Diaryl hydroxylamines as pan or dual inhibitors of indoleamine 2,3-dioxygenase-1, indoleamine 2,3-dioxygenase-2 and tryptophan dioxygenase

Article abstract of DOI:10.1016/j.ejmech.2018.11.010

Tryptophan (Trp) catabolizing enzymes play an important and complex role in the development of cancer. Significant evidence implicates them in a range of inflammatory and immunosuppressive activities. Whereas inhibitors of indoleamine 2,3-dioxygenase-1 (IDO1) have been reported and analyzed in the clinic, fewer inhibitors have been described for tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase-2 (IDO2) which also have been implicated more recently in cancer, inflammation and immune control. Consequently the development of dual or pan inhibitors of these Trp catabolizing enzymes may represent a therapeutically important area of research. This is the first report to describe the development of dual and pan inhibitors of IDO1, TDO and IDO2.

Full text of DOI:10.1016/j.ejmech.2018.11.010

Accepted Manuscript
Diaryl hydroxylamines as pan or dual inhibitors of indoleamine 2,3-dioxygenase-1,
indoleamine 2,3-dioxygenase-2 and tryptophan dioxygenase
Maria Winters, James B. DuHadaway, Khoa N. Pham, Ariel Lewis-Ballester, Shorouk
Badir, Jenny Wai, Eesha Sheikh, Syun-Ru Yeh, George C. Prendergast, Alexander J.
Muller, William P. Malachowski
PII:
S0223-5234(18)30964-4
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.010
EJMECH 10867
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 August 2018
Revised Date: 6 October 2018
Accepted Date: 5 November 2018
Please cite this article as: M. Winters, J.B. DuHadaway, K.N. Pham, A. Lewis-Ballester, S. Badir, J.
Wai, E. Sheikh, S.-R. Yeh, G.C. Prendergast, A.J. Muller, W.P. Malachowski, Diaryl hydroxylamines
as pan or dual inhibitors of indoleamine 2,3-dioxygenase-1, indoleamine 2,3-dioxygenase-2 and
tryptophan dioxygenase, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.11.010.
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ACCEPTED MANUSCRIPT
Diaryl Hydroxylamines as Pan or Dual Inhibitors of
Indoleamine 2,3-Dioxygenase-1, Indoleamine 2,3-
Dioxygenase-2 and Tryptophan Dioxygenase
Maria Winters1, James B. DuHadaway2, Khoa N. Pham5, Ariel Lewis-Ballester5, Shorouk Badir1,
Jenny Wai1, Eesha Sheikh1, Syun-Ru Yeh5, George C. Prendergast2,3,4, Alexander J. Muller2,4,
William P. Malachowski1*
1Department of Chemistry, Bryn Mawr College, Bryn Mawr, Pennsylvania 19010,
USA.2Lankenau Institute for Medical Research, Wynnewood, Pennsylvania 19096,
USA.3Department of Pathology, Anatomy & Cell Biology and 4Kimmel Cancer Center, Thomas
Jefferson University, Philadelphia, Pennsylvania 19104, USA.5Department of Physiology and
Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York
10461, United States
William P. Malachowski, email: wmalacho@brynmawr.edu; Alexander J. Muller, e-mail:
mullera@mlhs.org.
Address correspondence regarding the chemistry to William P. Malachowski at the Department
of Chemistry, Bryn Mawr College, 101 N. Merion Ave., Bryn Mawr, PA 19010-2899; phone:
610-526-5016; fax: 610-526-5086; e-mail: wmalacho@brynmawr.edu. Address correspondence
regarding the biology to Alexander J. Muller or George C. Prendergast at Lankenau Institute for

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Medical Research, Wynnewood, PA 19010; phone: 610-645-8034; fax: 610-645-2095; e-mail:
mullera@mlhs.org or prendergast@limr.org, respectively.
GRAPHICAL ABSTRACT
HIGHLIGHTS
•
•
•
Extension of monoaryl hydroxylamines to diaryl hydroxylamines illuminates activities as
potent pan and dual inhibitors of the tryptophan catabolizing enzymes, IDO1, IDO2 and
TDO.
This capability is unique given the lack of previous reports of dual and pan inhibitors for
these enzymes, which jointly regulate the kynurenine pathway of tryptophan catabolism
to modulate immune, inflammatory and neurological functions in the body.
Aryl halide substitution generated the most potent derivatives studied.
ABSTRACT Tryptophan (Trp) catabolizing enzymes play an important and complex role in the
development of cancer. Significant evidence implicates them in a range of inflammatory and
immunosuppressive activities. Whereas inhibitors of indoleamine 2,3-dioxygenase-1 (IDO1)

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have been reported and analyzed in the clinic, fewer inhibitors have been described for
tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase-2 (IDO2) which also have
been implicated more recently in cancer, inflammation and immune control. Consequently the
development of dual or pan inhibitors of these Trp catabolizing enzymes may represent a
therapeutically important area of research. This is the first report to describe the development of
dual and pan inhibitors of IDO1, TDO and IDO2.
Introduction
In mammals and other vertebrates, three enzymes, IDO1, IDO2 and TDO, catalyze the
rate-limiting first step of tryptophan catabolism down the kynurenine pathway which accounts
for ≥95% of tryptophan catabolism1, 2. Whereas TDO is a predominantly hepatic enzyme
functioning to maintain tryptophan homeostasis, IDO1 is induced by inflammation, particularly
by IFNγ, and can be found in lymphoid tissues and organs, along mucosal surfaces, and in the
placenta and epididymis3. IDO2 has been less thoroughly investigated than the other two
enzymes, but the available data indicate it has a more restricted expression than IDO13. In cancer
patients, IDO1 expression has been observed at varying levels and frequencies in most tumor
types while TDO is strongly expressed in hepatocarcinoma, but also weakly in many other tumor
types3Extensive preclinical evidence indicates that IDO1 inhibitors can relieve T cell suppression
and enhance cancer therapeutic responses4, 5, including when combined with cytotoxic
chemotherapy, radiotherapy or immune checkpoint therapy6-9. Nevertheless, the failure of the
IDO1-selective inhibitor epaccadostat to provide a benefit in combination with the immune
checkpoint agent pembrolizumab in a recent phase III clinical trial10 in melanoma patients
highlights the continuing challenges to exploit the role IDO1 has in regulating the immune
system.

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While this trial has raised many questions11, attendant with its failure has been the
growing recognition of the complex role and importance of the related dioxygenases, TDO and
IDO24, 5, which, like IDO1, also exert root functions in cancer inflammatory programming. It has
become clear that IDO1 drives both neovascularization along with immunosuppression in
tumors.12, 13 TDO promotes immunosuppression, but also other processes that promote metastatic
progression3, 14-17. IDO2 drives inflammation in autoimmune and cancer settings including
pancreas cancer18-20. Combo or pan IDO1/TDO/IDO2 inhibitors will potentially offer metabolic
adjuvants to illuminate key questions concerning the peculiar nature of chronic inflammatory
states that sustain cancer21. As metabolic adjuvants, IDO1-selective inhibitors do not plumb the
full potential of blunting Trp catabolism. Emerging evidence indicates that TDO and IDO2 are
jointly activated with IDO1 in many tumors3, 14-17, 22, arguing for independent as well as
overlapping functions. Whereas IDO1 modulates T-cell inflamed states, IDO2 influences B-cell
inflamed states critical for autoimmunity20 as well as certain cancers like pancreatic cancer23, 24
.
IDO2 targeting may pose an additional benefit in mitigating autoimmune side-effects of immune
checkpoint therapy based on evidence of a role in autoimmune inflammation20, 25. Lastly,
resistance to IDO1 blockade in preclinical models is associated with upregulated Trp catabolism
down the kynurenine pathway by TDO or IDO212. In the wake of these discoveries, the next
logical step would be the development of dual or broad spectrum inhibitors of tryptophan
catabolism to target IDO1, IDO2 and TDO.
Although a large number of IDO1 inhibitors have been communicated26, 27, there are
significantly fewer TDO22, 28-32 and IDO2 inhibitors33-35 described in the literature. To the best of
our knowledge, there is only one report of dual inhibition in the literature31, although a collection
of patents have appeared36-39. Our recent report of monoaryl hydroxylamine activity as an

