Synthesis of the isotopically labeled JAK1/3 inhibitor [D7]-R545 and its oxidative metabolite [D7]-R935: Protecting group-directed regioselective bromination to access 3,4,5-substituted anilines

Article abstract of DOI:10.1016/j.tet.2019.02.059

An extensive medicinal chemistry campaign and subsequent SAR studies led to the discovery of the highly potent and selective JAK1/3 inhibitor R545. To support advanced pre-clinical DMPK studies of R545, the isotopically labeled versions of the drug and its metabolite R935 were required. Herein, we describe the development of synthetic routes to deuterium-labeled [D₇]-R545 and its oxidative metabolite [D₇]-R935. Deuterium atoms were introduced at several sites in the target molecules by employing a convergent synthesis strategy. The desired regiochemistry at the right-hand side of [D₇]-R935 was established via a newly discovered protecting group-directed bromination. The described synthetic approach gave rise to the desired deuterium-labeled target compounds and sufficient quantities were synthesized to enable pre-clinical DMPK studies.

Full text of DOI:10.1016/j.tet.2019.02.059

Accepted Manuscript
Synthesis of the isotopically labeled JAK1/3 inhibitor [D ]-R545 and its oxidative
7
metabolite [D ]-R935: Protecting group-directed regioselective bromination to access
7
3,4,5-substituted anilines
Thilo J. Heckrodt, Yan Chen, Rajinder Singh
PII:
S0040-4020(19)30239-X
https://doi.org/10.1016/j.tet.2019.02.059
TET 30182
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 28 November 2018
Revised Date: 22 February 2019
Accepted Date: 25 February 2019
Please cite this article as: Heckrodt TJ, Chen Y, Singh R, Synthesis of the isotopically labeled JAK1/3
inhibitor [D ]-R545 and its oxidative metabolite [D ]-R935: Protecting group-directed regioselective
7
7
bromination to access 3,4,5-substituted anilines, Tetrahedron (2019), doi: https://doi.org/10.1016/
j.tet.2019.02.059.
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ACCEPTED MANUSCRIPT
protecting group-directed
bromination
Br
NBS
PMB
PMB
H2N
OCD3
D
D
3
N
OC
N
OC
NBS
3
H2N
OCD3
Br
Boc
Boc
undesired regioisomer
desired regioisomer
chemical
synthesis
D
biosynthesis
D3C
O
OH
N
O
P450
enzyme
N
H
D
OC
3
N
N
H
N
N
OCD3
H
H
[D7]-R545
JAK1/3 inhibitor
[D7]-R935
oxidative metabolite

