¼ 14.4, 7.2 Hz), 3.27e3.35 (m, 1H), 3.73 (s, 3H),
4.35e4.39 (m, 1H), 4.50 (br s, 1H), 5.07 (br s, 1H), 6.31 (d, 1H,
J ¼ 8.8 Hz), 6.92 (s, 1H), 7.12 (t, 1H, J ¼ 7.2 Hz), 7.22 (t, 1H, J ¼ 7.2 Hz),
7.28 (d, 1H, J ¼ 8.0 Hz), 7.64 (d, 1H, J ¼ 8.0 Hz).
4.1.6. General method for the derivatization of eCOOH group of
indoximod
DIPEA (9.42 mmol) was added to a solution of N-(tert-butox-
ycarbonyl)-1-methyl-D-tryptophan (3.14 mmol), the appropriate
alcohol or amine (3.14 mmol) and HATU (3.14 mmol) in acetonitrile
(30 mL) at 0 ꢀC, and the solution was allowed to warm to rt. After
stirring overnight (17 h), the reaction was diluted with water
(50 mL) and the product was extracted with CH2Cl2 (3 ꢃ 50 mL).
The combined organic extract was washed with water (25 mL),
brine (25 mL) dried over Na2SO4 and concentrated under reduced
pressure to afford the crude. Chromatographic purification afforded
the desired product.
4.1.7. General method for the synthesis of carbamate esters (21 and
22)
The appropriate chloroformate (1.37 mmol) was added drop-
wise to a stirred solution of indoximod (0.150 g, 0.687 mmol) in 1:1
THF/1 M NaHCO3 (2.75 mL, 2.75 mmol). The mixture was stirred for
30 min and the solution was diluted with water and extracted with
ether 2x. The aqueous layer was cooled to 0 ꢀC and conc. HCl so-
lution was added to adjust the pH to ~1. The cold aqueous layer was
immediately extracted with ethyl acetate and the combined
organic layers were washed with water, brine and dried over
Na2SO4. The solvent was removed under reduced pressure to afford
crude the carbamate. The crude was purified by column chroma-
tography to afford the pure carbamate.
4.1.6.1. (2,2-Dimethyl-1,3-dioxolan-4-yl)methyl,N-(tert-butox-
ycarbonyl)-1-methyl-
D
-tryptophanate (7). It was obtained in 78%
yield. 1H NMR:
d
1.27 (s, 3H), 1.33 (s, 3H), 1.35 (s, 9H), 3.21 (d, 2H,
J ¼ 5.6 Hz), 3.44e3.50 (m, 1H), 3.67 (s, 3H), 3.80e3.86 (m, 1H),
3.99e4.03 (m, 2H), 4.07e4.12 (m, 1H), 4.58 (q, 1H, J ¼ 6.5 Hz), 4.99
(d, 1H, J ¼ 8.2 Hz), 6.82 (s, 1H), 7.03 (t, 1H, J ¼ 7.4 Hz), 7.14 (t, 1H,
J ¼ 7.4 Hz), 7.21 (d, 1H, J ¼ 8.1 Hz), 7.47 (d, 1H, J ¼ 8.0 Hz).
4.1.7.1. Na-(Ethoxycarbonyl)-1-methyl-
D
-tryptophan
(21). Yield:
1.23 (t, 3H,
81%. LCMS (ESI, m/z): 291.21 [MþH]þ. 1H NMR:
d
J ¼ 6.8 Hz), 3.63e3.71 (m, 1H), 3.74 (s, 3H), 4.07e4.12 (m, 2H), 4.69
(dd, 1H, J ¼ 6.7, 11.6 Hz), 5.20 (dd, 1H, J ¼ 6.9, 11.5 Hz), 6.9 (s, 1H),
7.07 (t,1H, 6.9 Hz), 7.21e7.48 (m, 2H), 7.57 (d,1H, J ¼ 7.1 Hz), 9.07 (br
s, 1H).
4.1.6.2. (S)-3-(tert-Butoxy)-2-((tert-butoxycarbonyl)amino)-3-
oxopropyl-Na-(tert-butoxy carbonyl)-1-methyl-D-tryptophanate (9).
It was obtained in 40% yield. 1H NMR:
d 1.41 (s, 9H), 1.44 (s, 9H), 1.45
(s, 9H), 3.16 (dd, 1H, J ¼ 15.3, 4.8 Hz), 3.29 (dd, 1H, J ¼ 15.3, 4.8 Hz),
3.75 (s, 3H), 4.35e4.52 (m, 3H), 4.61 (d, 1H, J ¼ 6.3 Hz), 4.99 (d, 1H,
J ¼ 8.6 Hz), 5.28 (d,1H, J ¼ 8.7 Hz), 6.87 (s,1H), 7.11 (t,1H, J ¼ 7.3 Hz),
7.22 (t, 1H, J ¼ 7.3 Hz), 7.29 (d, 1H, J ¼ 8.2 Hz), 7.52 (d, 1H, J ¼ 7.8 Hz).
4.1.7.2. 1-Methyl-Na-((neopentyloxy)carbonyl)-
Yield: 72%. 1H NMR:
0.90 (s, 9H), 3.34 (s, 2H), 3.64 (s, 3H), 3.73 (t,
1H, J ¼ 6.8 Hz), 4.75 (d, 1H, J ¼ 7.8 Hz), 5.23 (d, 1H, J ¼ 7.9 Hz), 6.89
(s, 1H), 7.07 (t, 1H, J ¼ 8.2 Hz), 7.25e7.59 (m, 2H, overlapped with
CDCl3), 7.58 (d, 1H, 7.8 Hz), 8.4 (br s, 2H).
D
-tryptophan
(22).
d
4.1.6.3. tert-Butyl 4-(((Na-(tert-butoxycarbonyl)-1-methyl-D-trypto-
phyl)oxy)methyl)piperidine-1-carboxylate (11). It was obtained in
83% yield. 1H NMR:
d
0.93e1.10 (m, 2H), 1.29e1.32 (m, 1H), 1.45 (s,
4.1.8. General method for the derivatization of the eNH2 group of
18H), 1.63e1.69 (m, 2H), 2.59 (tt, 2H, J ¼ 2.4, 13.2 Hz), 3.25 (t, 2H,
J ¼ 5.4 Hz), 3.75 (s, 3H), 3.84e3.92 (m, 2H), 4.01e4.06 (m, 2H), 5.06
(d, 1H, J ¼ 8.0 Hz), 6.35 (br s, 1H), 6.86 (s, 1H), 7.10 (dt, 1H, J ¼ 1.2,
6.8 Hz), 7.24 (dt, 1H, J ¼ 1.2, 6.8 Hz), 7.28 (d, 1H, J ¼ 8.4 Hz), 7.53 (d,
1H, J ¼ 8.0 Hz).
indoximod esters
Appropriate D-tryptophanate hydrochloride ester (3.54 mmol)
and appropriate acid (3.54 mmol) were stirred in acetonitrile
(50 mL) at 0 ꢀC. HATU (1.48 g, 3.89 mmol) and iPr2NEt (2.46 mL,
14.15 mmol) were added and the reaction was stirred overnight at
rt. The solvent was removed under reduced pressure and the crude
was diluted with water (50 mL) and dichloromethane (50 mL). The
organic layer was separated, and the aqueous layer was extracted
4.1.6.4. tert-Butyl 4-(2-((Na-(tert-butoxycarbonyl)-1-methyl-D-tryp-
tophyl)oxy)ethyl)piperidine-1-carboxylate (13). It was obtained in

14
S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
with dichloromethane (3 ꢃ 50 mL). The combined organic layer
was washed with brine (30 mL), dried over Na2SO4, and concen-
trated under reduced pressure. The crude product was purified by
flash column chromatography to afford the desired product.
NMR:
d
1.25 (q, 2H, J ¼ 7.7 Hz), 1.39 (s, 9H), 1.44 (s, 9H), 1.47e1.55
(m, 1H), 1.67e1.80 (m, 2H), 3.02 (t, 2H, J ¼ 6.7 Hz), 3.29 (d, 2H,
J ¼ 5.5 Hz), 3.66 (s, 3H), 4.04 (s, 1H), 4.53 (s, 1H), 4.90 (q, 1H,
J ¼ 6.1 Hz), 4.97 (s, 1H), 5.09 (q, 2H, J ¼ 12.2 Hz), 6.57 (d, 1H,
J ¼ 7.8 Hz), 6.64 (s, 1H), 7.08 (t, 1H, J ¼ 7.4 Hz), 7.20 (t, 1H, J ¼ 7.6 Hz),
7.23e7.29 (m, 4H, overlapped with CDCl3), 7.30e7.39 (m, 3H), 7.49
(d, 1H, J ¼ 7.9 Hz).
4.1.8.1. Ethyl
phanate (23). It was obtained in 94% yield. 1H NMR:
Na-((tert-butoxycarbonyl)glycyl)-1-methyl-D-trypto-
1.22 (t, 3H,
d
J ¼ 7.2 Hz), 1.42 (s, 9H), 3.31 (d, 2H, J ¼ 5.2 Hz), 3.72e3.77 (m, 2H),
3.74 (s, 3H), 4.07e4.17 (m, 2H), 4.86e4.91 (m, 1H), 5.04 (br s, 1H),
6.50 (d, 1H, J ¼ 7.6 Hz), 6.86 (s, 1H), 7.10 (t, 1H, J ¼ 7.4 Hz), 7.21 (t, 1H,
J ¼ 7.4 Hz), 7.28 (d, 1H, J ¼ 8.0 Hz), 7.50 (d, 1H, J ¼ 7.6 Hz).
4.1.8.9. Ethyl Na-((tert-butoxycarbonyl)-L-phenylalanyl)-1-methyl-
D-tryptophanate (47). It was obtained in 80% yield. 1H NMR:
d 1.14
(t, 3H, J ¼ 7.1H), 1.29 (s, 9H), 2.82 (s, 2H), 2.91e3.02 (m, 1H),
3.03e3.10 (m, 2H), 3.25 (dd, 1H, J ¼ 14.78, 5.2 Hz), 3.67 (s, 3H),
3.99e4.07 (m, 2H), 4.33 (br s, 1H), 4.79 (q, 1H, J ¼ 6.2 Hz), 6.37 (d,
1H, J ¼ 7.8 Hz), 6.57 (s, 1H), 7.06 (ddd, 1H, J ¼ 8.0, 6.8, 1.2 Hz),
7.14e7.25 (m, 6H), 7.41 (d, 1H, J ¼ 7.9 Hz).