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impressive, structurally simple IDO1 inhibitor40 prompted us to explore this structural class of
compounds further for combinatorial activity against IDO/TDO enzymes. Cognizant of the
potential therapeutic benefits that might accrue from joint inhibition of IDO2 and TDO, we
therefore screened more broadly for the effects of diaryl hydroxylamines on IDO2 and TDO as
well as IDO1, to identify pan or selective inhibitors.
Based on literature defining two major pockets termed A and B in the active site of
IDO126, 41-43. The expansion of our previously reported monoaryl hydroxylamines to diaryl
derivatives was conceived initially as an effort to exploit both pockets in IDO1 to achieve more
potent and selective inhibition. In addition, IDO1’s substrate profile has always suggested greater
promiscuity relative to TDO44, 45 and the reported flexibility of the enzyme41 likely contributes to
the ability of IDO1 to accommodate a range of structures. We were cognizant of these challenges
and opportunities as we explored a new structural derivative of the monoaryl hydroxylamines.
Herein we report our results with regards to these studies.
Results and Discussion
Synthesis of diaryl inhibitors.
The synthesis of the diaryl inhibitors followed the pattern of the previously reported
monoaryl hydroxylamines40 in using the Mitsunobu reaction of an alcohol with N-
hydroxyphthalimide to create the critical hydroxylamine functional group. The alcohol
precursors of the Mitsunobu reaction were usually synthesized by adding phenyllithium or
phenyl Grignard to the appropriate aldehyde (Scheme 1). Details for the synthesis of all alcohol
precursors to the Mitsunobu reaction are shown in the Supplementary Data appendix. The

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Mitsunobu sequence shown in Scheme 2 illustrates the transformation of the alcohol substrates
to O-alkylhydroxylamines.
Scheme 1. Synthesis of Diaryl Alcohols with One Ring Substituted
Scheme 2.Mitsunobu Reaction to Convert Alcohols to O-Alkyl Hydroxylamines
Enzyme inhibition data analysis.
Analysis of the diaryl compounds with isolated enzyme assays for IDO1, IDO2 and TDO
activity allowed a preliminary ranking of the derivatives (Table 1). These results illustrated an
array of activity and selectivity in this class of dioxygenase inhibitors. In all cases, compounds
were tested as racemic mixtures, so it is possible that one enantiomer is the preferred and more
potent stereoisomer. IDO1, which is the primary focus of most therapeutic work, was most
potently inhibited by compounds with bromo and chloro substituents on the aromatic rings
(compounds 1-7) with a potency in the range of 1-4 µM. This was also consistent with the results
from our studies with the monoaryl hydroxylamines40. Methoxy or hydroxy substituted
derivatives (20, 23, 25) were noticeably less potent (23 µM, 85% inhibition and 18% inhibition,

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respectively). The preference for bromo and chloro substitution on IDO1 inhibitors has also been
found with the most potent compounds that have entered the clinic4.
In addition to the potency enhancing effects of halogenated derivatives towards IDO1
inhibition, we found two modifications that often led to IDO1 selective inhibitors: 2-fluoro
substitution of one aryl ring (10, 12, 13, 16) and diaryl derivatives with a longer chain of atoms
between the two aryl rings (5, 11, 17). In the ortho-fluoro substitution cases, the two most potent
examples also have a trifluoromethyl group on the same ring and therefore the highly electron
deficient aryl ring may be critical for IDO1 selectivity. With respect to the diaryls with greater
distance between the two aryl rings, their selectivity is likely the result of the extended second
aryl group better occupying the B pocket of IDO1. Inhibitors that occupy both the A and B
pockets of IDO1 often have greater potency42, 43, although, to the best of our knowledge, our
discovery is the first to illustrate selectivity from an extended binding inhibitor. This could be
critical to the development of more selective inhibitors of the dioxygenase family.
New pan, dual and selective dioxygenase inhibitors were also identified. For example,
compound 3 with the 3,4-dichloro aryl ring substitution, was a pan inhibitor (IDO1/IDO2/TDO
potency: 2, 32, 4 µM, respectively). Several of the chlorinated and fluorinated derivatives
demonstrated dual IDO1/TDO inhibition, including 4 (4,4’-dichloro), 6 (2,4-dichloro), 8 (4-
chloro), 9 (3-fluoro), 14 (2,3-dichloro), and 15 (4,4’-difluoro). All six had IDO1 and TDO IC50
values in the single digit micromolar range. One dual IDO1/IDO2 inhibitor was identified in the
2-chloro substituted diaryl derivative, compound 19, which had IC50 values of 18 and 25 µM for
IDO1 and IDO2, respectively. There was one diaryl compound (26) tested that was a selective
TDO inhibitor (12 µM). Twelve of the other compounds were IDO1 selective: 1, 2, 5, 7, 10-13,
16-18, and 20. As noted earlier, seven of these derivatives fit into one of two categories: (1) 2-

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fluoro substitution (10, 12, 13, 16) or (2) extended distance between the two aromatic rings (5,
11, 17). Four of the IDO1 selective inhibitors had 3,5-dihalo (2 and 7) or 2-position substitution
(18 and 20). The most potent compound, 1, fit none of these patterns as it had a 3-bromo
substitution, thereby demonstrating the empirical nature of much of this analysis.
Table 1. Enzyme Inhibition Data for Diaryl Hydroxylamines with Ring Substitutiona
Compound
no.
Structurea
IDO1 (µM or %
IDO2 (µM or %
TDO (µM or %
inhibition)b
inhibition)b
inhibition)b
3-bromo
77.6%
69.8%
32 µM
69.3%
92.6%
83.4%
4 µM
2 µM
1
2
3
4
1 µM
2 µM
3,5-dichloro
3,4-dichloro
4,4’-dichloro
2 µM
3 µM
3 µM
84.3%
75.5%
5
2,4-dichloro
3,5-difluoro
73%
83%
6
7
4 µM
4 µM
6 µM
92.6%
4-chloro
3-fluoro
74.3%
82%
8
9
5 µM
5 µM
6 µM
7 µM
5 µM
6 µM
93.2%
2-fluoro-3-trifluoromethyl
81.1%
10
11
78.1%
93.1%
2,4-difluoro
46%
90.3%
73.4%
12
13
7 µM
8 µM
2-fluoro-5-trifluoromethyl
73.8%

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8 µM
9 µM
2,3-dichloro
72.4%
14
4,4’-difluoro
2-fluoro
73.6%
77.8%
15
16
9 µM
9 µM
87.2%
12 µM
76.0%
89.9%
17
16 µM
2-bromo
2-chloro
81.0%
25 µM
80.0%
90.4%
89.8%
80.3%
18
19
20
17 µM
18 µM
23 µM
2-methoxy
83%
76%
76.5%
88.6%
93.3%
43.6%
21
22
2-methoxy-4-methyl-4’chloro
3,5-bis(trifuoromethyl)
4-hydroxy
85%
56%
73.8%
53.3%
72.3%
36.1%
66%
23
24
25
26
50.8%
48.7%
12 µM
18.3%
3-trifluoromethyl
18%
a In all structures, n=0 unless otherwise shown.
bThe inhibition activity of each compound as listed was first tested with 100 µM inhibitor (except 24 and
25, which were tested with 200
>95% inhibition activity.
µM inhibitor), IC50 values were then determined for those exhibiting
Figure 1. Venn diagram of selectivities seen with diaryl hydroxylamines in enzyme inhibition
assay.