ACCEPTED MANUSCRIPT
Synthesis of the isotopically labeled JAK1/3 inhibitor [D7]-R545 and its oxidative
metabolite [D7]-R935: Protecting group-directed regioselective bromination to access 3,4,5-
substituted anilines
Thilo J. Heckrodta,*, Yan Chena and Rajinder Singhb
* Corresponding author.
E-mail address: theckrodt@rigel.com (T.J. Heckrodt).
a
Rigel Pharmaceuticals, Inc.
Department of Medicinal Chemistry
South San Francisco, CA 94080
USA
b
ChemoCentryx, Inc.
Mountain View, CA 94043
USA
Abstract: To support advanced pre-clinical DMPK studies of the JAK1/3 inhibitor R545, the
isotopically labeled versions of the drug and its metabolite R935 were required. Herein, we
describe the development of synthetic routes to deuterium-labeled [D7]-R545 and its oxidative
metabolite [D7]-R935. Deuterium atoms were introduced at several sites in the target molecules
by employing a convergent synthesis strategy. The desired regiochemistry at the right-hand side
of [D7]-R935 was established via a newly discovered protecting group-directed bromination.
Keywords: JAK1/3 inhibitor, kinase, oxidative metabolite, protecting group-directed
halogenation, aryne reaction, oxidative olefin cleavage
INTRODUCTION AND BACKGROUND
Kinases of the JAK (Janus kinase) family have become important targets for small-molecule
inhibitors. Blocking or attenuating the activity of these kinases has emerged as a viable strategy
for the treatment of rheumatoid arthritis (RA) and other inflammatory diseases. To date, several
small-molecule inhibitors of the JAK pathway have gained FDA approval. For instance, they are
marketed for the treatment of RA (tofacitinib, trade name Xeljanz) and myelofibrosis
(ruxolitinib, trade name Jakafi).[1] As the result of an extensive medicinal chemistry and lead
optimization campaign, we discovered the highly potent and selective small-molecule JAK1/3
inhibitor R545.[2] This compound inhibits the JAK1/3 enzymes at nanomolar concentrations in
cell-based assays while maintaining good selectivity against JAK1/2. It has only minimal
activity in cell-based kinase counter assays e.g. SYK and VEGFR2. A 200-member kinase panel
did not turn up any significant other activities. R545 also exhibited favorable pharmacokinetic
characteristics as evident in its excellent PK in higher species (e.g. dog, monkey). In addition,
the in vitro safety assessment was encouraging: biochemical hERG channel inhibition was low
and R545 had no activity in the manual patch clamp assay. A four-strain Ames test did not
produce any signal indicative of potential mutagenicity. Only weak direct P450 inhibition was
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observed (3A4, 1A2) and R545 was found not to be a mechanism-based P450 inhibitor. Initial
animal studies using an acute PK-PD model (IL-2 stimulation of IFNγ) indicated high inhibition
rates. Further confirmatory studies in the CIA (collagen induced arthritis) model[3] showed that
R545 is capable of completely suppressing arthritis disease. Moreover, it was demonstrated that
significant reduction of acute cardiac allograft rejection was achieved after heterotopic rat heart
transplantation. Subtherapeutic and therapeutic doses of R545 prolonged the mean graft
survival.[2]
In preparation for human clinical trials with R545, more detailed DMPK studies revealed that an
oxidative metabolite (R935) was formed in several species (Scheme 1). The oxidation of R545
to the benzyl alcohol R935 appears to be mediated by CYP 3A4 and partly by 1A2. The blood
levels of the oxidative metabolite R935 were species dependent (rat > dog > monkey).
Scheme 1 P450-mediated formation of the metabolite R935.
Screening of the oxidative metabolite R935 in cell-based JAK assays indicated that the
metabolite R935 retained potency in the JAK1/3 assay but was significantly less selective over
JAK1/2 compared to the parent R545. Since the goal of the program was to develop a JAK1/3
selective inhibitor, a circulating metabolite with residual JAK1/2 activity was not desirable.
These findings prompted an in-depth DMPK evaluation of R545 and the significance of its
oxidative metabolite R935. In order to support these studies, as well as to further pre-clinical
development, the isotopically labeled versions of R545 and its oxidative metabolite R935 were
needed as internal standards to improve the accuracy of quantitation in complex matrices.
Internal standards work by normalizing for differences in extraction, injection, chromatography,
ionization and detection between samples. To facilitate the analysis of the reference standards
by bioanalytical mass spectrometry, it was aimed for a high mass delta of +7 relative to the
parent. This paper describes the synthesis of the isotopically labeled JAK1/3 inhibitor and its
oxidative metabolite.
RESULTS AND DISCUSSION
General synthetic plan
The synthesis of the isotopically labeled compounds followed the general synthetic route used to
access the di-amino pyrimidine inhibitors during our MedChem campaign: 1st SNAr reaction of
the left-hand side (LHS) aniline with the pyrimidine core followed by a 2nd SNAr reaction with
the right-hand side (RHS) aniline (Scheme 2). The preparation of unlabeled R545 is described in
the literature.[4] In order to achieve the incorporation of the deuterium labels, the existing
synthetic strategy was modified to generate two key building blocks: the “[D4]-mono-SNAr
2