4.1.8.2. Ethyl Na-((tert-butoxycarbonyl)-L-alanyl)-1-methyl-D-tryp-
tophanate (26). It was obtained in 95% yield. 1H NMR:
d 1.20 (t, 3H,
J ¼ 7.0 Hz), 1.29 (d, 3H, J ¼ 7.2 Hz), 1.40 (s, 9H), 3.30 (d, 1H,
J ¼ 5.6 Hz), 3.75 (s, 3H), 4.09e4.16 (m, 3H), 4.81e4.86 (m, 1H), 4.93
(br s, 1H), 6.61 (br s, 1H), 6.87 (s, 1H), 7.09 (t, 1H, J ¼ 7.4 Hz), 7.21 (t,
1H, J ¼ 7.6 Hz), 7.27 (d, 1H, J ¼ 8.4 Hz, overlapped with CDCl3), 7.52
(d, 1H, J ¼ 8.0 Hz).
4.1.8.10. Ethyl Na-((tert-butoxycarbonyl)-L-tryptophyl)-1-methyl-D-
tryptophanate (50). It was obtained in 97% yield. 1H NMR:
d 1.12 (t,
3H, J ¼ 7.1 Hz), 1.39 (s, 9H), 2.90 (d, 1H, J ¼ 15.2 Hz), 3.05e3.32 (m,
3H), 3.56 (s, 3H), 3.91e4.10 (m, 2H), 4.44 (br s, 1H), 4.75 (br s, 1H),
5.15 (br s, 1H), 6.18 (d, 1H, J ¼ 7.8 Hz), 6.27 (s, 1H), 6.86 (d, 1H,
J ¼ 2.3 Hz), 7.04 (ddd, 1H, J ¼ 8.0, 6.8, 1.2 Hz), 7.14 (ddd, 1H, J ¼ 8.0,
7.1,1.2 Hz), 7.16e7.27 (m, 3H), 7.30 (dt,1H, J ¼ 8.1,1.0 Hz), 7.37 (d,1H,
J ¼ 8.2 Hz), 7.68 (d, 1H, J ¼ 7.7 Hz), 7.80 (s, 1H).
4.1.8.3. Ethyl Na-((tert-butoxycarbonyl)-L-valyl)-1-methyl-D-trypto-
phanate (29). It was obtained in 95% yield. 1H NMR:
d 0.80 (d, 3H,
J ¼ 6.8 Hz), 0.87 (d, 3H, J ¼ 6.8 Hz),1.19 (t, 3H, J ¼ 7.2 Hz),1.40 (s, 9H),
2.09e2.17 (m, 1H), 3.25e3.32 (m, 2H), 3.74 (s, 3H), 3.94e3.97 (m,
1H), 4.09e4.15 (m, 2H), 4.84e4.89 (m, 1H), 4.93e4.95 (m, 1H), 6.45
(d, 1H, J ¼ 7.6 Hz), 6.87 (s, 1H), 7.10 (t, 1H, J ¼ 7.4 Hz), 7.21 (t, 1H,
J ¼ 7.6 Hz), 7.27 (d, 1H, J ¼ 7.6 Hz), 7.53 (dd, 1H, J ¼ 8.0, 1.2 Hz).
4.1.8.11. Ethyl Na-((tert-butoxycarbonyl)-D-tryptophyl)-1-methyl-D-
tryptophanate (53). It was obtained in 97% yield. 1H NMR:
d 1.18 (t,
3H, J ¼ 7.1 Hz), 1.38 (s, 9H), 1.73 (br s, 1H), 3.13 (dd, 2H, J ¼ 5.4,
2.5 Hz), 3.32 (s, 1H), 3.57 (s, 3H), 4.05 (dd, 2H, J ¼ 17.2, 7.2 Hz), 4.43
(s, 1H), 4.72e4.80 (m, 1H), 5.07 (s, 1H), 6.22 (s, 1H), 6.42 (s, 1H), 6.90
(s, 1H), 6.97 (s, 1H), 7.04e7.25 (m, 5H), 7.33 (d, J ¼ 8.2 Hz, 1H), 7.66
(d, J ¼ 7.8 Hz, 1H), 7.87 (s, 1H).
4.1.8.4. tert-Butyl (S)-5-(((R)-1-(benzyloxy)-3-(1-methyl-1H-indol-
3-yl)-1-oxopropan-2-yl)amino) -4-((tert-butoxycarbonyl)amino)-5-
1
oxopentanoate (32). It was obtained in 93% yield. H NMR:
d 1.38
(s, 9H), 1.43 (s, 9H), 1.76e1.91 (m, 1H), 1.94e2.09 (m, 1H), 2.20 (dt,
1H, J ¼ 16.6, 7.0 Hz), 2.31 (dt, 1H, J ¼ 16.6, 7.3 Hz), 3.19e3.36 (m, 2H),
3.67 (s, 3H), 4.90 (dt, ¼ 8.1, 5.6 Hz), 5.00e5.14 (m, 2H), 5.19 (s, 1H),
6.68e6.75 (m, 2H), 7.08 (ddd, 1H, J ¼ 8.0, 6.9, 1.2 Hz), 7.18e7.28 (m,
4H), 7.29e7.37 (m, 2H), 7.50 (dt, 1H, J ¼ 8.0, 1.0 Hz).
4.1.8.12. Ethyl
phyl)-1-methyl-D-tryptophanate (56). It was obtained in 95% yield.
1H NMR:
3.35 (d, 1H, J ¼ 15.0 Hz), 3.57 (s, 3H), 3.68 (s, 3H), 3.94e4.10 (m, 2H),
4.42 (br s, 1H), 4.75 (d, 1H, J ¼ 6.8 Hz), 5.04 (s, 1H), 6.24 (br s, 1H),
6.37 (s, 1H), 6.84 (br s, 1H), 6.94 (s, 1H), 7.08e7.18 (m, 3H), 7.17e7.25
(m, 2H), 7.27e7.33 (m, 1H), 7.65 (d, 1H, J ¼ 7.9 Hz).
Na-(Na-(tert-butoxycarbonyl)-1-methyl-D-trypto-
d
1.16 (t, 3H, J ¼ 7.1 Hz), 1.37 (s, 9H), 3.02e3.20 (m, 3H),
4.1.8.5. Ethyl Na-((tert-butoxycarbonyl)-L-glutaminyl)-1-methyl-D-
tryptophanate (35). It was obtained in 90% yield. 1H NMR:
d 1.16 (t,
3H, J ¼ 7.1H), 1.33 (s, 9H), 1.79e1.99 (m, 2H), 2.05 (ddd, 1H, J ¼ 15.2,
6.9, 5.7 Hz), 2.18 (ddd, 1H, J ¼ 14.8, 8.6, 5.9 Hz), 3.21 (d, 2H,
J ¼ 5.9 Hz), 3.68 (s, 3H), 4.00e4.14 (m, 3H), 4.75 (dt, 1H, J ¼ 7.7,
5.9 Hz), 5.22 (s, 1H), 5.55 (d, 1H, J ¼ 7.0 Hz), 5.90 (s, 1H), 6.85 (s, 1H),
6.87e6.93 (m, 1H), 7.04 (ddd, 1H, J ¼ 8.0, 6.9, 1.1 Hz), 7.14 (ddd, 1H,
J ¼ 8.2, 6.9, 1.1 Hz), 7.17e7.21 (m, 1H), 7.45 (d, 1H, J ¼ 7.9 Hz).
4.1.8.13. Ethyl Na-((tert-butoxycarbonyl)-L-methionyl)-1-methyl-D-
tryptophanate (63). It was obtained in 84% yield. 1H NMR:
d 1.21 (t,
3H, J ¼ 7.2 Hz), 1.40 (s, 9H), 1.79e1.89 (m, 1H), 1.94e2.00 (m, 1H),
2.01 (s, 3H), 2.31e2.36 (m, 1H), 2.36e2.46 (m, 1H), 3.30 (dd, 2H,
J ¼ 5.7, 3.6 Hz), 3.75 (s, 3H), 4.12 (q, 2H, J ¼ 7.2 Hz), 4.26 (d, 1H,
J ¼ 7.5 Hz), 4.84 (q,1H, J ¼ 6.4 Hz), 5.17 (d,1H, J ¼ 8.3 Hz), 6.67 (d,1H,
J ¼ 7.2 Hz), 6.89 (s, 1H), 7.10 (t, 1H, J ¼ 7.4 Hz), 7.21 (t, 1H, J ¼ 7.2 Hz),
7.28 (d, 1H, J ¼ 7.5 Hz), 7.53 (d, 1H, J ¼ 7.9 Hz).
4.1.8.6. Ethyl
tryptophanate (38). It was obtained in 93% yield. 1H NMR:
Na-((tert-butoxycarbonyl)-L-isoleucyl)-1-methyl-D-
d
0.80e0.84 (m, 6H), 1.02e0.91 (m, 2H), 1.19 (t, 3H, J ¼ 7.1 Hz), 1.40
(s, 9H), 1.87 (m, 1H), 3.28 (t, 2H, J ¼ 5.4 Hz), 3.72 (s, 3H), 4.00e4.04
(m, 1H), 4.05e4.16 (m, 2H), 4.85 (q, 1H, J ¼ 6.4 Hz), 4.95 (d, 1H,
J ¼ 9.0 Hz), 6.46 (d, 1H, J ¼ 7.7 Hz), 6.87 (s, 1H), 7.10 (ddd, 1H, J ¼ 8.0,
6.8, 1.1 Hz), 7.20 (ddd, 1H, J ¼ 8.2, 6.9, 1.2 Hz), 7.26 (d, 1H, J ¼ 8.0 Hz),
7.53 (dt, 1H, J ¼ 7.9, 1.0 Hz).
4.1.9. General method for the hydrolysis of substituted indoximod
ethyl esters
Water (3 mL) and lithium hydroxide monohydrate (67 mg,
1.59 mmol) was added to
a solution of appropriate amide
(0.991 mmol) in THF (10 mL) and the mixture was stirred under
ambient temperature for 2 h. The mixture was neutralized with 1 M
HCl (at 0 ꢀC) and poured into ice cold water (20 mL). The aqueous
layer was extracted with EtOAc (3 ꢃ 35 mL). The combined organic
layers were dried over Na2SO4 and concentrated. The crude product
was purified by flash column chromatography to afford the desired
product.
4.1.8.7. Ethyl Na-((tert-butoxycarbonyl)-L-leucyl)-1-methyl-D-tryp-
tophanate (41). It was obtained in 92% yield. 1H NMR:
d 0.82e0.89
(m, 6H), 1.20 (t, 3H, J ¼ 7.2 Hz), 1.36e1.41 (m, 1H, overlapped with
Boc), 1.39 (s, 9H), 1.52e1.60 (m, 2H), 3.29 (d, 2H, J ¼ 5.7 Hz), 3.74 (s,
3H), 4.04e4.17 (m, 3H), 4.81e4.86 (m, 2H), 6.59e6.61 (m, 1H), 6.87
(s, 1H), 7.07e7.11 (m, 1H), 7.16e7.22 (m, 1H), 7.24e7.32 (m, 1H), 7.52
(dt, 1H, J ¼ 7.9, 1.1 Hz).