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Cell-based data analysis.
The first point that was noticeable in the cell-based IC50 data (Table 2) was that almost all
inhibitors demonstrated more potent inhibition than in the isolated enzyme assay. This is quite
common with IDO1 screens46, 47 and seems reasonable to expect for the similarly challenging
isolated enzyme assays for TDO and IDO2. In particular, the IDO1 enzyme assay uses a
surrogate reduction system (typically methylene blue and ascorbic acid) which is not the native
reduction operation, to maintain the IDO1 in its catalytically active ferrous state; for although
there is no redox cycling in the catalytic reaction, IDO1 is quite prone to oxidation to the ferric
heme in the presence of oxygen. Also, as with the isolated enzyme assays, all compounds were
tested as racemic mixtures.
The cell-based and isolated enzyme screens generally demonstrated similar relative
potency for IDO1, IDO2 and TDO in most cases, although some differences in selectivity
profiles were observed. For example, the same bromo and dichloro substituted derivatives (1-4)
that were potent IDO1 inhibitors in the isolated enzyme assay were also potent IDO1 inhibitors

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in the cell-based assays (0.6-2 µM). However, the cell-based data for these compounds exhibited
more significant potency with IDO2 and TDO as well. Indeed, most of the diaryl compounds
demonstrated pan inhibition with high nanomolar or low micromolar inhibition of all three
tryptophan catabolizing enzymes (Figure 2) . In all these cases, the potency for IDO1 was the
strongest, albeit in some examples only marginally so or within experimental error. For
comparison and as proof of the significant pan inhibitor potency of the diaryl hydroxylamines,
two clinical IDO1 inhibitor candidates, epacadostat (INCB24360) and PF-068400003, were
tested and are included in Table 2 and the Venn Diagram Figure 2. Although INCB24360 shows
pan inhibition at roughly the same level as many of the more IDO1 selective diaryl inhibitors, the
structural simplicity of the diaryl hydroxylamine inhibitors and their ease of synthesis (3 steps
versus 14 steps for epacadostat) makes them an important discovery and an exciting new lead
compound series.
As with the enzyme assays, the halogenated derivatives (fluoro, chloro and bromo) were
the most potent and, within these examples, the dihalogenated versions (2-4, 6, 7, 12, 14, 15)
demonstrated stronger pan inhibition than the monohalogenated analogs (5, 8, 11, 16, 18, 19),
which had at least one inhibition constant above 20
demonstrated IDO1 selective inhibition, along with three ortho monosubstituted derivatives 18-
20, which had roughly five- to ten-fold greater potency with IDO1 (~1 M) than either IDO2
(~10 M) or TDO (>20 M). The power of ring halogenation was also witnessed in our previous
µM. The meta monofluorinated 9
µ
µ
µ
report40 on monoaryl O-alkylhydoxylamine derivatives as IDO1 inhibitors.
As with the isolated enzyme assay, the extended binding inhibitor 5 with the greater
distance between the two aryl rings demonstrated more than 20-fold selectivity for IDO1 over

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IDO2 and TDO. However, the other extended binding inhibitors, 11 and 17, were less potent and
less selective, illustrating that compound 5 has the better distance between the two aryl rings to
generate selective and maximum IDO1 inhibition. Interestingly, there were two compounds, 16
and 21, which showed dual IDO1/TDO inhibitor activity with roughly 10-fold greater potency
for IDO1 and TDO (~1-5 µM) over IDO2 (~30-50 µM). Notably, the loss of the second aryl
ring, as in compound 22, resulted in significant attenuation in overall inhibition activity and for
TDO in particular. This example serves as an important illustration of the requisite diaryl
character in these inhibitors. Surprisingly, the four least potent compounds in the isolated
enzyme assay (23-26) showed single micromolar potency towards IDO1 in the cell-based assay
and retained low micromolar inhibition of IDO2 and TDO. Again, this is likely attributed to
differences in the isolated enzyme assay procedure versus the cell-based assay.
Table 2. Inhibition Data (IC50 values) for Cell-based Assays of Diaryl Hydroxylamines with
Ring Substitutiona
IDO1 (µM)b
IDO2 (µM)
TDO (µM)
Compound
no.
Structurea
Epacadostat
(INCB24360)
0.07
18.9
25.2
0.21
327
378
PF-06840003
3-bromo
0.590
0.870
11.8
8.66
7.15
5.70
1
2
3,5-dichloro

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3,4-dichloro
4,4’-dichloro
1.91
1.09
1.43
19.5
8.11
4.53
123
3
4
12.5
36.1
5
6
1.99
7.68
4.82
2,4-dichloro
3,5-difluoro
4-chloro
0.357
7.15
8.75
27.8
9.99
16.8
20.5
3.48
23.7
6.21
5.62
58.7
7
8
3-fluoro
0.939
0.933
6.74
9
2-fluoro-3-trifluoromethyl
10
11
2,4-difluoro
2-fluoro-5-trifluoromethyl
2,3-dichloro
1.21
1.09
1.87
1.68
1.26
14.7
18.9
8.94
8.95
12.1
30.5
19.3
6.37
9.02
4.54
5.15
3.92
49.7
12
13
14
15
16
4,4’-difluoro
2-fluoro
17
2-bromo
2-chloro
1.46
2.48
1.11
3.36
12.3
10.7
16.5
49.9
22.5
21.8
48.8
4.41
18
19
20
2-methoxy
21
22
21.0
40.2
2770
2-methoxy-4-methyl-4’chloro
3,5-bis(trifuoromethyl)
4-hydroxy
1.76
3.35
2.89
1.73
8.77
14.5
12.6
24.7
19.1
77.4
29.8
13.6
23
24
25
26
3-trifluoromethyl
a In all structures, n=0 unless otherwise shown.

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bThe IDO1 assay used HeLa cells, the IDO2 assay used mouse Trex cells and the TDO assay used Trex
cells. Data is from a single data point except for the most potent compounds, which were done in
triplicate and the results averaged.
H
N
Cl
ONH2
hIDO1 selective
NH
O
F3C
O
S
ONH2
F
OMe
HO
ONH2
O
F
hIDO1-hTDO
ONH2
ONH2
N
H
Cl
ONH2
Me
F
OMe
Cl
F
F
PF-06840003
ONH2
F
F
ONH2
ONH2
Br
Cl
Cl
Cl
ONH2
Cl
F
ONH2
Cl
Cl
F
ONH2
CF3
ONH2
Br
Cl
ONH2
ONH2
Cl
ONH2
F3C
Cl
Cl
ONH2
ONH2
F
ONH2
hIDO1-hIDO2
ONH2
CF3
OH
HN
F3C
ONH2
N
O
H
H2N
Br
F
S
N
N
N
O
O
H
N
F
ONH2
pan inhibitors
Figure 2. Venn diagram of selectivities seen with diaryl hydroxylamines in cell-based assay.
Compound associated cytotoxicity could be a possible complicating factor for
interpreting the cell-based enzyme inhibition data and a potential concern with regard to safety
and tolerability. Therefore, a viability screen with HeLa cells was undertaken which
demonstrated roughly
(See Supplementary Data appendix, Figure S3). For these three compounds, the cell viability
dropped to approximately 60% around 100 M. The overall lack of cytotoxicity apparent at the
effective inhibitory dose range for these compounds suggests that general cytotoxicity is not
≥80% viability up to 100 µM for all compounds except for 1, 12 and 24
µ