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product” and a “[D3]-RHS aniline” – coupling these two fragments allowed the synthesis of [D7]-
R545 and [D7]-R935.
Scheme 2 General synthetic approach to deuterium labeled di-amino pyrimidine JAK
inhibitors.
Synthesis of a common intermediate: The “[D4]-mono-SNAr product”
The synthesis of the [D4]-mono-SNAr product 4 started from commercially available [D4]-
thymine[5] (1) (Scheme 3). [D4]-thymine (1) was heated in a sealed tube at 130 °C with
phosphoryl chloride which resulted in clean conversion to the di-chloro pyrimidine 2. Reaction
of the carbamate aniline 3[6] and pyrimidine 2 at room temperature provided the desired [D4]-
mono-SNAr product 4. This compound served as a common intermediate for the preparation of
both target molecules [D7]-R545 and [D7]-R935.
Scheme 3 Synthesis of a common intermediate: [D4]-mono-SNAr product 4. Reagents and
conditions: (a) POCl3 (solvent), 130 ºC, 3 h, 99%; (b) 3 (0.95 eq), NaHCO3 (0.95 eq),
MeOH/H2O (4/1), RT, 1 d, 79%.
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In addition to utilizing the commercial [D4]-thymine (1), we also devised a synthetic route to a
mixed-label [D3,13C,15N2]-thymine (7) (mass delta: M+6) as shown in Scheme 4.
Scheme 4 Synthesis of the mixed label thymine 7. Reagents and conditions: (a) LDA (2.05 eq),
13CD3I (1.05 eq), THF, -78 ºC to RT, ON, 49%; (b) [15N2]-urea (1.0 eq), H2SO4 (fuming), 80 ºC,
22 h. (LDA = lithium diisopropylamide).
Malate 5 was methylated via Fráter–Seebach alkylation[7] with [13CD3]-methyl iodide to give
racemic 6 which was condensed with commercially available [15N]-labeled urea using fuming
sulfuric acid. The formation of the mixed label thymine (7) proceeded smoothly, however,
isolation of the product proved to be challenging. Due to the poor solubility of thymine in
organic solvents, it was difficult to extract the product 7 from the aqueous phase and separate it
from the inorganic salts that ensued upon neutralization (e.g. Na2SO4). Since [D4]-thymine (1) is
available at reasonable cost, it was decided to deprioritize the preparation of mixed-label thymine
(7).
Synthesis of the labeled JAK1/3 inhibitor [D7]-R545
The labeled inhibitor [D7]-R545 was quickly accessed utilizing intermediate 8[4] obtained from
the synthetic route leading to the unlabeled JAK1/3 inhibitor R545 (Scheme 5). Efficient
demethylation of hexa-substituted 8 was achieved using BBr3 in THF.[8] Subsequent re-
alkylation with CD3I provided compound 9 which was converted to aniline 10 via global
reduction using a Parr hydrogenator. An SNAr reaction under acidic conditions with TFA
furnished the desired [D7]-R545 (Scheme 5).
4

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Scheme 5 Preparation of [D7]-R545. Reagents and conditions: (a) BBr3 (1.3 eq), DCM, 45 ºC,
2 d, 65%; (b) CD3I (1.5 eq), K2CO3,(1.5 eq), MeCN, 40 ºC, ON, 62%; (c) H2 (80 psi), Pd/C (10
wt %), MeOH, 21 h, 97%; (d) 4 (0.7 eq), TFA (2 eq), iPrOH, 100 ºC, ON, 83%.
Synthesis of the labeled oxidative metabolite [D7]-R935
The synthesis of the labeled metabolite [D7]-R935 and its right-hand side methoxy aniline
building block with the required 1,3,4,5-substitution pattern was more challenging to
accomplish. The general synthetic strategy is outlined in Scheme 6.
X
OCD3
aryne
reaction
R1N
R2
OCD3
regioselective
halogenation
R1N
R2
OC
D
3
LG
olefination
OH
oxidative
cleavage
R1N
R2
OCD3
R1N
R2
OCD3
Scheme 6 General synthetic plan to access 3,4,5-substituted anilines.
Our synthetic approach called for an aryne reaction to establish the desired 1,3,5-substitution
pattern on the aromatic ring followed by a regioselective halogenation in para. The benzyl
alcohol is then introduced by olefination and subsequent oxidative cleavage (Scheme 6). This
synthetic strategy ultimately yielded the desired [D7]-R935, however, several synthesis
generations were needed to successfully implement the proposed synthetic approach.
First–generation approach: Achieving the desired ring regiochemistry via aryne reaction and
protecting group-directed bromination
5

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2-Chloro-5-methyl phenol (11) was chosen as starting material. The phenol allowed an easy
introduction of three deuterium atoms via methylation with CD3I to give intermediate 12
(Scheme 7). At the center of our strategy to establish the required regiochemistry at the aromatic
ring was an aryne reaction using the strong base NaNH2.[9] Although it was possible to isolate
the desired product 13, the reaction was low yielding and many by-products had to be separated
by extensive column chromatography. Bromination of 13 with NBS did not produce a separable
mixture of regioisomers. Instead, the reaction yielded exclusively the undesired product 14. The
regiochemistry of bromide 14 was unambiguously assigned by NOE experiments correlating the
methyl group to the two aromatic protons (Scheme 7).
Scheme 7 Aryne reaction and formation of undesired product 14. Reagents and conditions: (a)
CD3I (1.2 eq), K2CO3 (1.3 eq), acetone, reflux, ON, 91%; (b) NaNH2 (3.0 eq), THF, 60 ºC, 16 h,
17%; (c) NBS (1.0 eq), MeCN, -5 ºC to RT, 2 h, 43%.
To drive the bromination to the para position, a bulkier protecting group was thought to be
beneficial. Therefore, the aryne reaction was carried out with tritylamine
(triphenylmethylamine) in order to introduce the large trityl protecting group[10] (Scheme 8). The
reaction produced the desired product albeit in low yield. TMS2NH and Ph3SiNH2 were also
employed in the aryne reaction but failed to yield the desired product. When the trityl aniline 15
was treated with one equivalent of NBS the desired para-product was observed, however, in the
form of a ~ 2:1 mixture of ortho- and para-product (Scheme 8). To increase bulkiness and steer
the bromination in para, it was attempted to introduce a Boc group in Tr-aniline 15, however,
doubly protected aniline 16 was not observed.
6