4.1.9.1. Na-((tert-Butoxycarbonyl)glycyl)-1-methyl-
(24). It was obtained in 83% yield. 1H NMR:
1.39 (s, 9H), 3.25e3.35
(m, 2H), 3.2e3.74 (m, 5H), 4.85e4.90 (m, 1H), 5.21 (br s, 1H), 6.63
D-tryptophan
4.1.8.8. Benzyl Na-(N2,N6-bis(tert-butoxycarbonyl)-L-lysyl)-1-
d
methyl-D-tryptophanate (44). It was obtained in 91% yield. 1H

S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
15
(br s, 1H), 6.90 (s, 1H), 7.08 (t, 1H, J ¼ 7.4 Hz), 7.17e7.27 (m, 2H,
overlapped with CDCl3), 7.55 (d, 1H, J ¼ 7.6 Hz).
3.05e3.13 (m, 3H), 3.29 (s, 1H), 3.55 (s, 3H), 4.44 (s, 1H), 4.75 (q,
J ¼ 6.1 Hz, 1H), 5.10 (s, 1H), 6.26 (s, 1H), 6.58 (s, 1H), 6.89 (s, 2H),
7.07e7.24 (m, 5H), 7.31 (d, 1H, J ¼ 8.0 Hz), 7.64 (d, 1H, J ¼ 6.6 Hz),
8.09e8.35 (m, 1H).
4.1.9.2. Na-((tert-Butoxycarbonyl)-L-alanyl)-1-methyl-
(27). It was obtained in 86% yield. 1H NMR:
1.38 (s, 9H), 3.19e3.38 (m, 3H), 3.73 (s, 3H), 4.22e4.27 (m, 1H), 4.84
(br s, 1H), 6.77 (br s, 1H), 6.87 (s, 1H), 7.08 (t, 1H, J ¼ 7.4 Hz), 7.19 (t,
1H, J ¼ 7.4 Hz), 7.24 (d,1H, J ¼ 8.8 Hz, merged with chloroform), 7.57
(d, 1H, J ¼ 7.6 Hz).
D
-tryptophan
d
1.21 (d, 3H, J ¼ 7.2 Hz),
4.1.9.11. Na-(Na-(tert-Butoxycarbonyl)-1-methyl-D-tryptophyl)-1-
methyl-
-tryptophan (57). It was obtained in 40% yield. 1H NMR:
D
d
1.27 (s, 9H), 2.99 (dd, 1H, J ¼ 14.7, 5.4 Hz), 3.09 (dd, 1H, J ¼ 14.3,
6.7 Hz), 3.16 (dd, 1H, J ¼ 14.8, 5.2 Hz), 3.25e3.44 (m, 1H), 3.57 (s,
3H), 3.69 (s, 3H), 4.39 (br s, 1H), 4.76 (dt, 1H, J ¼ 8.1, 5.5 Hz), 5.01 (br
s, 1H), 6.29 (br s, 1H), 6.53 (s, 1H), 6.79 (br s, 1H), 6.91 (s, 1H), 6.97
(br s, 2H), 7.07e7.18 (m, 2H), 7.20 (d, 1H, J ¼ 8.2 Hz), 7.21e7.34 (m,
2H, overlapped with CDCl3), 7.62 (d, 1H, J ¼ 7.9 Hz).
4.1.9.3. Na-((tert-Butoxycarbonyl)-L-valyl)-1-methyl-
(30). It was obtained in 99% yield. 1H NMR:
D
-tryptophan
d
0.77 (d, 3H,
J ¼ 6.8 Hz), 0.81 (d, 3H, J ¼ 6.4 Hz), 1.38 (s, 9H), 1.84e1.92 (m, 1H),
3.30e3.32 (m, 1H), 3.66e3.77 (m, 4H), 4.08e4.12 (m, 1H),
4.88e4.92 (m, 1H), 5.23 (d, 1H, J ¼ 9.2 Hz), 6.66 (d, 1H, J ¼ 7.2 Hz),
6.92 (s, 1H), 7.09 (t,1H, J ¼ 7.4 Hz), 7.20 (t,1H, J ¼ 7.6 Hz), 7.26 (d, 1H,
J ¼ 8.4 Hz, overlapped with CDCl3), 7.62 (d, 1H, J ¼ 8.0 Hz).
4.1.10. Synthesis of Na-((S)-5-(tert-butoxy)-2-((tert-
butoxycarbonyl)amino)-5-oxopentanoyl)-1-methyl-D-tryptophan
(33)
tert-Butyl (S)-5-(((R)-1-(benzyloxy)-3-(1-methyl-1H-indol-3-
yl)-1-oxopropan-2-yl)amino)-4-((tert-butoxycarbonyl)amino)-5-
oxopentanoate (32) (800 mg, 1.38 mmol) was suspended in MeOH
(8 mL) and THF (8 mL). After cooling to 0 ꢀC, NaOH solution (2.4 mL,
2 M) was added and the reaction mixture was stirred for 1 h. The
solution was acidified with 1 M HCl to pH ¼ 4 and the solvents were
concentrated under reduced pressure (40 ꢀC). The solution was
partitioned between water and DCM in a separatory funnel and the
organic layer was collected. The aqueous layer was extracted with
DCM (2 ꢃ 15 mL) and the combined organic layer was washed with
water and brine. Chromatographic purification afforded the desired
4.1.9.4. Na-((tert-Butoxycarbonyl)-D-glutaminyl)-1-methyl-
phan (36). It was obtained in 83% yield. 1H NMR:
1.34 (s, 9H), 1.59
D-trypto-
d
(dd, 1H, J ¼ 14.1, 7.9 Hz), 1.73e1.77 (m, 1H), 1.94e2.04 (m, 2H), 3.02
(dd, 1H, J ¼ 14.6, 7.9 Hz), 3.13 (dd, 1H, J ¼ 14.5, 5.2 Hz), 3.69 (s, 3H),
3.90e3.96 (m, 1H), 4.40e4.45 (m, 1H), 6.72 (s, 1H), 6.80 (d, 1H,
J ¼ 8.3 Hz), 6.96e7.02 (m, 1H), 7.05 (s, 1H), 7.10 (ddd, 1H, J ¼ 8.2, 7.0,
1.1 Hz), 7.18 (s, 1H), 7.34 (d, 1H, J ¼ 8.2 Hz), 7.51 (d, 1H, J ¼ 7.9 Hz),
7.98 (d, 1H, J ¼ 7.9 Hz), 12.70 (br s, 1H).
4.1.9.5. Na-((tert-Butoxycarbonyl)-L-isoleucyl)-1-methyl-
phan (39). It was obtained in 92% yield. 1H NMR:
0.75e0.88 (m,
D-trypto-
d
product (0.502 g, 72%). 1H NMR:
d 1.38 (s, 9H), 1.44 (s, 9H),
8H), 1.37 (s, 9H), 1.62e1.70 (m, 1H), 3.13e3.17 and 3.30e3.32 (two
m, 2H), 3.65 and 3.70 (2 s, 3H), 4.89e4.92 (m, 1H), 5.33 (d, 1H,
J ¼ 9.2 Hz), 6.79 (t, 1H, J ¼ 7.1 Hz), 6.92 (s, 1H), 7.08 (t, 1H, J ¼ 7.4H),
7.19 (t, 1H, J ¼ 7.7 Hz), 7.25 (d, 1H, J ¼ 6.8 Hz), 7.56 and 7.62 (2 d, 1H,
J ¼ 8.0 Hz).
1.68e1.81 (m, 1H), 1.84e1.99 (m, 1H), 2.12e2.33 (m, 3H), 3.23e3.42
(m, 2H), 4.23 (s, 3H), 4.86 (d, 1H, J ¼ 6.9 Hz), 5.41 (d, 1H, J ¼ 8.6 Hz),
6.83 (d, 1H, J ¼ 7.5 Hz), 6.93 (s, 1H), 7.09 (dt, 1H, J ¼ 8.0, 1.2 Hz), 7.18
(t, 1H, J ¼ 7.8 Hz), 7.23 (apparent d overlapped with CDCl3, 1H), 7.60
(d, 1H, J ¼ 7.9 Hz).
4.1.9.6. Na-((tert-Butoxycarbonyl)-L-leucyl)-1-methyl-
(42). It was obtained in 87% yield. 1H NMR:
0.76e0.96 (m, 6H),
1.39 (s, 9H), 1.40e1.54 (m, 3H), 3.29 (dd, 1H, J ¼ 15.1, 5.3 Hz), 3.40
(dd, 1H, J ¼ 14.9, 5.7 Hz), 3.70 (s, 3H), 4.41 (td, 1H, J ¼ 9.3, 5.4 Hz),
4.86 (q, 1H, J ¼ 6.7, 5.8 Hz), 5.26 (d, 1H, J ¼ 9.1 Hz), 6.88 (br s, 1H),
7.05e7.11 (m, 1H), 7.14e7.28 (m, 3H), 7.59 (d, 1H, J ¼ 7.9 Hz).
D
-tryptophan
4.1.11. General method for Boc deprotection
d
HCl solution (1.77 mL, 4.0 M solution in dioxane) was added to a
solution of appropriate Boc protected amine (0.707 mmol) in
dioxane (2 mL) at 0 ꢀC. The solution was allowed to warm to rt and
stirred vigorously for 2.5e18 h. The solvent was removed using
rotary evaporator. The solid was diluted with dry ether (15 mL) and
the product was filtered to afford the crude product. The crude was
dried under high vacuum to afford the desired product.
4.1.9.7. Na-(N2,N6-Bis(tert-butoxycarbonyl)-L-lysyl)-1-methyl-
tryptophan (45). It was obtained in 91% yield. 1H NMR:
1.05e1.20
D-
d
(m, 2H), 1.37 (s, 9H), 1.44 (s, 9H), 1.65e1.80 (m, 2H), 2.948e3.06 (m,
2H), 3.15e3.51 (m, 2H), 3.69 (s, 3H), 3.84e4.04 (m, 1H), 4.15 (d, 1H,
J ¼ 7.6 Hz), 4.69 (s, 1H), 4.85 (d, 1H, J ¼ 6.6 Hz), 5.43 (s, 1H),
5.73e6.18 (m, 2H), 6.91 (s, 1H), 7.06 (t, 1H, J ¼ 7.4 Hz), 7.18 (t, 1H,
J ¼ 7.5 Hz), 7.24 (d, 1H, J ¼ 8.3 Hz), 7.60 (d, 1H, J ¼ 7.9 Hz).