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likely to be an issue for advancing the diaryl hydroxylamine series. It was clear that the diaryl
hydroxylamines, like the monoaryl hydroxylamines, have little to suggest that they would be
problematic. Assessment of impact of human serum protein binding indicated a substantial
reduction in inhibitory activity for one of the compounds, 6, while the inhibitory activity of 2
was relatively unaffected (see Supplementary Data appendix, Figure S4). This analysis indicates
that it should be possible to develop diaryl hydroxylamines inhibitors that are not unduly
compromised by serum protein binding.
Conclusions
Several important discoveries were made in the studies reported here. First, diaryl
hydroxylamines demonstrated strong potency in isolated enzyme assays and were even stronger
in cell-based assays. This reaffirms a common pattern for IDO1 inhibition studies and alerts
researchers in the field that isolated enzyme studies should be interpreted with caution. This is
understandable because the isolated enzyme assay for these Trp catabolizing enzymes is not an
accurate replication of the actual cell environment. Second, the value of halogenation (fluoro,
chloro and bromo) to potency in all three enzymes is a critical recognition, echoing the most
potent IDO1 inhibitors that have entered the clinic. Third, as seen with 5, extended binding
inhibitors do offer selectivity for IDO1 over the two other dioxygenase enzyme targets. Most
notably, this study illustrates a rare example of potent pan and IDO1/TDO selective inhibitors.
Although IDO1 inhibitors have been developed and reported for over a decade26, the
development of pan or variably selective inhibitors is still in its infancy. Indeed, given the recent
failure of the IDO1-selective inhibitor epacadostat in the ECHO-301 Phase 3 trial in melanoma,
it seems likely that dual or pan inhibitors may be important to the development of effective
therapeutic inhibitors of tryptophan catabolism11. The illustration in this report that these

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relatively simple inhibitors have significant potency and unique selectivity is an important
development in the field that should benefit other researchers. Future studies will seek to further
advance these understandings in the creation of more therapeutically relevant structures.
Experimental Section
General Synthetic Chemistry Experimental Parameters
All reactants and reagents were commercially available and were used without further
purification unless otherwise indicated. Anhydrous CH2Cl2 was obtained by distillation from
calcium hydride under nitrogen. Anhydrous THF was freshly distilled from Na and
benzophenone. All reactions were carried out under an inert atmosphere of argon or nitrogen
unless otherwise indicated. Concentrated refers to the removal of solvent with a rotary
evaporator at normal water aspirator pressure followed by further evacuation with a direct-drive
rotary vane vacuum pump. Thin layer chromatography was performed using silica gel 60 Å
precoated glass or aluminum backed plates (0.25 mm thickness) with fluorescent indicator,
which were cut. Developed TLC plates were visualized with UV light (254 nm), iodine, or
ninhydrin. Flash column chromatography was conducted with the indicated solvent system using
normal phase silica gel 60 Å, 230-400 mesh. Yields refer to chromatographically and
spectroscopically pure (>95%) compounds except as otherwise indicated. Melting points were
1
determined using an open capillary and are uncorrected. H NMR spectra were recorded at 400
MHz. Chemical shifts are reported in
δ
values (ppm) relative to an internal reference (0.05% v/v)
1
1
of tetramethylsilane (TMS) for H NMR. Peak splitting patterns in the H NMR are reported as
follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br,broad. 13C NMR experiments
were conducted with the attached proton test (APT) pulse sequence. 13C multiplicities are

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reported as δu (up) for methyl and methine, and δd (down) for methylene and quaternary carbons.
HPLC was conducted on an Agilent 1100 with an Ascentis Express C-18 column (100 x 4.6 mm,
2.7
µm) and a mobile phase of 80:20 MeCN:H2O. GC analyses were performed on the free
hydroxylamine with an EI-MS detector fitted with a 30 m x 0.25 mm column filled with cross-
linked 5% PH ME siloxane (0.25 µm film thickness); gas pressure 7.63 psi He. Analysis of
samples involved heating from 70 to 250°C (10°C/min) and finally holding at 250°C for 7 min.
All compounds were found to be >95% purity by elemental analysis or GC as indicated.
General Synthesis of O-Alkyl Hydroxylamines.
To a solution of alcohol (1 mmol) in freshly distilled THF (5 ml) was added
triphenylphosphine (1.1 mmol) and N-hydroxylphthalimide (1.1 mmol). After the solution was
cooled to 0oC diisopropylazodicarboxylate (1.1 mmol) was added dropwise. The solution was
allowed to warm to room temperature over 3 hours. Reaction progress was monitored by TLC
(1:1 heptanes:ethyl acetate). Hydrazine monohydrate (1.1 mmol) was then added and the
solution was allowed to stir for 30 min. Water was added to dissolve the white precipitate. The
aqueous layer was then extracted with ethyl acetate 3 times, washed with brine, and dried over
Na2SO4. Removal of the solvent under reduced pressure afforded a yellow oil that was purified
by flash chromatography (1:1 heptanes/ethyl acetate with 0.1% triethylamine).
HCl salt formation: The O-alkyl hydroxylamine was dissolved in a minimal amount of diethyl
ether. HCl in anhydrous diethyl ether (1.0 M, 1 eq) was added slowly. The white precipitate was
filtered and washed with diethyl ether and dried under high vacuum.
O-(3-Bromo-α-phenylbenzyl)hydroxylamine hydrochloride (1). Synthesized from 3-bromo-α-
phenyl-benzenemethanol according to the general procedure to afford 1 as white crystals in 66%
1
yield. Mp=159-162oC. H NMR (CD3OD)
δ 7.57-7.60 (m, 2H, ArH), 7.33-7.52 (m, 7H, ArH),