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Scheme 8 Tritylamine aryne reaction and non-selective bromination. Reagents and conditions:
(a) NaNH2 (3.0 eq), TrNH2 (2.0 eq), THF, -78 ºC to RT, ON, 5%; (b) NBS (1.0 eq), CHCl3, 0 ºC
to RT, ON; (c) Boc2O (2.5 eq), Et3N (2.0 eq), THF, RT to 80 ºC, ON, no rct. (Tr =
triphenylmethyl, NBS = N-bromosuccinimide, Boc = tert-butyloxycarbonyl).
Finally, it was discovered that double protection of the aniline nitrogen is essential to achieve a
completely para-selective bromination. Since the trityl protecting group proved to be too bulky
for a subsequent introduction of a Boc group, it was decided to switch to a benzylamine aryne
reaction. Benzyl protected aniline 17 was then treated with Boc2O to furnish the two-fold
protected aniline 18. In contrast to the previous bromination attempts, we were pleased to see
that this reaction delivered exclusively the desired para-bromination product 19. In addition to
the bulky aniline substitution, the electron withdrawing effects of the Boc group might have
played a role to drive the high para-selectivity of this bromination. The regiochemistry of the
aromatic bromide 19 was unambiguously assigned by NOESY experiments (observed NOEs are
shown as blue arrows, Scheme 9).
(a) NaNH2
BnNH2
(b) Et3N
OCD3
Boc2O
N
OCD3
N
OCD3
12
H
O
O
18
Cl
17
(c) NBS
NOE
O
Br
OCD3
N
O
19
Scheme 9 Benzyl amine aryne reaction and regio selective bromination. Reagents and
conditions: (a) NaNH2 (3.0 eq), BnNH2 (2.0 eq), THF, -78 ºC to RT, 2 d, 43%; (b) Boc2O (2.5
eq), Et3N (1.5 eq), THF, 60 ºC, ON, 74%; (c) NBS (1.05 eq), DCM, RT, 1 d, 90%.
7

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Having established the desired regiochemistry in aniline 19, we turned our attention to
converting 19 into a suitable anilino substrate for a SNAr or Buchwald reaction. Hence, bromide
19 was reacted with vinylboronic acid pinacol ester using a Suzuki–Miyaura coupling[11] to give
olefin 20. The double bond was subsequently dihydroxylated with OsO4 to 21 and cleaved in
situ under oxidative conditions using NaIO4.[12] First, it was attempted to remove the benzyl
group by hydrogenation of aldehyde 22 to give compound 23. However, benzyl protected 22
was resistant to reductive cleavage. To eliminate steric bulk, and thus facilitate the
hydrogenation of the benzyl group, the Boc protecting group was hydrolyzed with HCl.
Unfortunately, an attempted one-pot hydrogenation/reduction using Pd/C in combination with
NaBH4 resulted in over-reduction leading to dimethyl aniline 25 and other by-products.
Compound 25 is the free base of the HBr-salt 10 shown in Scheme 5. The desired anilino
compound 24 was not observed (Scheme 10).
Scheme 10 Attempted synthesis of anilino SNAr substrates 23 & 24: Suzuki coupling and
oxidative olefin cleavage. Reagents and conditions: (a) vinyl-B(pin) (2.2 eq), PdCl2(PPh3)2 (12
mol %), Na2CO3 (2.6 eq), DME/EtOH/H2O (7/3/2), 110 ºC, 5 h, 83%; (b) OsO4 (0.1 eq), 2,6-
lutidine (2.0 eq), dioxane/H2O, then NaIO4 (4.0 eq), H2O, RT, 90 min, 94%; (c) H2 (80 psi), Pd/C
8