4.1.11.1. Piperidin-4-ylmethyl 1-methyl-D-tryptophanate dihydro-
chloride (12-Cl). It was obtained in 50% yield. LCMS (ESI, m/z):
316.21 [M þ H]þ. 1H NMR (DMSO‑d6):
d 1.16e1.34 (m, 2H), 1.41 (d,
1H, J ¼ 13.6 Hz), 1.53 (d, 1H, J ¼ 13.6 Hz), 1.61e1.66 (m, 1H),
2.66e2.70 (m, 2H), 3.08e3.16 (m, 2H), 3.22e3.28 (m, 1H),
3.36e3.44 (m, 1H), 3.74 (s, 3H), 3.78e3.88 (m, 2H), 4.12e4.17 (m,
1H), 7.05 (t,1H, J ¼ 7.4 Hz), 7.15 (t,1H, J ¼ 7.4 Hz), 7.24 (s,1H), 7.40 (d,
1H, J ¼ 8.0 Hz), 7.55 (d, 1H, J ¼ 7.6 Hz), 8.83 (br s, 3H), 9.06 (br s, 1H),
9.34 (br s, 1H).
4.1.9.8. Na-((tert-Butoxycarbonyl)-L-phenylalanyl)-1-methyl-
tophan (48). It was obtained in 75% yield. 1H NMR:
1.30 (s, 9H),
D-tryp-
d
2.81e2.88 (m, 1H), 2.94e3.00 (m, 1H), 3.08 (dd, 1H, J ¼ 14.8, 5.8 Hz),
3.21e3.25 (m, 1H), 3.66 (s, 3H), 4.41 (d, 1H, J ¼ 6.7 Hz), 4.79e4.86
(m, 1H), 5.13 (d, 1H, J ¼ 8.3 Hz), 6.56 (d, 1H, J ¼ 6.5 Hz), 6.63 (s, 1H),
6.95e7.25 (m, 8H), 7.46 (d, 1H, J ¼ 7.9 Hz).
4.1.11.2. 2-(Piperidin-4-yl)ethyl 1-methyl-D-tryptophanate dihydro-
chloride (14-Cl). It was obtained in 64% yield. LCMS (ESI, m/z):
330.41 [M þ H]þ. 1H NMR (DMSO‑d6):
d 1.24e1.45 (m, 5H), 1.60 (d,
4.1.9.9. Na-((tert-Butoxycarbonyl)-L-tryptophyl)-1-methyl-
phan (51). It was obtained in 91% yield. 1H NMR:
1.35 (s, 9H),
D
-trypto-
2H, J ¼ 13.2 Hz), 2.64e2.72 (m, 2H), 3.11e3.14 (m, 2H), 3.25 (dd, 1H,
J ¼ 14.4, 7.6 Hz), 3.33e3.83 (m, 1H, merged with H2O from DMSO),
3.75 (s, 3H), 3.99e4.08 (m, 2H), 4.15 (t, 1H, J ¼ 6.6 Hz), 7.04 (t, 1H,
J ¼ 7.4 Hz), 7.16 (t, 1H, J ¼ 7.6 Hz), 7.24 (s, 1H), 7.42 (d, 1H, J ¼ 8.0 Hz),
7.53 (d, 1H, J ¼ 8.0 Hz), 8.75 (br s, 3H), 8.95 (br s, 1H), 9.16 (br s, 1H).
d
2.90e3.25 (m, 4H), 3.50 (s, 3H), 3.71e3.79 (m, 1H), 4.31e4.55 (m,
1H), 4.62e4.76 (m, 1H), 6.45 (s, 1H), 6.70e6.91 (m, 1H), 6.98e7.57
(m, 8H), 8.02 (br s, 1H).
4.1.9.10. Na-((tert-Butoxycarbonyl)-D-tryptophyl)-1-methyl-
tophan (54). It was obtained in 84% yield. 1H NMR:
1.31 (s, 9H),
D
-tryp-
4.1.11.3. 2-(Dimethylamino)ethyl 1-methyl-D-tryptophanate dihy-
drochloride (16-Cl). It was obtained in 42% yield. LCMS (ESI, m/z):
d

16
S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
289.4 [M þ H]þ. 1H NMR (MeOH-d4):
d
2.69 (s, 3H), 2.77 (s, 3H),
4.1.11.11. Na-(L-Isoleucyl)-1-methyl-
Cl). It was obtained in 94% yield. LCMS (ESI, m/z): 332.41 [M þ
H]þ. 1H NMR (DMSO‑d6):
0.55e0.65 (m, 6H), 0.71e0.75 (m, 1H),
1.03e1.12 (m, 1H), 1.57e1.63 (m, 1H), 2.99 (dd, 1H, J ¼ 14.6, 9.8 Hz),
3.19 (dd, 1H, J ¼ 14.6, 4.7 Hz), 3.61e3.63 (m, 1H), 3.69 (s, 3H),
4.58e4.64 (m, 1H), 7.0 (t, 1H, J ¼ 7.6 Hz), 7.08e7.13 (m, 2H), 7.35 (d,
1H, J ¼ 8.2 Hz), 7.53 (d, 1H, J ¼ 7.9 Hz), 8.10 (br s, 3H), 8.72 (d, 1H,
J ¼ 8.1 Hz).
D
-tryptophan hydrochloride (40-
3.30e3.31 (m, 4H), 3.46 (dd, 2H, J ¼ 6.7, 2.1 Hz), 3.81 (s, 3H),
4.33e4.38 (m, 1H), 4.46 (t, 1H, J ¼ 6.6 Hz), 4.51e4.57 (m, 1H),
7.09e7.13 (m, 1H), 7.20e7.24 (m, 2H), 7.40 (d, 1H, J ¼ 8.0 Hz), 7.58 (d,
1H, J ¼ 8.0).
d
4.1.11.4. 2-(Tetrahydro-2H-pyran-4-yl)ethyl 1-methyl-D-tryptopha-
nate hydrochloride (18-Cl). It was obtained in 94% yield. LCMS (ESI,
m/z): 331.41 [M þ H]þ. 1H NMR(DMSO‑d6):
d 0.93e1.11 (m, 2H), 1.18
4.1.11.12. Na-(L-Leucyl)-1-methyl-
Cl). It was obtained in 95% yield. LCMS (ESI, m/z): 332.31 [M þ
H]þ. 1H NMR (DMSO‑d6):
D
-tryptophan hydrochloride (43-
(d, 1H, J ¼ 6.2 Hz), 1.26e1.43 (m, 4H), 3.14 (d, 2H, J ¼ 11.2 Hz), 3.23
(dd, 1H, J ¼ 14.7, 7.7 Hz), 3.29e3.39 (m, 2H), 3.69e3.78 (m, 4H), 4.04
(d, 2H, J ¼ 6.2 Hz), 4.17 (t, 1H, J ¼ 6.6 Hz), 7.04 (ddd, 1H, J ¼ 8.0, 7.1,
1.0 Hz), 7.16 (ddd, 1H, J ¼ 8.3, 7.0,1.2 Hz), 7.23 (s, 1H), 7.42 (d, 1H,
J ¼ 8.2 Hz), 7.53 (dd, 1H, J ¼ 8.1, 1.4 Hz), 8.69 (br s, 3H).
d
0.68 (t, 6H, J ¼ 5.5 Hz).1.17e1.34 (m, 3H),
2.99 (dd, 1H, J ¼ 14.5, 9.6 Hz), 3.20 (dd, 1H, J ¼ 14.6, 4.7 Hz),
3.34e3.40 (m, 3H), 3.68 (s, 3H), 4.52e4.62 (m, 1H), 6.99 (t, 1H,
J ¼ 7.4 Hz), 7.08e7.16 (m, 2H), 7.35 (d, 1H, J ¼ 8.2 Hz), 7.54 (d, 1H,
J ¼ 7.9 Hz), 8.17 (br s, 2H), 8.85 (d, 1H, J ¼ 8.3 Hz).
4.1.11.5. 1-Methyl-D-tryptophyl-
L
-valine
hydrochloride
(20-Cl).
It was obtained in 40% yield. LCMS (ESI, m/z): 378.42 [M þ H]þ. 1H
4.1.11.13. Na-(L-Lysyl)-1-methyl-
Cl). It was obtained in 87% yield. LCMS (ESI, m/z): 347.42 [M þ
H]þ. 1H NMR (DMSO‑d6):
0.88e1.13 (m, 2H), 1.33e1.56 (m, 4H),
D
-tryptophan dihydrochloride (46-
NMR (DMSO‑d6):
d 0.71e0.77 (m, 6H), 1.91e2.00 (m, 1H), 3.08 (dd,
1H, J ¼ 14.4, 8.4 Hz), 3.23 (dd, 1H, J ¼ 14.4, 8.4 Hz), 3.73 (s, 3H),
4.12e4.17 (m, 2H), 7.06 (t, 1H, J ¼ 7.4 Hz), 7.17 (t, 1H, J ¼ 7.8 Hz), 7.20
(s, 1H), 7.40 (d, 1H, J ¼ 8.4 Hz), 7.74 (d, 1H, J ¼ 8.0 Hz), 8.2 (br s, 3H),
8.74 (d, 1H, J ¼ 8.4 Hz).
d
2.54 (t, 2H, J ¼ 7.1 Hz), 2.95e3.10 (m, 1H), 3.15e3.24 (m, 1H), 3.42
(apparent q overlapping with H2O, 1H, J ¼ 7.0 Hz), 3.73 (s, 3H),
4.50e4.67 (m, 1H), 7.01 (t, 1H, J ¼ 7.5 Hz), 7.06e7.18 (m, 2H), 7.38 (d,
1H, J ¼ 8.3 Hz), 7.55 (d, 1H, J ¼ 7.9 Hz), 8.02 (br s, 3H), 8.20 (br s, 3H),
8.83 (d, 1H, J ¼ 8.1 Hz), 12.93 (br s, 1H).
4.1.11.6. Na-Glycyl-1-methyl-
-tryptophan hydrochloride (25-Cl).
D
It was obtained in 87% yield. LCMS (ESI, m/z): 276.31 [M þ H]þ. 1H
4.1.11.14. Na-(L-Phenylalanyl)-1-methyl-
(49-Cl). It was obtained in 91% yield. LCMS (ESI, m/z): 366.32 [M þ
H]þ. 1H NMR (DMSO‑d6):
D-tryptophan hydrochloride
NMR (DMSO‑d6): d 3.02e3.08 (m, 1H), 3.17e3.22 (m,1H), 3.48e3.60
(m, 2H), 3.74 (s, 3H), 4.55e4.58 (m, 1H), 7.03 (t, 1H, J ¼ 7.8 Hz),
7.12e7.18 (m, 2H), 7.38 (d, 1H, J ¼ 8.0 Hz), 7.55 (d, 1H, J ¼ 8.0 Hz),
8.13 (br s, 3H), 8.76 (d, 1H, J ¼ 8.0 Hz), 12.87 (br s, 1H).
d
2.78 (dd, 1H, J ¼ 13.9, 7.1 Hz), 2.89e2.97
(m, 2H), 3.10 (dd, 1H, J ¼ 14.5, 5.3 Hz), 3.35 (br s, 3H), 3.47 (s, 3H),
4.05 (dd, 1H, J ¼ 7.1, 5.6 Hz), 4.51 (td, 1H, J ¼ 8.2, 5.3 Hz), 6.92e6.94
(m, 2H), 6.99e7.18 (m, 6H), 7.36 (dt, 1H, J ¼ 8.3, 0.9 Hz), 7.56 (dt,
J ¼ 8.0, 0.9 Hz, 1H), 8.89 (d, 1H, J ¼ 8.1 Hz).