ACCEPTED MANUSCRIPT
6.11 (s, 1H, ArC
H)
.
13C NMR (CD3OD) δu 132.0, 130.5, 130.0, 129.3, 128.9, 127.3, 126.0, 87.0;
δd 139.8, 136.5, 122.5. Anal. calcd. for C13H13ClBrNO C=49.63, H=4.17, N=4.45.Found:
C=49.63%, H =4.48%, N =4.38%.
O-(3,5-Dichloro-α-phenylbenzyl)hydroxylamine hydrochloride (2). Synthesized from 3,5-
dichloro-
crystals in 88%yield. Mp= 172-177 C. H NMR (CD3OD)
2H, ArH, J=8.6 Hz), 6.84-6.87 (d, 2H, Ar
α-phenyl-benzenemethanolaccording to the general procedure to afford 2 as white
o
1
δ
7.38-7.44 (m, 4H, Ar
H), 7.24(d,
H, J=6.68 Hz), 6.01 (s, 1H, ArCH
). 13C NMR (CD3OD)
δu 131.7, 131.2, 130.9, 129.5, 127.8, 88.5; δd 143.6, 138.4, 137.6. Anal. calcd. for C13H12Cl3NO:
C=51.26; H=3.97; N=4.60. Found: C=51.54%, H = 3.69%, N =4.57%.
O-(3,4-Dichlorophenylbenzyl)hydroxylamine hydrochloride (3). Synthesized from respective
alcohol according to the general procedure to afford 3 as white crystals in 67% yield. Mp=155-
156oC.1H NMR (CD3OD)
δ
7.61 (d, 2H, ArH, J=8.0Hz),δ 7.50-7.44 (m, 4H, ArH),δ 7.36-7.34
(d, 1H, ArH, J=8.0Hz),
δ 6.18 (s, 1H, ArCH
(ONH2)). 13C NMR (CD3OD) δu 130.8, 129.4, 129.1,
129.0, 127.2, 126.9, 86.4; δd 138.0, 136.2, 132.8, 132.6. Anal. calcd. for C13H12Cl3NO:C=51.26;
H=3.97; N=4.60, Found: C=51.26%, H = 3.65%, N =4.41%.
O-[Bis(4-chlorophenyl)methyl]-hydroxylamine hydrochloride monohydrate (4). Synthesized
from bis-4-chlorophenyl methanol according to the general procedure to afford 4 as white
crystals in 49% yield. Mp=201-202oC. 1H NMR (CD3OD)
δ
7.46-7.48 (d, 2H, ArH, J=8.40 Hz),
13
7.39-7.41 (d, 2H, ArH, J=8.48 Hz), 6.08 (s, 1H, ArCH). C NMR (CD3OD) δu 128.9, 86.3; δd
135.6, 135.0. Anal. calcd. for C13H14Cl2NO-0.5 HCl C=48.36, H=3.91, N=4.34. Found:
C=48.52%, H =3.87%, N =4.19%.
5-(Aminooxy)-5-(3-chlorophenyl)-N-phenylpentanamide hydrochlorie (5). Synthesized from
the respective alcohol according to the general procedure with the following modifications: A

ACCEPTED MANUSCRIPT
resin-bound triphenylphosphine was used and the intermediate N-hydroxy-phthalimide
intermediate was purified by column chromatography and then used immediately in the next
1
hydrazinolysis step. The product was obtained as a white solid in 62% yield. Mp=182-184oC. H
NMR (d6-DMSO)
δ
10.97 (br s, 3 H, NH3), 10.05 (s, 1 H, N
H-C(=O)), 7.6 (d, 1 H, ArH, J=8.0
Hz), 7.47 (d, 3 H, ArH, J=4.0 Hz), 7.39-7.37 (m, 1 H, Ar
H), 7.27 (t, 2 H, ArH, J=8.0 Hz), 7.01 (t,
1 H, ArH, J=8.0 Hz), 5.19 (t, 1 H, NO-CH, J=6.4 Hz), 2.37 (t, 2 H, O=CCH2, J=4.0 Hz), 1.97-
1.88 (m, 1 H, CH2), 1.78-1.60 (m, 2 H, CH2), 1.58-1.47 (m, 1 H, CH2). 13C NMR (d6-DMSO) δu
131.1, 129.3, 129.1, 127.6, 126.4, 123.4, 119.5, 85.0; δd 171.3, 141.0, 139.8, 133.8, 36.1, 34.8,
21.6. GC tR = 22.528 min.
O-(2,4-Dichlorophenylbenzyl)hydroxylamine hydrochloride (6). Synthesized from 2,4-
dichlorophenylbenzhydrol according to the general procedure to afford 6 as white crystals in
1
76% yield. Mp=170-171.5oC. H NMR (CD3OD)
δ 7.56-7.57 (m, 2H, ArH),7.47-7.51 (m, 6H,
ArH) 6.46 (s, 1H, ArCH
). 13C NMR (CD3OD) δu 129.6, 128.9, 128.8, 127.8, 127.7, 84.0; δd
135.4, 135.0, 134.0, 133.7. Anal. calcd. for C13H12Cl3NO-0.25 HCl: C=49.77, H=3.94, N=4.47.
Found: C=49.66%, H =4.17%, N =4.40%.
O-(3,5-Diflouro-α-phenylbenzyl)hydroxylamine hydrochloride (7). Synthesized from 3,5-
diflouro-
crystals in 89% yield. Mp=177-181oC.1H NMR (CD3OD)
). 13C NMR (CD3OD) δu 129.5, 128.9,
α-phenyl benzenemethanol according to the general procedure to afford 7 as white
δ
7.65-7.68 (m, 3H, ArH) 7.45-7.48
(m, 1H, ArH),7.36-7.44 (m, 4H, ArH), 6.52 (s, 1H, ArCH
127.3, 110.0 (q, 2JC-F=19, 8 Hz), 103.9 (t, 2JC-F=25 Hz) 86.4; δd 163.3 (dd, 1JC-F=250 Hz, 3JC-F=13
Hz), 141.7 (d, JC-F=8 Hz), 136.1. GC tR = 10.764 min.
O-(4-Chloro-α-phenylbenzyl)hydroxylamine hydrochloride (8). Synthesized from 4-chloro-α-
phenyl-benzenemethanol according to the general procedure to afford 8 as white crystals in 54%

ACCEPTED MANUSCRIPT
yield. Mp-171-175oC.1H NMR (CD3OD)
, J=6.7 Hz), 6.01 (s, 1H, ArCH2). 13C NMR (CD3OD) δu 129.2, 128.8,
δ 7.38-7.44 (m, 4H, ArH), 7.24 (d, 2H, ArH, J=8.6
Hz), 6.85 (t, 1H, Ar
H
128.4, 127.2, 87.2; δd 136.6, 136.0, 134.9. Anal. calcd. for C13H13Cl2NO: C=57.80; H=4.85;
N=5.18. Found: C=58.02%, H = 4.58%, N =5.14%.
O-(3-Flouro-α-phenylbenzyl)hydroxylamine hydrochloride (9). Synthesized from 3-flouro-α-
phenyl-benzenemethanol according to the general procedure to afford 9 as white crystals in 77%
yield. Mp=169-170oC.1H NMR (CD3OD)
7.43-7.44 (m, 6H, ArH) 7.24-7.26 (m, 1H,
δ
ArH),7.14-7.20 (m, 2H, Ar
H), 6.15 (s, 1H, ArC
H
). 13C NMR (CD3OD) δu 130.6 (d, 3JC-F=8 Hz),
3
2
129.2, 128.8, 127.3, 123.0 (d, JC-F=3 Hz), 115.6 (d, 2JC-F=21 Hz), 113.8 (d, JC-F=23 Hz), 87.0;
δd 163.0 (d, 1JC-F=245 Hz), 139.9 (d, 3JC-F=7 Hz), 136.6. Anal. calcd. for C13H13ClFNO C=61.55;
H=5.17; N=5.52. Found: C=61.21%, H = 4.79%, N =5.48%.
O-(2-Fluoro-3-trifluoromethyl-α-phenylbenzyl)hydroxylamine hydrochloride (10). Synthesized
from 2-fluoro-4-trifluoromethyl)-
α-phenyl benzenemethanol according to the general procedure
to afford 10 as white crystals in 61% yield. Mp=157-158oC. 1H NMR (CD3OD)
δ
7.38-7.44 (m,
4H, ArH), 7.24 (d, 2H, Ar
H, J=8.6), 6.85 (t, 1H, ArH, J=6.7), 6.01 (s,1H, ArC
H
(ONH2)). 13C
3
3
NMR (CD3OD) δu 132.1 (d, JC-F=3 Hz), 129.6, 129.0, 127.9 (m), 127.2, 124.9 (d, JC-F=5 Hz),
81.1 (d, 3JC-F=3 Hz); δd 157.2 (d, 1JC-F=250 Hz), 135.2, 126.7 (t, 2JC-F=13 Hz), 122.5 (d, 1JC-F=270
2
Hz), 118.4 (q, JC-F=33, 12 Hz). Anal. calcd. for C14H12ClF4NO: C=52.27, H=3.76, N=4.35.
Found: C=52.39%, H =3.49%, N =4.29%.
O-(3-(3-chlorophenyl)-1-phenylpropyl)hydroxylamine hydrochloride (11) Synthesized from
1
the respective alcohol according to the general procedure to afford 11. Mp=140.5-141.5oC. H
NMR (CD3OD)
δ
7.44-7.54 (m, 5 H, Ar
H), 7.28 (t, 1 H, Ar
H, J=8.0 Hz), 7.22 (s, 2 H, ArH), 7.13
(d, 1 H, Ar , J=8.0 Hz), 4.98 (t, 1 H, ArC
H
H
(ONH2), J=6.9 Hz), 2.59-2.76 (m, 2 H, ArCH2),