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(10 wt %), EtOH, RT, 18 h, no rct; (d) HCl (1 eq), MeOH, RT, 2 h; (e) H2 (60 psi), Pd/C (5 wt
%), NaBH4 (3.0 eq), 3 h, EtOH.
Second–generation approach: Attempted synthesis of a vinyl or benzyl alcohol aniline as
substrate for the SNAr reaction
Since the hydrogenation conditions to remove the benzyl group in compound 22 led to the over-
reduction of the benzyl alcohol, a new protecting group (para-methoxybenzyl, PMB) was
employed that could potentially be removed under oxidative conditions. The PMB group was
introduced by running the aryne reaction with para-methoxybenzylamine to provide the PMB-
aniline 26 (Scheme 11). Compound 26 was then treated with Boc2O and regioselectively
brominated in para to give bromide 27. It should be noted that no bromination occurred on the
electron-rich methoxybenzyl group under these conditions. This sequence followed the
procedures established in the 1st generation approach. The PMB bromide 27 was vinylated in the
same fashion as the before with the analogous benzyl compound using a Suzuki coupling
(Scheme 11).
Scheme 11 PMB aryne reaction, regio selective bromination, Suzuki coupling and attempted
oxidative PMB-deprotection. Reagents and conditions: (a) NaNH2 (3.0 eq), pOMeBnNH2 (2.0
eq), THF, -78 ºC to RT, 1 d, 34%; (b) Boc2O (2.0 eq), Et3N (2.0 eq), THF, 85 ºC, ON, 66%; (c)
NBS (1.05 eq), DCM/MeCN, RT to 40 ºC, 1 d, 83%; (d) Vinyl-B(pin) (2.2 eq), PdCl2(PPh3)2 (15
mol %), Na2CO3 (3.0 eq), DME/EtOH/H2O (7/3/2), 110 ºC, 5 h, 83%.
It was then attempted to deprotect the system to the free vinyl aniline in preparation for the
subsequent SNAr reaction with pyrimidine chloride 4. First, oxidative conditions for the PMB
removal were tried (DDQ and CAN).[13] After considerable experimentation it was clear that
these conditions did not result in a clean removal of the PMB group. The use of CAN (2.0 eq) in
MeCN/H2O led to a very messy reaction with only one product identified as the phenol resulting
from the demethylation of the PMB group. DDQ (1.2 eq) in DCM/H2O produced a sluggish
9

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reaction that was accompanied by many side products. It was then tried to remove the Boc group
first. The desired product was observed by TLC and HPLC upon treatment with HCl (5–10 eq)
in dioxane, however, during neutralization and aqueous work-up the product decomposed. It
was possible to precipitate out the HCl salt of Boc-deprotected 28. Treating this salt with DDQ
or CAN did not generate the desired vinyl aniline 29. Selective removal of the PMB group by
hydrogenation in the presence of the double bond was not possible: hydrogenation with Pd/C led
only to the reduction of the double bond while keeping the PMB group in place. In contrast,
using Pt/H2 did produce ~ 5-10% of the desired product (Boc-protected 29) with the double bond
intact, however, due to the low yield this transformation was not considered a viable route for
moving forward. It was concluded that the desired product 29 can be considered as an electron-
rich phenylogous enamine which is highly reactive and prone to decomposition. This conclusion
is also supported by the fact that vinyl methoxy anilines with the substitution pattern of 29 are
unknown in the literature to date (Scheme 11).
Given that the desired vinyl methoxy aniline 29 was inaccessible, it was decided to attempt to
convert olefin 28 to the benzyl alcohol 24 (Scheme 12). It was hoped, that a complete
deprotection of the aniline could be achieved at the benzyl alcohol stage. Although amino
benzyl alcohol 24 is likely a poor substrate in an acid-catalyzed SNAr reaction (reactive
carbocation formation), it could be used in a Buchwald-type coupling.
Scheme 12 Salvaging intermediate 28: 4-amino benzyl alcohol 24 as new target.
Oxidative cleavage[12] of the vinyl group in 28 was achieved using OsO4/NaIO4 followed by
treatment with NaBH4 to give benzyl alcohol 30 in good yield. It was then tried to remove the
PMB group by hydrogenation with Pd/C. However, once again, under these conditions the
benzyl alcohol was also reduced leading to undesired product 31. The attempted oxidative
removal of the PMB group by employing DDQ caused quantitative oxidation of the benzyl
alcohol 30 to the corresponding aldehyde 32. At this stage, it was attempted to remove the PMB
group by hydrogenation since the aldehyde 32 should be less prone to over-reduction than the
corresponding benzyl alcohol 30. However, in contrast to alcohol 30 the aldehyde 32 was
resistant to hydrogenation leaving the PMB group mostly intact (similar reactivity as seen in
Scheme 10, 22 ꢀ 23). Attempted cleavage of the Boc group in 30 with 4M HCl also failed to
produce any meaningful quantities of the target compound 33 – probably due to the formation of
a highly reactive carbocation species at the benzylic position under strongly acidic conditions
(Scheme 13).
10