4.1.11.7. Na-(L-Alanyl)-1-methyl-
-tryptophan hydrochloride (28-Cl).
D
It was obtained in 44% yield. LCMS (ESI, m/z): 290.31 [M þ H]þ. 1H
NMR (DMSO‑d6):
d 1.18 (d, 3H), 3.02e3.06 (m, 1H), 3.17e3.23 (m,
4.1.11.15. Na-(L-Tryptophyl)-1-methyl-
(52-Cl). It was obtained in 90% yield. LCMS (ESI, m/z): 405.42 [M þ
H]þ. 1H NMR (DMSO‑d6):
D-tryptophan
hydrochloride
1H), 3.72 (s, 3H), 4.05e4.09 (m, 1H), 4.57e4.62 (m, 1H), 7.02 (t, 1H,
J ¼ 7.6 Hz), 7.12e7.15 (m, 2H), 7.38 (d, 1H, J ¼ 8.0 Hz), 7.52 (d, 1H,
J ¼ 7.6 Hz), 8.16 (br s, 3H), 8.88e8.92 (m, 1H).
d
2.88 (dd, 1H, J ¼ 14.7, 8.2 Hz), 2.98 (dd,
1H, J ¼ 14.5, 7.9 Hz), 3.08 (dt, 2H, J ¼ 14.7, 5.0 Hz), 3.63 (s, 3H), 4.06
(br s, 1H), 4.55 (q, 1H, J ¼ 7.9), 6.87 (dd, 1H, J ¼ 8.0, 7.0 Hz), 6.97 (s,
1H), 7.01 (t, 1H, J ¼ 7.4 Hz), 7.06 (t, 1H, J ¼ 7.4 Hz), 7.08e7.15 (m, 2H),
7.34 (d, 2H, J ¼ 8.2 Hz), 7.56 (dd, 2H, J ¼ 8.0, 5.1 Hz), 8.09 (s, 3H), 8.95
(d, 1H, J ¼ 8.1 Hz), 11.02 (s, 1H).
4.1.11.8. Na-(L-Valyl)-1-methyl-
-tryptophan hydrochloride (31-Cl).
D
It was obtained in 92% yield. LCMS (ESI, m/z): 318.42 [M þ H]þ. 1H
NMR (DMSO‑d6):
d
0.54 (d, 3H, J ¼ 7.2 Hz), 0.72 (d, 3H, J ¼ 6.8 Hz),
1.89e1.94 (m, 1H), 3.01 (dd, 1H, J ¼ 14.8, 9.6 Hz), 3.22 (dd, 1H,
J ¼ 14.6, 5.0 Hz), 3.56e3.65 (m, 1H), 3.70 (s, 3H), 4.61e4.66 (m, 1H),
7.01 (t, 1H, J ¼ 7.6 Hz), 7.12 (s, 1H), 7.12 (t, 1H, J ¼ 7.6 Hz), 7.36 (t, 1H,
J ¼ 8.0 Hz), 7.56 (d, 1H, J ¼ 8.0 Hz), 8.09 (br s, 3H), 8.78 (d, 1H,
J ¼ 8.4 Hz), 12.8 (br s, 1H).
4.1.11.16. Na-(D-Tryptophyl)-1-methyl-
D-tryptophan hydrochloride
(55-Cl). It was obtained in 95% yield. LCMS (ESI, m/z): 405.42
[MþH]þ. 1H NMR (Methanol-d4):
d
3.15 (d, 1H, J ¼ 8.5 Hz), 3.19 (d,
1H, J ¼ 8.5 Hz), 3.36 (d, 1H, J ¼ 4.9 Hz), 3.37e3.41 (m, 1H), 3.71 (s,
3H), 4.06 (t, 1H, J ¼ 3.6 Hz), 4.74 (s, 1H), 6.93 (s, 1H), 7.02 (t, 1H,
J ¼ 6.2 Hz), 7.04e7.07 (m, 1H), 7.14 (td, 2H, J ¼ 7.9, 1.7 Hz), 7.20 (s,
1H), 7.22 (d, J ¼ 8.1 Hz, 1H), 7.30 (d, 1H, J ¼ 8.2 Hz), 7.38 (d, 1H,
J ¼ 8.1 Hz), 7.56 (d,1H, J ¼ 8.0 Hz), 7.65 (d,1H, J ¼ 7.9 Hz), 7.70 (d,1H,
J ¼ 8.2 Hz).
4.1.11.9. (S)-4-Amino-5-(((R)-1-carboxy-2-(1-methyl-1H-indol-3-yl)
ethyl)amino)-5-oxopentanoic acid hydrochloride (34-Cl). It was ob-
tained in 85% yield. LCMS (ESI, m/z): 348.42 [M þ H]þ. 1H NMR
(DMSO‑d6):
d (mixture of rotamers) 1.73e2.21 (m, 4H), 2.93e3.12
(m, 1H), 3.14e3.27 (m, 1H), 3.70 (s, 3H), 3.83 (q, 1H, J ¼ 5.8 Hz),
4.53e4.72 (m, 1H), 7.01 (tt, 1H, J ¼ 7.3, 3.7 Hz), 7.07e7.19 (m, 2H),
7.35 (dt, 1H, J ¼ 7.5, 3.5 Hz), 7.44e7.61 (m, 1H), 8.42 (br s, 3H),
8.83e9.10 (m, 1H).
4.1.11.17. 1-Methyl-Na-(1-methyl-D-tryptophyl)-
D
-tryptophan
hy-
drochloride (58-Cl). It was obtained in 92% yield. LCMS (ESI, m/z):
419.42 [M þ H]þ. 1H NMR (DMSO‑d6):
3.10 (td, 2H, J ¼ 15.5, 7.9 Hz),
d
3.24 (ddd, 2H, J ¼ 17.5, 15.1, 5.9 Hz), 3.72 (s, 2H), 3.73 (s, 4H), 4.02
(dd, 1H, J ¼ 8.3, 5.1 Hz), 4.58 (q, 1H, J ¼ 7.0 Hz), 7.04 (td, 2H, J ¼ 7.4,
4.2 Hz), 7.09e7.23 (m, 4H), 7.40 (t, 2H, J ¼ 8.1 Hz), 7.58 (d, 1H,
J ¼ 7.9 Hz), 7.74 (d,1H, J ¼ 7.9 Hz), 8.11 (s,1H), 8.97 (d,1H, J ¼ 7.7 Hz),
12.82 (br s, 1H).
4.1.11.10. Na-(L-Glutaminyl)-1-methyl-
(37-Cl). It was obtained in 97% yield. LCMS (ESI, m/z): 347.32 [M þ
H]þ. 1H NMR (DMSO‑d6):
1.79e1.84 (m, 2H), 1.95e2.06 (m, 2H),
D-tryptophan hydrochloride
d
3.04 (dd, 1H, J ¼ 14.6, 8.5 Hz), 3.19 (dd, 1H, J ¼ 14.6, 5.2 Hz),
3.49e3.35 (m, 2H), 3.70 (s, 3H), 3.78e3.88 (m, 1H), 4.53 (td, 1H,
J ¼ 8.3, 5.2 Hz), 6.93 (s, 1H), 7.00 (ddd, 1H, J ¼ 8.0, 7.0, 1.0 Hz),
7.16e7.07 (m, 2H), 7.35 (dt, 1H, J ¼ 8.3, 0.9 Hz), 7.38 (s, 1H), 7.54 (dt,
1H, J ¼ 7.9, 1.0 Hz), 8.28 (d, 2H, J ¼ 4.2 Hz), 8.87 (d, 1H, J ¼ 8.1 Hz).
4.1.11.18. Ethyl Na-(L-glutaminyl)-1-methyl-D-tryptophanate hydro-
chloride (59-Cl). It was obtained in 59% yield. LCMS (ESI, m/z):
375.42 [M
þ
H]þ. 1H NMR (DMSO‑d6):
d 1.08 (t, 3H,

S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
17
J ¼ 7.1 Hz),1.81e1.97 (m, 2H), 2.01e2.12 (m, 2H), 3.07 (dd, 1H,
J ¼ 14.4, 8.4 Hz), 3.16 (dd, 1H, J ¼ 14.4, 6.0 Hz), 3.70 (s, 3H), 3.82 (t,
1H, J ¼ 6.0 Hz), 4.03 (q, 2H, J ¼ 7.1 Hz), 4.53 (q,1H, J ¼ 7.0 Hz), 6.93 (s,
1H), 7.02 (ddd, 1H, J ¼ 7.9, 7.0, 1.0 Hz), 7.09e7.14 (m, 2H), 7.35 (d, 1H,
J ¼ 8.2 Hz), 7.40 (s, 1H), 8.24 (br s, 3H), 9.01 (d, 1H, J ¼ 7.2 Hz).
trifluoroacetic acid the reaction mixture was diluted with dry HCl
solution (1 mL, 4 M solution in dioxane) and the mixture was
stirred for 10 min. The solvent was evaporated under reduced
pressure, the product was triturated with ethanol:ether (10:90,
15 mL) and the product was filtered and washed with dry ether
(10 mL).The product was dried under reduced pressure to afford 10-
Cl (0.190 g, 61%). LCMS (ESI, m/z): 306.31 [M þ H]þ. 1H NMR (MeOH-
4.1.11.19. Ethyl Na -(L-isoleucyl)-1-methyl-D-tryptophanate hydro-
chloride (60-Cl). It was obtained in 93% yield. LCMS (ESI, m/z):
d4):
d
3.22e3.28 (m, 1H), 3.43 (dd, 1H, J ¼ 15.4, 4.7 Hz), 3.70 (s, 3H),
360.42 [M þ H]þ. 1H NMR (DMSO‑d6):
d
0.60e0.66 (m, 6H),
4.23 (t, 1H, J ¼ 3.9 Hz), 4.35 (dd, 1H, J ¼ 8.0, 4.9 Hz), 4.60 (d, 2H,
J ¼ 3.8 Hz), 6.99e7.04 (m, 1H), 7.05 (s, 1H), 7.09e7.16 (m, 1H), 7.29
(d, 1H, J ¼ 8.3 Hz), 7.50 (d, 1H, J ¼ 7.9 Hz).
0.75e0.82 (m, 2H), 1.12 (t, 3H, J ¼ 7.1 Hz, 4H), 1.63 (br s, 1H), 3.02
(dd, 1H, J ¼ 14.6, 9.4 Hz), 3.17 (dd, 1H, J ¼ 14.6, 5.2 Hz), 3.61 (br s,
1H), 3.69 (s, 3H), 4.07 (q, 2H, J ¼ 7.1 Hz), 4.62 (br s, 1H), 7.01 (t, 1H,
J ¼ 7.5 Hz), 7.10e7.14 (m, 2H), 7.36 (d, 1H, J ¼ 8.2 Hz), 7.49 (d, 1H,
J ¼ 7.9 Hz), 8.00 (br s, 2H), 8.85 (br s, 1H).