ACCEPTED MANUSCRIPT
13
2.27-2.37 (m, 1 H, ArCH2CH2), 2.05-2.15 (m, 1 H, ArCH2CH2). C NMR (CD3OD) δu 129.7,
129.6, 129.0, 128.1, 127.2, 126.5, 126.0, 86.5; δd 143.0, 136.6, 134.0, 36.6, 30.8. GC tR =14.656
min.
O-(2,4-Difluorophenylbenzyl)hydroxylamine hydrochloride (12). Synthesized from respective
alcohol according to the general procedure to afford 12 as white crystals in 62% yield. Mp=167-
1
168oC. H NMR (CD3OD)
δ
7.46-7.43 (overlapping signal, 5H, Ar
H
),
δ
7.11-7.05 (overlapping
3
signal, 2H, ArH),
δ
6.38 (s, 2H, ArC
H
(ONH2)). 13C NMR (CD3OD) δu 129.6 (d, JC-F=4 Hz),
3
2
2
129.5 (d, JC-F=4 Hz), 129.3, 128.8, 127.1, 111.7 (q, JC-F=22 Hz, 3JC-F=4 Hz), 104.0 (t, JC-F=25
3
1
3
1
3
Hz), 81.4 (d, JC-F=3 Hz); δd 163.6 (d, JC-F=249 Hz, JC-F=13 Hz), 160.8 (d, JC-F=249 Hz, JC-
F=12 Hz), 135.6, 120.9 (q, 2JC-F=13 Hz, 4JC-F=4 Hz). GC tR = 10.835 min.
O-((2-Fluoro-5-trifluoromethyl-α-phenylbenzyl)hydroxylamine
hydrochloride
(13).
Synthesized from the respective alcohol according to the general procedure to afford 13 as white
1
crystals in 91% yield. Mp=157-158.5oC. H NMR (CD3OD)
δ 7.78-7.82 (m, 2H, ArH ), 7.40-
7.51(m, 6H, ArH),6.45(1H, ArC
H
(ONH2)). 13C NMR (CD3OD) δu 129.6, 129.0, 128.5, 127.3,
3
2
1
125.0 (t, JC-F=4 Hz), 116.9 (d, JC-F=23 Hz), 81.3; δd 162.1 (d, JC-F=254 Hz), 135.1, 129.6,
126.3 (d, 2JC-F=14 Hz). GC tR = 10.494 min.
O-(2,3-Dichloro-α-phenylbenzyl)hydroxylamine hydrochloride (14). Synthesized from 2,3-
dichloro-
α
-phenyl-benzenemethanol according to the general procedure to afford 14 as white
1
crystals in 63%yield. Mp=170-175oC. H NMR (CD3OD)
δ
7.64 (dd, 1H, Ar
H
, J=2.1, 8.0 Hz),
13C NMR
7.60 (dd, 1H, Ar
H
, J=1.4 Hz, 7.8 Hz), 7.42-7.80 (m, 6H, Ar
H), 6.53 (s,1H, ArC
H)
.
(CD3OD) δu 130.8, 129.5, 128.8, 128.2, 128.0, 125.9, 84.8; δd 137.2, 135.0, 133.6, 131.1. Anal.
calcd. for C13H12Cl3NO: C=51.26; H=3.97; N=4.60. Found: C=51.27%, H = 3.90%, N =4.52%.

ACCEPTED MANUSCRIPT
O-[Bis(4-fluorophenyl)methyl]-hydroxylamine hydrochloride (15). Synthesized from bis-4-
fluorophenyl methanol according to the general procedure to afford 15 as white crystals in 58%
yield. Mp=164-165.5oC. 1H NMR (CD3OD)
). 13C NMR (CD3OD) δu 129.5 (d, 3JC-F=8 Hz), 115.5 (d, 2JC-F=22 Hz), 86.4; δd
δ 7.44-7.46 (m, 4H, ArH), 7.18-7.23 (m, 4H, ArH),
6.12 (s,1H, ArC
H
163.2 (d, 1JC-F=246 Hz), 133.0 (d, 4JC-F=3 Hz). Anal. calcd. for C13H12ClF2NO C=57.47, H=4.45;
N=5.16. Found: C=57.29%, H = 4.51%, N =5.16%.
O-(2-Fluoro-α-phenylbenzyl)hydroxylamine hydrochloride (16). Synthesized from 2-fluoro-α-
phenyl benzenemethanol according to the general procedure to afford 16 as white crystals in
85%yield. Mp=184-185oC. 1H NMR (CD3OD)
δ
7.42-7.50 (m, 7H, Ar
H), 7.27-7.31 (t, 1H, ArH,
J=6.60 Hz), 7.18-7.23 (t, 1H, Ar
H
,
J=8.64 Hz), 6.44 (s, 1H, ArCH
). 13C NMR (CD3OD) δu 131.0
3
4
3
2
(d, JC-F=8 Hz), 129.2, 128.8, 127.8 (d, JC-F=3 Hz), 127.2, 124.6 (d, JC-F=4 Hz), 115.6 (d, JC-
3
1
2
F=22 Hz), 81.8 (d, JC-F=4 Hz); δd 160.4 (d, JC-F=246 Hz), 135.9, 124.5 (d, JC-F=13 Hz). Anal.
Calcd. for C13H13ClFNO C=61.83; H=5.17; N=5.52. Found: C=61.83%, H = 4.89%, N =5.48%.
O-(1,3-diphenylpropyl)hydroxylamine hydrochloride (17). Synthesized from 1,3-
diphenylpropan-1-ol according to the general procedure to afford 17 in 40% yield. Mp=156-
159oC. 1H NMR (CD3OD)
δ
7.45-7.54 (m, 5 H, Ar
H), 7.30 (t, 2 H, ArH, J=7.9 Hz), 7.20 (t, 2 H,
Ar , J=6.2 Hz), 4.97 (t, 1 H, ArC
H
H(ONH2), J=6.9 Hz), 2.60-2.74 (m, 2 H, ArCH2), 2.28-2.37
(m, 1 H, ArCH2CH2), 2.05-2.14 (m, 1 H, ArCH2CH2). 13C NMR (CD3OD) δu 129.5, 129.0,
128.2, 128.0, 127.2, 126.8, 125.9, 86.9; δd 140.5, 136.8, 37.0, 31.1. GC tR = 12.784 min.
O-(2-Bromo-α-phenylbenzyl)hydroxylamine hydrochloride (18). Synthesized from 2-bromo-α-
phenyl-benzenemethanol according to the general procedure to afford 18 as white crystals in
1
34% yield. Mp=166-167oC. H NMR (CD3OD)
δ
7.71 (dd, 1H, ArH, J=8.0, 1.6 Hz) 7.61 (dd,
), 7.36 (dt,
1H, ArH, J=8.0, 1.6 Hz), 77.53 (dt, 1H, ArH, J=8.0, 1.6 Hz), 7.40-7.48 (m, 5H, Ar
H

ACCEPTED MANUSCRIPT
1H, J=8.0, 1.6 Hz), 6.47 (s, 1H, ArCH
). 13C NMR (CD3OD) δu 135.4, 130.6, 129.3, 129.0, 128.7,
128.0, 127.9, 127.2, 86.6; δd 136.2, 135.4, 123.1. Anal. calcd. for C13H13ClBrNO: C=49.63;
H=4.17; N=4.45. Found: C=49.78%, H =4.38%, N =4.63%.
O-(2-Chloro-α-phenylbenzyl)-hydroxylamine hydrochloride (19). Synthesized from 3-chloro-
α
-phenyl-benzenepropanol according to the general procedure to afford 19 as white crystals in
84%yield. Mp=169-174oC.1H NMR (CD3OD)
). 13C NMR (CD3OD) δu 130.3, 129.9, 129.3, 128.9, 128.7, 127.9,
δ
7.50 (d, 1H, ArH, J=4.0 Hz), 7.41-7.48 (m, 8H,
ArH), 6.51 (s, 1H, ArCH
127.6, 127.5, 84.5; δd 135.5, 134.7, 133.2. GC tR = 13.022 min.
O-(2-Methoxy-α-phenylbenzyl)hydroxylamine hydrochloride (20). Synthesized from 2-
methoxy-
α-phenyl-benzenemethanol according to the general procedure to afford 20 as white
crystals in 89%yield. Mp=134-134.5oC. 1H NMR (CD3OD)
δ
7.35-7.46 (m, 6H, ArH), 6.98-7.01
13
(m, 3H, Ar
H
), 6.08 (s, 1H, ArC
H
), 3.81 (s, 3H, ArOCH3). C NMR (CD3OD) δu 129.9, 129.0,
128.6, 127.2, 119.3, 114.2, 112.9, 87.8, 54.4; δd 160.2, 138.4, 137.0. Anal. calcd. for
C14H16ClNO2: C=63.28, H=6.07, N=5.27. Found: C=63.44%, H = 6.23%, N =5.31%.
O-(phenyl(thiophen-3-yl)methyl)hydroxylamine hydrochloride (21). Synthesized from 2-
methoxy-
crystals in 61% yield. Mp=138-139.5oC. 1H NMR (CD3OD)
(ONH2)). 13C NMR (CD3OD) δu 129.3, 128.7,
α-phenyl-benzenemethanol according to the general procedure to afford 21 as white
δ
7.41-7.53 (m, 7 H, ArH), 7.14 (dd,
1 H, ArH, J=5.0, 1.0 Hz), 6.27 (s, 1 H, ArCH
128.6, 127.2, 127.0, 126.1, 125.2, 84.2; δd 138.1, 136.8. GC tR =10.857 min.
O-(cyclohexyl(phenyl)methyl)hydroxylamine hydrochloride (22). Synthesized from
cyclohexyl(phenyl)methanol according to the general procedure to afford 22 in 87% yield.
1
Mp=193-196.5oC. H NMR (CD3OD)
δ
7.38-7.52 (m, 5 H, Ar
H), 4.70 (d, 1 H, ArCH(ONH2),
J=8.2 Hz), 2.14 (d, 1 H, C
HCHONH2, J=6.4 Hz), 1.76-1.85 (m, 2 H, CH2), 1.65-1.68 (m, 2 H,

ACCEPTED MANUSCRIPT
C
H2), 1.12-1.37 (m, 5 H, CH2), 0.9-1.03 (m, 1 H, CH2). 13C NMR (CD3OD) δu 129.3, 128.8,
127.6, 91.7, 42.7 δd 135.9, 29.2, 28.5, 25,8, 25.4, 25.2. GC tR =10.232 min.
O-[4-Chloro-α-(2’-methyl,4’-methoxyphenyl)benzenemethyl]hydroxylamine
hydrochloride
(23). Synthesized from the respective alcohol according to the general procedure to afford 23 as
1
white crystals in 72% yield. Mp=139.5-140.5oC. H NMR (CD3OD)
δ
7.36-7.43 (m, 4H, Ar
H),
7.19-7.21 (d, 1H, ArH J=7.76 Hz), 6.88-6.93 (m, 2H, Ar
H), 6.43(s, 1H, ArCHONH2), 3.87 (s,
3H, ArOCH3), 2.39 (s, 3H, ArCH3). 13C NMR (CD3OD) δu 129.2, 128.5, 126.7, 121.3, 111.8,
81.6, 54.7, 20.3; δd 157.3, 141.2, 136.0, 134.4, 121.6. Anal. calcd. for C15H17Cl2NO2: C=57.34,
H=5.45, N=4.46. Found: C=57.60%, H = 5.52%, N =4.36%.
O-(3,5-Bis-trifluoromethyl-α-phenyl-benzyl)hydroxylamine hydrochloride (24). Synthesized
from 3,5-bis-trifluoromethyl-α-phenyl benzenemethanol according to the general procedure to
afford 24 as white crystals in 88%yield. Mp=158-160oC.1H NMR (CD3OD)
δ 7.90 (m, 3H,
13
ArH), 7.38-7.56 (m, 5H, Ar
H), 6.31(s, 1H, ArC
H
). C NMR (CD3OD) δu 129.8, 129.2, 127.5,
122.5 (m), 86.0; δd 141.0, 135.7, 132.0 (q, 2JC-F=33 Hz), 123.2 (q, 1JC-F=270 Hz). Anal. calcd. for
C15H12ClF6NO: C= 48.47, H=3.25, 3.77. Found: C=48.90%, H =2.98%, N =3.76%.
O-(4-Hydroxy-α-phenyl)-benzyl hydroxylamine hydrochloride.(25) Synthesized from the 4-
[[(1,1-dimethylethyl)dimethylsilyl]oxy]-
general procedure to afford
α
-phenyl-benzenemethanol (25s) according to the
O
-[4-[[(1,1-dimethylethyl)dimethylsilyl]oxy]- -phenyl-
α
benzyl]hydroxylamine. To a solution of the protected phenol (0.3-3.5mmol) in anhydrous
tetrahydrofuran (5-25 mL) was added tetrabutylammonium fluoride (1 M, 1.0 equiv per
TBDMS), and the pale yellow solution was stirred for 45 min. The mixture was poured into
water and extracted with ethyl acetate. Removal of the solvent in vacuo from the organic phase
afforded a tan oil that was subjected to gravity column chromatography (9:1 hexane/ethyl