4.1.14. General method for the generation of free base form of
indoximod prodrugs
Conditions A: Appropriate prodrug 8-Cl, 12-Cl 59-Cl, 62-Cl or
64-Cl, (1.25 mmol) was stirred in aqueous saturated NaHCO3
(25 mL) solution for 5e10 min at 0 ꢀC and the product was extracted
with CH2Cl2 (3 ꢃ 35 mL) or CF3CH2OH:CH2Cl2 (5:95; 3 ꢃ 35 mL).
The combined organic extract was dried over Na2SO4 and concen-
trated to afford the desired free base.
Conditions B: 25-Cl and 46-Cl were converted to the corre-
sponding free bases with Dowex sulfonic acid resin. The compound
was eluted with 20% aqueous NH4OH solution and the aqueous
solution was concentrated under reduced pressure, the crude was
co-evaporated with ethanol three times to yield the desired free
base.
4.1.11.20. Ethyl Na-(L-phenylalanyl)-1-methyl-D-tryptophanate hy-
drochloride (61-Cl). It was obtained in 60% yield. LCMS (ESI, m/z):
394.42 [M þ H]þ. 1H NMR (DMSO‑d6):
d
1.15 (t, 3H, J ¼ 7.1 Hz), 2.52
(dd, 1H, J ¼ 13.7, 9.9 Hz), 3.17e3.23 (m, 3H), 3.46 (dd, 1H, J ¼ 9.9,
4.1 Hz), 3.64 (s, 3H), 4.03e4.11 (m, 2H), 4.83 (dt, 1H, J ¼ 8.4, 5.6 Hz),
6.72 (s, 1H), 6.99 (ddd, 1H, J ¼ 8.0, 6.9, 1.1 Hz), 7.31e7.05 (m, 7H),
7.45 (d, 1H, J ¼ 7.9 Hz), 7.61 (d, 1H, J ¼ 8.4 Hz).
4.1.11.21. Ethyl Na -(L-leucyl)-1-methyl-D-tryptophanate hydrochlo-
ride (62-Cl, NLG802). It was obtained in 93% yield. HRMS (ESI, m/z):
[M þ H]þ calculated for C20H30N3Oþ3 , 360.2282; found 360.2283.
Elemental analysis: C 60.67%, H 7.71%, N 10.71%, Cl 8.98%. 1H NMR
(DMSO‑d6):
d
0.73 (t, 6H, J ¼ 5.7 Hz),1.16 (t, 3H, J ¼ 7.1 Hz),1.23e1.37
4.1.15. General method for the generation of mono and di phosphate
salts of indoximod prodrugs
(m, 3H), 3.03 (dd, 1H, J ¼ 14.5, 9.4 Hz), 3.20 (dd, 1H, J ¼ 14.5, 5.3 Hz),
3.72 (s, 4H), 4.10 (q, 2H, J ¼ 7.1 Hz), 4.57e4.62 (m, 1H), 7.0e7.04 (m,
1H), 7.12e7.16 (m, 2H), 7.38 (d, 1H, J ¼ 8.2 Hz), 7.52 (d, 1H,
J ¼ 7.9 Hz), 8.20 (3H, s), 9.01 (d, 1H, J ¼ 8.1 Hz). 13C NMR (100 MHz,
To a solution of prodrug in its free base form (0.747 mmol) in
EtOH (5 mL) at 0 ꢀC, was added phosphoric acid (0.747 mmol; a
solution in EtOH 1 mL) or (1.494 mmol in case of diamine) and the
mixture was allowed to warm to RT and stirred for 5e18 h. The
solvent was removed under reduced pressure and the residue was
diluted with methyl tert-butylether (10 mL), after stirring for 1e5 h
the solid was filtered and dried under reduced pressure to afford
the desired product.
DMSO‑d6):
d 14.0, 22.0, 22.3, 23.3, 27.0, 32.3, 50.7, 53.2, 60.7, 108.5,
109.6, 118.2, 118.6, 121.1, 127.3, 128.5, 136.6, 169.0, 171.3.
4.1.11.22. Ethyl Na-(L-methionyl)-1-methyl-D-tryptophanate hydro-
chloride (64-Cl). It was obtained in 90% yield. LCMS (ESI, m/z):
378.42 [M þ H]þ. 1H NMR (DMSO‑d6):
1.69 (t, 3H, J ¼ 7.1 Hz), 2.44
d
(s, 3H), 2.61e2.82 (m, 2H), 3.59 (dd, 1H, J ¼ 14.5, 9.5 Hz), 3.74 (dd,
1H, J ¼ 14.6, 5.0 Hz), 4.27 (s, 3H), 4.37 (s, 1H), 4.63 (q, 2H, J ¼ 7.1 Hz),
5.05e5.22 (m, 1H), 7.56 (t,1H, J ¼ 7.4 Hz), 7.62e7.75 (m, 2H), 7.91 (d,
1H, J ¼ 8.2 Hz), 8.05 (d, 1H, J ¼ 7.8 Hz), 8.86 (s, 2H), 9.60 (d, 1H,
J ¼ 7.8 Hz).
4.1.15.1. (2R)-1-(2,3-Dihydroxypropoxy)-3-(1-methyl-1H-indol-3-
yl)-1-oxopropan-2-aminium
dihydrogen
phosphate
(8-P).
Obtained with 44% yield. 1H NMR (DMSO‑d6):
d
3.07e3.15 (m, 2H),
3.27e3.38 and 3.43e3.50 (m, 2H), 3.60e3.68 (m, 1H), 3.73 (s, 3H),
3.84 (br s, 1H), 3.90e3.96 (m, 1H), 4.02e4.12 (m, 1H), 6.95 (br s, 3H),
7.02 (ddd, 1H, J ¼ 8.0.70.1.0 Hz), 7.11e7.19 (m, 2H), 7.38 (dt, 1H,
J ¼ 8.3, 0.9 Hz), 7.49e7.56 (m, 1H).
4.1.12. Synthesis of 2,3-dihydroxypropyl 1-methyl-D-tryptophanate
hydrochloride (8-Cl)
TFA (16.3 mL, 212.7 mmol) and water (0.958 g, 53.18 mmol) was
added to a solution of 7 (11.5 g, 26.59 mmol) in THF (100 mL) at 0 ꢀC,
the cooling bath was removed, and the mixture was stirred at rt for
2 h. HCl (13.3 mL, 53.18 mmol; 4.0 M solution in dioxane) was
added and continued stirring for 1 h. The reaction was stirred at
40 ꢀC for 45 min. The precipitated white solid was filtered and
washed with MTBE to afford 8-Cl (4.5 g, 51%). LCMS (ESI, m/z):
4.1.15.2. Piperidin-4-ylmethyl 1-methyl-D-tryptophanate dihydrogen
phosphate (12-P). Obtained with 79% yield. 1H NMR (DMSO‑d6):
d
1.35e1.56 (m, 4H), 1.63e1.68 (m, 1H), 2.61e2.73 (m, 2H),
3.09e3.26 (m, 4H), 3.73 (s, 3H), 3.81 (dd, 1H, J ¼ 5.1, 10.9 Hz), 3.88
(dd,1H, J ¼ 5.1,11.1 Hz), 3.95 (t,1H, J ¼ 6.7 Hz), 7.02 (t,1H, J ¼ 7.4 Hz),
7.09e7.17 (m, 1H), 7.21 (s, 1H), 7.38 (d, 1H, J ¼ 8.2 Hz), 7.49 (d, 1H,
J ¼ 7.9 Hz), 8.44 (br s, 10H).
293.31 [M þ H]þ. 1H NMR (DMSO‑d6):
d 3.32e3.40 (m, 1H),
3.44e3.52 (m, 3H), 3.76e3.86 (m, 4H), 4.16e4.37 (m, 3H), 7.10 (t,
1H, J ¼ 7.4 Hz), 7.14 (s, 1H), 7.19 (t, 1H, J ¼ 7.6 Hz), 7.38 (d, 1H,
J ¼ 8.2 Hz), 7.58 (d, 1H, J ¼ 7.9 Hz).
4.1.15.3. (R)-2-((1-Carboxy-2-(1-methyl-1H-indol-3-yl)ethyl)
amino)-2-oxoethan-1-aminium dihydrogen phosphate (25-P).
Obtained with 80% yield. 1H NMR(DMSO‑d6):
d 3.01e3.05 (m, 1H),
3.18e3.22 (m, 1H), 3.42e3.56 (m, 2H), 3.72 (s, 3H), 4.42e4.50 (m,
1H), 7.01e7.14 (m, 3H), 7.33e7.37 (m, 1H), 7.51e7.55 (m, 1H), 8.44
(br s, 9H), 8.65 (s, 1H).
4.1.13. Synthesis of O-(1-methyl-D-tryptophyl)-L-serine
dihydrochloride (10-Cl)
HCl (2 mL, 4 M solution in dioxane) was added to a solution of 9
(0.450 g, 824.66 mmol) in CH2Cl2 (10 mL) at 0 ꢀC and the solution
was allowed to warm to rt. After stirring for 5 h, the solvent was
evaporated, and the reaction was diluted with trifluoroacetic acid
(8 mL) and the solution was stirred for 7 h at rt. After evaporating
4.1.15.4. (S)-6-(((R)-1-Carboxy-2-(1-methyl-1H-indol-3-yl)ethyl)
amino)-6-oxohexane-1,5-diaminium dihydrogen phosphate (46-P).