ACCEPTED MANUSCRIPT
acetate). Formation of the HCl salt followed the general procedure to afford 25 as white crystals
1
in 57% yield. Mp=168.5-169.5oC. H NMR (CD3OD)
δ
7.38-7.46 (m, 5H, ArH), 7.24 (d, 2H,
ArH, J=8.6 Hz), 6.86 (d, 2H, ArH, J=8.6 Hz), 6.01 (s, 1H, ArC
H
). ). 13C NMR (CD3OD) δu
129.8, 128.8, 127.7, 115.6, 69.1; δd 158.5, 134.1, 123.9. Anal. calcd. for C13H14ClNO2-H2O:
C=57.89; H=5.98; N=5.19. Found: C=58.03% H=6.01% N= 5.20% .
O-(3-Trifluoromethyl-α-phenylbenzyl)hydroxylamine hydrochloride (26). Synthesized from
phenyl(3-(trifluoromethyl)phenyl)methanol according to the general procedure to afford 26 as
white crystals in 77%yield. Mp=199-199.5oC. 1H NMR (CD3OD)
7.61 (d, 1H, ArH, J=2.3 Hz),
7.58 (s, 1H, Ar ), 7.50 (dd, 1H, ArH, J=8.6, 2.2 Hz), 6.47 (s, 1H, ArCH
). ). 13C NMR (CD3OD)
δ
H
δu 132.8, 129.6, 129.3, 128.8, 128.7, 127.6, 126.2 (q, 3JC-F=6 Hz), 83.4; δd 136.1, 135.0, 128.3 (d,
1
2JC-F=30 Hz), 124.2 (q, JC-F=272 Hz). Anal. calcd. for C14H13ClF3NO: C=55.37; H=4.31;
N=4.61. Found: C=55.52%, H = 4.58%, N =5.04%.
Enzyme-based IDO1, IDO2 and TDO inhibition assays. The steady-state activity of each
enzyme was measured in the absence or presence of an inhibitor at 20
℃ with a protocol reported
previously48, 49 (with small modifications). For IDO1, each reaction was initiated by mixing 50
µM L-tryptophan with 100 nM enzyme in 50 mM Tris buffer (pH 7.4) that contained 200 nM
catalase, 12 µM methylene blue, 20 mM ascorbate and 100 µM inhibitor (unless otherwise
indicated). The same protocol was used for IDO2 except that 4 mM L-tryptophan and 1 µM
enzyme were used; in addition pyocyanin (39 µM) instead of methylene blue was used as an
electron mediator. For TDO, the experiment was initiated by mixing 100 µM L-tryptophan with
250 nM enzyme in 50 mM Tris buffer (pH 7.4) that contained 100 µM ascorbate and 100
µM
inhibitor (unless otherwise indicated). The inhibitor stocks were prepared in dimethyl sulfoxide

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(DMSO), the final DMSO concentration for all reactions was kept at 3.5% (v/v). The initial
linear velocities of the reactions,
v, were obtained by monitoring the formation of the product N-
formyl kynurenine at 321 nm (
ε
= 3750 M-1cm-1) as a function of time with a UV2400
spectrophotometer (Shimadzu Scientific Instruments, Inc.) with a spectral slit width of 2 nm.
The % inhibition for each inhibitor was calculated based on (1- vo )x100, where vo was obtained
in the absence of any inhibitor. For each inhibitor that exhibited 95% inhibition activity for
IDO1 or TDO, or 85% inhibition activity for IDO2, the IC50 value was determined by
v/
≥
≥
measuring the inhibition activity as a function of inhibitor concentration. It is noted that the
threshold for IDO2 was set lower (85%) for the initial screening due to the generally lower
inhibition activity of the inhibitors. All the data were analyzed with Origin 6.1 software
(Microcal Software, Inc., MA).
Cell-based IDO1, IDO2 and TDO inhibition assays. Compounds were evaluated for inhibitory
activity against human IDO1 expressed endogenously in HeLa cells and human TDO and mouse
IDO2 expressed exogenously in T-Rex cells. HeLa cells were seeded in a 96 well plate at a
density of 10,000 cells per well in 100
streptomycin with L-tryptophan being adjusted to 100
the addition of IFN to a final concentration of 100 ng/mL. T-Rex cells containing a tet-regulated
human TDO were seeded in a 96-well plate at a density of 10,000 cells per well in 100 L of
DMEM + 10% FBS + 1% penicillin-streptomycin with L-tryptophan adjusted to 100 M and
TDO expression was induced by the addition of 100 L of media containing 2 ng/mL
doxycycline with adjusted L-tryptophan. T-Rex cells containing a tet-regulated mouse IDO2
cDNA were seeded in a 96-well plate at a density of 10,000 cells per well in 100 L of DMEM +
ꢀl DMEM + 10% fetal bovine serum + 1% penicillin-
ꢀ
M and IDO1 expression was induced by
γ
ꢀ
ꢀ
ꢀ
ꢀ

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10% FBS + 1% penicillin-streptomycin and IDO2 expression was induced by the addition of 100
ꢀ
L of media containing 2 ng/mL doxycycline + 8 mM 5-aminolevulinic acid + 20 ꢀM hemin + 4
mM L-tryptophan. The IDO1 and TDO induction was allowed to proceed for 24 hours, the
IDO2 was induced for 48 hours. After induction, the media was discarded, the wells rinsed once,
and serial dilutions of compound in 200
ꢀL of appropriate media with the final concentration of
tryptophan adjusted to 100 M for IDO1 and TDO and to 2mM for IDO2. Following incubation
ꢀ
at 37 °C for an additional 20 hrs, the assay was stopped by the addition of 50
TCA to each well, and the cells were fixed by incubating for 1 hr at 4 °C.
ꢀL of 50% (w/v)
Assessment of IDO1, IDO2 and TDO activity
Compound IC50 values were assessed from single point dilution series with the most potent
compounds subsequently retested two or more times and the results reported as averages.
Following the TCA fixation step, the supernatants were transferred to a round-bottomed 96-well
plate and incubated at 65 °C for 15 min. The plates were then centrifuged at 1250 × g for 10 min,
and 100 ꢀL of clarified supernatant was transferred to a new flat-bottomed 96-well plate and
mixed at equal volume with 2% (w/v) p-dimethylaminobenzaldehyde in acetic acid. The yellow
reaction was measured at 490 nm using a Synergy HT microtiter plate reader (Bio-Tek,
Winooski, VT). Graphs of inhibition curves with IC50 values were generated using Prism v.5.0
(GraphPad Software, Inc.).
Supplementary Data
Full experimental details for the synthesis of alcohols that were not commercially available are
1
13
described. Copies of H and C NMR spectra for all tested hydroxylamine compounds are also

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available. Raw IC50 data for top inhibitors in the enzyme and cell-based inhibition assays are
shown. Toxicity screen with HeLa cells for diaryl hydroxylamines, epacadostat and PF-0684003
are available. In addition, an assay assessing the impact of human serum protein binding on the
apparent IC50 values is also shown. This material is available at …
Acknowledgements
Financial support for this work was provided by NIH grants R01 CA109542 (GCP, AJM and
WPM) and R01 GM086482 (SRY). AJM and GCP acknowledge additional support from the
Lankenau Medical Center Foundation and Main Line Health. GCP holds the Havens Chair for
Biomedical Research at the Lankenau Institute for Medical Research. WPM acknowledges
additional support from NIH grant GM087291 and Bryn Mawr College. A generous award
(CHE-0958996) from the National Science Foundation enabling acquisition of the 400 MHz
NMR spectrometer used in these studies is gratefully acknowledged
Conflict of Interest Statement
AJM, WPM and GCP declare interests as inventors of IDO technology licensed to New Link
Genetics Corporation, a biopharmaceutical company involved in the clinical development of
IDO inhibitors, as described in U.S. Patents Nos. 7705022, 7714139, 8008281, 8058416,
8383613, 8389568, 8436151, 8476454 and 8586636. WPM and GCP are also former scientific
advisors and GCP a former grants recipient of New Link Genetics. GCP is currently a scientific
advisor for Kyn Therapeutics, which is developing IDO/TDO/AhR small molecule antagonists
for cancer treatment. AJM is a scientific advisor and grant recipient of I-O Biotech AG, which is
developing IDO vaccines for cancer treatment.

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