Obtained with 81% yield. 1H NMR (D2O):
d
0.39e0.78 (m, 2H), 1.21
(ddd, 2H, J ¼ 9.1, 6.8, 2.6 Hz), 1.28e1.49 (m, 2H), 2.39 (td, 2H, J ¼ 7.4,

18
S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
3.8 Hz), 3.08 (dd, 1H, J ¼ 15.0, 10.9 Hz), 3.45 (ddd, 1H, J ¼ 15.1, 4.5,
1.0 Hz), 3.74 (s, 3H), 3.79 (t, 1H, J ¼ 6.7 Hz), 4.68e4.77 (m, 1H), 7.14
(d, 1H, J ¼ 0.8 Hz), 7.14e7.20 (m, 1H), 7.28 (ddd, 1H, J ¼ 8.3, 7.1,
1.1 Hz), 7.41e7.47 (m, 1H), 7.70 (dd, 1H, J ¼ 7.9, 0.9 Hz).
4.1.16.3. (S)-1-(((R)-1-Ethoxy-3-(1-methyl-1H-indol-3-yl)-1-
oxopropan-2-yl)amino)-4-methyl-1-oxopentan-2-aminium meth-
anesulfonate (62-M). It was obtained in 69% yield. LCMS (ESI, m/z):
360.42 [M þ H]þ. 1H NMR (DMSO‑d6):
0.73 (dd, 6H, J ¼ 8.2, 6.3 Hz,
d
6H), 1.16 (t, 3H, J ¼ 7.1, 7.1 Hz), 1.24 (t, 2H, J ¼ 7.1, 7.1 Hz), 1.32 (dt, 1H,
J ¼ 13.0, 6.7, 6.7 Hz), 2.29 (s, 3H), 3.03 (dd, 1H, J ¼ 14.5, 9.3 Hz), 3.20
(dd,1H, J ¼ 14.5, 5.3 Hz), 3.72 (s, 3H), 4.11 (q, 2H, J ¼ 7.1 Hz), 4.64 (td,
1H, J ¼ 8.8, 5.5 Hz), 7.02 (t, 1H, J ¼ 7.5 Hz), 7.13 (d, 2H, J ¼ 9.8 Hz),
7.38 (d, 1H, J ¼ 8.2 Hz), 7.52 (d, 1H, J ¼ 7.9 Hz), 8.01 (s, 3H), 8.92 (d,
1H, J ¼ 8.2 Hz, 1H).
4.1.15.5. (S)-5-Amino-1-(((R)-1-ethoxy-3-(1-methyl-1H-indol-3-yl)-
1-oxopropan-2-yl)amino)-1,5-dioxopentan-2-aminium dihydrogen
phosphate (59-P). Obtained with 59% yield. 1H NMR (DMSO‑d6):
d
1.10 (t, 3H, J ¼ 7.0 Hz),1.64e1.70 (m, 1H),1.75e1.85 (m,1H), 2.06 (t,
2H, J ¼ 7.9 Hz), 3.06e3.18 (m, 2H), 3.44 (br s, 1H), 3.72 (s, 3H), 4.04
(q, 2H, J ¼ 7.1 Hz), 4.52 (q, 1H, J ¼ 7.1 Hz), 6.80 (s, 1H), 7.02 (t, 1H,
J ¼ 7.5 Hz), 7.11e7.16 (m, 2H), 7.32e7.38 (m, 2H), 7.50 (d, 1H,
J ¼ 7.9 Hz), 7.82 (br s, 3H), 8.57 (s, 1H).
4.1.16.4. Ethyl Na-((S)-2-( 4-azanyl)-4-methylpentanoyl)-1-methyl-
l
D-tryptophanate besylate (62-B). It was obtained in 68% yield.
LCMS (ESI, m/z): 360.42 [M þ H]þ. 1H NMR (DMSO‑d6):
d 0.73 (dd,
6H, J ¼ 8.2, 6.3 Hz), 1.16 (t, 3H, J ¼ 7.1 Hz), 1.24 (t, 2H, J ¼ 7.3 Hz), 1.32
(dt, 1H, J ¼ 13.0, 6.5 Hz), 2.98e3.09 (m, 1H), 3.20 (dd, 1H, J ¼ 14.5,
5.2 Hz), 3.72 (s, 3H), 4.11 (q, 2H, J ¼ 7.1 Hz), 4.64 (td, 1H, J ¼ 8.9,
5.4 Hz), 6.99e7.05 (m, 1H), 7.09e7.17 (m, 2H), 7.26e7.35 (m, 3H),
7.38 (d, 1H, J ¼ 8.2 Hz), 7.52 (d, 1H, J ¼ 8.0 Hz), 7.59 (dd, 2H, J ¼ 7.7,
1.9 Hz), 8.00 (s, 3H), 8.92 (d, 1H, J ¼ 8.2 Hz).
4.1.15.6. (S)-1-(((R)-1-Ethoxy-3-(1-methyl-1H-indol-3-yl)-1-
oxopropan-2-yl)amino)-4-methyl-1-oxopentan-2-aminium
dihy-
drogen phosphate (62-P). Obtained in 59% yield. LCMS (ESI, m/z):
360.42 [M þ H]þ. 1H NMR (DMSO‑d6):
d
0.77 (dd, 6H, J ¼ 6.5, 6H,
2.2 Hz), 1.1 (t, 3H, J ¼ 7.1, 7.1 Hz), 1.18e1.32 (m, 1H), 1.39e1.50 (m,
1H), 1.39e1.49 (m, 1H), 3.06 (dd, 1H, J ¼ 14.5, 8.4 Hz), 3.17 (dd, 1H,
J ¼ 14.4, 5.4 Hz), 3.40 (dd, 1H, J ¼ 8.6, 5.7 Hz), 3.72 (s, 3H), 4.06 (q,
2H, J ¼ 7.1, 7.1, 7.1 Hz), 4.55 (td, 1H, J ¼ 8.1, 8.1, 5.5 Hz), 5.52 (bs, 8H),
7.02 (t,1H, J ¼ 7.2 Hz), 7.10e7.15 (m, 2H), 7.38 (d, 1H, J ¼ 8.3 Hz), 7.51
(d, 1H, J ¼ 7.9 Hz), 8.62 (d, 1H, J ¼ 7.9 Hz).
4.1.17. General method for the generation of mono, disulfate and
hydrogen sulfate salts of indoximod and indoximod prodrugs
To a solution of each prodrug in it free base form (1.22 mmol) in
dry THF (10 mL) at 0 ꢀC was added sulfuric acid (0.611 mmol or
1.22 mmol) as a solution in THF (2 mL) and the solution was allowed
to warm to rt. After stirring for 2e6 h, the solvent was distilled-off
and the crude was stirred with methyl tert-butyl ether, the solid
was filtered and dried under vacuum to yield the desired product.
4.1.15.7. (S)-1-(((R)-1-Ethoxy-3-(1-methyl-1H-indol-3-yl)-1-
oxopropan-2-yl)amino)-4-(methylthio)-1-oxobutan-2-aminium
dihydrogen phosphate (64-P). Obtained with 70% yield. 1H
NMR(DMSO‑d6):
d
1.13 (t, 3H, J ¼ 7.1 Hz), 1.64e1.72 (m, 1H),
1.73e1.84 (m, 1H), 1.93 (s, 3H), 2.28 (t, 2H, J ¼ 7.9 Hz), 3.08 (dd, 1H,
J ¼ 14.6, 8.5 Hz), 3.18 (dd,1H, J ¼ 14.5, 5.2 Hz), 3.54 (t,1H, J ¼ 6.0 Hz),
3.73 (s, 3H), 4.07 (q, 2H, J ¼ 7.1 Hz), 4.56 (q, 1H, J ¼ 6.8, 6.1 Hz), 7.02
(t, 1H, J ¼ 7.4 Hz), 7.07e7.23 (m, 2H), 7.38 (d, 1H, J ¼ 8.2 Hz), 7.51 (d,
1H, J ¼ 7.9 Hz), 7.98 (br s, 5H), 8.68 (d, 1H, J ¼ 7.7 Hz).
4.1.17.1. (2R)-1-(2,3-Dihydroxypropoxy)-3-(1-methyl-1H-indol-3-
yl)-1-oxopropan-2-aminium sulfate (8-S). Obtained with 43% yield.
1H NMR (DMSO‑d6):
d 3.05e3.19 (m, 2H), 3.29e3.40 and 3.44e3.55
(two m, 2H), 3.62e3.69 (m, 1H), 3.74 (s, 3H), 3.89e3.99 (m, 2H),
4.07e4.12 (m, 1H), 6.25 (br s, 2H), 7.03 (t, 1H, J ¼ 7.7 Hz), 7.11e7.21
(m, 2H), 7.40 (d, 1H, J ¼ 8.1 Hz), 7.51e7.57 (m, 1H).
4.1.16. General method for the generation of mono and di
methanesulfonate and benzenesulfonate salts of indoximod
prodrugs
To a solution of each prodrug in its free base form (0.25 g,
0.723 mmol) in ethanol (10 mL) at rt, was added methanesulfonic
or benzenesulfonic acid (0.723 mmol or 1.446 mmol in case of di-
amines) and the mixture was stirred at rt overnight. Ethanol was
evaporated and the crude product was stirred in methyl tert-butyl
ether for 1e5 h. The precipitate was filtered and dried to yield the
corresponding methanesulfonate or benzenesulfonate salt.
4.1.17.2. (S)-6-(((R)-1-Carboxy-2-(1-methyl-1H-indol-3-yl)ethyl)
amino)-6-oxohexane-1,5-diaminium sulfate (46-S). Obtained with
82% yield. 1H NMR (DMSO‑d6):
d 1.08e1.58 (m, 7H), 2.55e2.71 (m,
2H), 3.03 (dd, 1H, J ¼ 14.6, 8.8 Hz), 3.21 (dd, 1H, J ¼ 14.6, 4.9 Hz),
3.63 (s, 1H), 3.72 (s, 3H), 4.53 (d, 1H, J ¼ 7.9 Hz), 7.02 (t, 1H,
J ¼ 7.4 Hz), 7.09e7.18 (m, 2H), 7.37 (d, 1H, J ¼ 8.2 Hz), 7.56 (d, 1H,
J ¼ 7.9 Hz), 8.25 (br s, 6H).
4.1.17.3. (S)-5-Amino-1-(((R)-1-ethoxy-3-(1-methyl-1H-indol-3-yl)-
4.1.16.1. (2R)-1-(2,3-Dihydroxypropoxy)-3-(1-methyl-1H-indol-3-
1-oxopropan-2-yl)amino)-1,5-dioxopentan-2-aminium sulfate (59-
yl)-1-oxopropan-2-aminium methanesulfonate (8-M). Obtained
S). Obtained with 83% yield. 1H NMR (DMSO‑d6):
d 1.10 (t, 3H,
with 41% yield. 1H NMR (DMSO‑d6):
d 2.31 (s, 3H), 3.24e3.29 (m,
J ¼ 7.1 Hz), 1.63e1.74 (m, 1H), 1.75e1.86 (m, 1H), 2.02e2.07 (m, 2H),
3.13 (qd, 2H, J ¼ 14.5, 6.8 Hz), 3.52 (dd, 1H, J ¼ 7.4, 5.0 Hz), 3.72 (s,
3H), 4.04 (q, 2H, J ¼ 7.1 Hz), 4.55 (q, 1H, J ¼ 1.6 Hz), 6.47 (br s, 2H),
6.85 (s, 1H), 7.03 (t, 1H, J ¼ 7.5 Hz), 7.10e7.19 (m, 2H), 7.29 (s, 1H),
7.38 (d,1H, J ¼ 8.2 Hz), 7.51 (d,1H, J ¼ 7.9 Hz), 8.59 (d,1H, J ¼ 7.9 Hz).
2H), 3.29e3.41 (m, 2H), 3.65e3.68 (m, 1H), 3.75 (s, 3H), 4.04 (dd,
1H, J ¼ 11.1, 6.3 Hz), 4.16 (dd,1H, J ¼ 11.0, 4.0 Hz), 4.28 (br s,1H), 7.06
(ddd, 1H, J ¼ 8.0, 7.1, 1.0 Hz), 7.17 (ddd, 1H, J ¼ 8.2, 7.1, 1.1 Hz), 7.21 (s,
1H), 7.39e7.46 (m, 1H), 7.54 (dt, 1H, J ¼ 8.1, 0.9 Hz), 8.29 (br s, 3H).
4.1.16.2. ((S)-5-Amino-1-(((R)-1-ethoxy-3-(1-methyl-1H-indol-3-
yl)-1-oxopropan-2-yl)amino)-1,5-dioxopentan-2-aminium
meth-
4.1.17.4. Ethyl Na-((S)-2-(
D-tryptophanate sulfate (62-S). Obtained with 29% yield. 1H NMR
(DMSO‑d6):
(m, 1H), 1.22e1.30 (m, 1H), 1.45 (dt, 1H, J ¼ 13.5, 6.8, 6.8 Hz),
3.00e3.08 (m, 1H), 3.15 (dd, 1H, J ¼ 14.5, 5.6 Hz), 3.70 (s, 3H), 4.05
(q, 2H, J ¼ 7.1, 7.1, 7.1 Hz), 4.54 (q, 1H, J ¼ 7.5, 7.5, 7.4 Hz), 7.00 (t, 1H,
J ¼ 7.5, 7.5 Hz), 7.11 (m, 2H), 7.36 (d, 1H, J ¼ 8.2 Hz), 7.49 (d, 1H,
J ¼ 7.9 Hz), 8.48 (d, 1H, J ¼ 7.9 Hz).
l
4-azanyl)-4-methylpentanoyl)-1-methyl-
anesulfonate (59-M). Obtained with 78% yield. 1H NMR (DMSO‑d6):
d
1.11 (t, 3H, J ¼ 7.1 Hz),1.80e1.86 (m, 2H),1.97e2.13 (m, 2H), 2.31 (s,
d
0.72e0.78 (m, 6H),1.11 (t, 3H, J ¼ 7.2, 7.2 Hz),1.14e1.18
3H), 3.08 (dd,1H, J ¼ 14.5, 8.2 Hz), 3.18 (dd,1H, J ¼ 14.5, 6.0 Hz), 3.72
(s, 3H), 3.85 (q, 1H, J ¼ 5.6 Hz), 4.06 (q, 2H, J ¼ 7.1 Hz), 4.59 (td, 1H,
J ¼ 8.0, 6.0 Hz), 6.98 (s, 1H), 7.03 (ddd, 1H, J ¼ 8.0, 6.9, 1.0 Hz),
7.09e7.18 (m, 2H), 7.34e7.42 (m, 2H), 7.52 (dt, 1H, J ¼ 7.9, 1.0 Hz),
8.12 (d, 3H, J ¼ 5.6 Hz), 8.93 (d, 1H, J ¼ 7.9 Hz).

S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
19
4.2. Stability studies of prodrugs
different dose levels. Each group orally was dosed at a different
dose level. Group 1 animals were dosed with 1 Capsule A at the
Prodrugs were dissolved in DMSO at 200
test matrices at final concentration of 5
m
M and added to the
target dose of 92
Capsules A at the target dose of 275
were dosed with 4 Capsules B at target dose of 825
m
mol/kg; Group 2 animals were dosed with 3
mol/kg and Group 3 animals
mol/kg.
m
M. An aliquot was
m
immediately taken at t ¼ 0, flash frozen in liquid nitrogen and
stored at ꢁ80 ꢀC until analysis. The rest of the sample was incu-
bated at 37 ꢀC for the indicated time periods, aliquots taken and
frozen at ꢁ80C. Analysis was performed by precipitating aliquots
with 4 vol acetonitrile, centrifugation and followed by LC-MS/MS
analysis using a specific analytical method for each compound.
Matrix solutions were: 1) saline solution, 2) 0.1 N HCl (pH 1)in
water, 3) PBS buffer pH 7.2, 4) Simulated Gastric Fluid (SGF): 2 mg/
mL NaCl, 3.2 mg/mL pepsin, HCl to pH 1.2, 5) Simulated Intestinal
Fluid (SIF): 6.8 mg/mL K2HPO4, NaOH to pH 6.8 (~38 mM), 10 mg/
mL pancreatin.
m
Capsule compositions are described in Table 2, Supplementary
Materials. Blood samples were collected at pre-dose, 0.083, 0.25,
0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h post-dose. Concentrations of
indoximod in plasma samples were determined by LC-MS/MS.
For PK studies with NLG802 and determination of oral
bioavailability, a group of 3 males and 3 females were administered
a single dose of 15 mg/kg NLG802 (2 mL/kg of a 7.5 mg/mL) by
intravenous (IV) injection, followed by blood collections up to 24 h
post dose for pharmacokinetic analysis. Two weeks later, the same
animals were dosed orally by nasal gastric gavage a single dose of
75 mg/kg, followed by blood collections up to 24 h post dose from
all animals and urine and feces collections up to 12 h post dose for
pharmacokinetic and excretion analysis. Additional groups of ani-
mals (3 males and 3 females per group) were dosed by nasal gastric
gavage at 0, 225 and 675 mg/kg NLG802, followed by blood
collection for 24 h. The concentration of the parent prodrug 62, the
intermediate metabolite M9 and the active metabolite indoximod
were determined from plasma samples using a validated bio-
analytical method. Pharmacokinetic parameters were calculated by
non-compartmental analysis using WinNonLin software (Certara).
For plasma stability measurements, 450
was mixed with 50 L of 50 M solution of the test compound.
Aliquots (50 L) were taken at 0, 15, 30, 45, 60, 90 and 120 min and
m
L of plasma at 37 ꢀC
m
m
m
mixed with 4 vol of cold acetonitrile, centrifuged and analyzed by
LC-MS/MS using a specific method for each prodrug. The relative
concentrations (areas) were plotted vs time, and the percentage of
area under the curve was used as a measure of the stability of each
prodrug in plasma.
4.3. Pharmacokinetics
4.3.1. Preparation of capsules and PK in rats
4.3.3. Metabolite identification in hepatocytes from different
species
Metabolite identification of NLG802 was conducted by incuba-
tion of 10 mM NLG802 with hepatocytes from 5 different species
(CD-1 mouse, Sprague Dawley rats, Beagle dogs, Cynomolgus
monkey and human) at 37 ꢀC for 120 min, and samples analyzed by
using UPLC-QTOF-PDA system. MetaboLynx™ software was used
for the post-acquisition data processing and the structures of the
metabolites were elucidated based on the characteristics of their
MS and MS2 data.
When testing pharmacokinetics of indoximod after dosing
different prodrugs in solution, prodrugs were formulated in either
saline, Cremaphor®: EtOH: saline (10:10:80), or Chremaphor:
EtOH: saline: HCl (10:10:80:0.1 N) at 2 mg/mL (mg of free base of
each prodrug), dosed at 5 mL/kg to achieve a dose of 10 mg/kg.
To test PK in rats by dosing capsules, gelatin capsules (Torpac,
20 mg capacity) were manually prepared containing 11
mmol/
capsule A, 28 mol/capsule B or 50 mol/capsule C of indoximod
m
m
free base (2.5, 6.3 or 11.4 mg/capsule, respectively) or its prodrugs
in their various salt forms, in an excipient blend consisting of
microcrystalline cellulose, lactose monohydrate, croscarmellose
sodium and magnesium stearate in the proportions shown in
Table 1, Supplementary Methods. The composition and dose uni-
formity of a representative sample of capsules from each batch was
verified by weight and by LC-MS/MS to determine the average
indoximod or prodrug content. To test the pharmacokinetic profile
achieved by dosing indoximod prodrugs in different salt forms, 1
4.4. Antitumor activity
C57Bl/6 female mice, 8e10 weeks of age, weighing 20e25 g
were inoculated with 105 B16F10 cells subcutaneously in the flank
on Day 1. NLG802 (99.7% purity), was formulated in 0.5% (w/v) HEC,
0.25% Tween 80 and 0.01% simethicone, in water, at 7.04, 14.06,
28.35 and 56.69 mg/mL, and administered by oral gavage at 0.2 mL
per animal, twice a day, from days 6e11. Spleen cells for pmel-1
mice [55] were harvested, made into simple cell suspension by
using mesh, sorted by magnetic beads using EasySep Mouse CD8þT
cell isolation kit (StemCell Technologies). Purified CD8aþ T cells
were injected intravenously by tail vein injection at 2 ꢃ 106 T cells
per mouse, on Day 7. Vaccination to stimulate adoptively trans-
ferred pmel-1 cells was performed as described [56]. Briefly, an
emulsion of gp100 peptide (synthesized by HP Chemicals, sequence
capsule A (11
mmol/capsule) or 2 capsules B (28
mmol/capsule) or 3
capsules C (50
delivery.
mmol/capsule) were dosed to rats by intra-stomach
Rats were fasted 16 h prior to dosing to eliminate any con-
founding food effects, and food was returned 2 h after dosing. Blood
samples were obtained from each rat at 0, 15 min, 30 min, 1 h, 2 h,
4 h, 6 h, 10 h, 24 h, 48 h and 72 h after dosing. The concentration of
indoximod in plasma was determined by LC-MS/MS, and pharma-
cokinetic parameters were calculated using the software Win-
NonLin (Certara). An individual set of pharmacokinetic parameters
were calculated for each animal.
KVPRNQDWL, 25
Inc, 50 TCC ATG ACG TTC CTG ACG TT 3’, 50
with use of two-way syringes. Vaccine in total volume of 50
(containing 25 g gp100 peptide and 50 g CpG ODN) was injected
mg) and CpG ODN (from TriLink Biotechnologie
mg), and IFA was made
m
L
m
m
4.3.2. Preparation of capsules and PK in cynomolgus monkeys
For comparison of pharmacokinetics of indoximod with that of
prodrugs 62-Cl and 64-Cl, the plasma pharmacokinetics of indox-
imod capsule and test formulations 62-Cl and 64-Cl capsules was
studied following single oral administrations to male Cynomolgus
monkeys in a crossover study design. Nine non-naïve male Cyn-
omolgus monkeys were assigned to 3 treatment groups, and each
group had three male animals. In this study with crossover design,
the animals were dosed with indoximod, 62-Cl or 64-Cl capsules at
into the foot pad of experimental mice on the same side as the
established tumor, once on Day 7. Tumors were measured with
caliper on Day 11 and tumor volume was calculated by multiplying
the dimensions of the tumor.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have

20
S. Kumar et al. / European Journal of Medicinal Chemistry 198 (2020) 112373
GCN2 kinase in T cells mediates proliferative arrest and anergy induction in
response to indoleamine 2,3-dioxygenase, Immunity 22 (2005) 633e642.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112373.
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