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Scheme 13 Vinyl to alcohol conversion and attempts to remove the PMB or Boc protecting
group. Reagents and conditions: (a) OsO4 (0.05 eq), 2,6-lutidine (2.0 eq), dioxane/H2O, then
NaIO4 (4.0 eq), H2O, RT, 1 h; (b) NaBH4 (2.0 eq), EtOH/iPrOH, RT, ON, 41% over two steps;
(c) H2 balloon, Pd/C (5 wt %), iPrOH, RT, ON, 91%; (d) DDQ (3.0 eq), DCM/H2O, RT, ON,
85%. (DDQ = 2,3-dichloro-5,6-dicyano-p-benzoquinone).
Third–generation approach: Late-stage introduction of the benzyl alcohol
Given that envisioned anilino SNAr substrates (compounds 23 & 24) lacked synthetic
accessibility, it was decided to carry out the second SNAr reaction with the protected bromo
aniline 19 (Scheme 14). The bromide group could then serve as a handle for introducing the
desired benzyl alcohol. Although highly hindered, the bromo aniline 19 (from the first-
generation approach) was surprisingly efficient in delivering compound 34 in the displacement
reaction with chloro-pyrimidine 4. It was not necessary to remove the Boc group before the
SNAr reaction since it was cleaved in situ under the acidic reaction conditions. It should be noted
that only PTSA was an effective catalyst for this SNAr reaction. Other acids, such as TFA or
HCl, furnished only traces of product. Next, the olefin was attached via Suzuki coupling; to
improve the efficiency of this transformation, Burke’s slow-release protocol[14] in connection
with vinyl MIDA boronate was employed. Unexpectedly, the reaction was quite sluggish and
delivered olefin 35 in only 47% yield. As in the previous sequences, the oxidative cleavage of
the vinyl group in 35 and reduction to benzyl alcohol 36 was achieved using OsO4/NaIO4
followed by addition of NaBH4. Compound 36 constitutes the benzyl protected form of the
target molecule [D7]-R935. Again, removing the benzyl protecting group proved to be a major
obstacle. Several methods were tried to no avail and only Lewis acid conditions using BBr3
furnished traces of product. However, this was not sufficient to generate meaningful quantities
for advanced DMPK studies (Scheme 14).
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Br
OCD3
O
O
N
D
D
D3C
19
Br
O
D3C
N
O
N
O
O
N
H
N
N
OCD3
N
H
N
H
(a) PTSA
N
Cl
N
H
4
34
N
(b) Pd(OAc)2. S-Phos
K3PO4
B
O
O
O
O
D
D
(c) 2,6-lutidine, OsO4
then NaIO4
D3C
D3C
O
O
N
N
OH
O
O
N
H
N
H
(d) NaBH4
N
N
N
OCD3
N
OCD3
N
H
N
H
36
35
X
D
D3C
O
N
OH
O
N
H
N
N
H
OCD3
N
H
[D7]-R935 not observed
Scheme 14 SNAr reaction with double-protected aniline, MIDA boronate cross-coupling and
oxidative olefin cleavage/reduction. Reagents and conditions: (a) 19 (1.5 eq), PTSA•H2O (1.5
eq), iPrOH, 100 ºC, 21 h, 49%; (b) vinyl-MIDA (1.2 eq), Pd(OAc)2 (5 mol %), S-Phos (10 mol
%), K3PO4, dioxane/H2O (5:1), 60 ºC, 23 h, 47%; (c) OsO4 (0.3 eq), 2,6-lutidine (2.0 eq),
dioxane/H2O, then NaIO4 (4.0 eq), H2O, RT, 2 h, 74%; (d) NaBH4 (2.0 eq), EtOH/iPrOH, RT, 2
h, 86%. (PTSA = p-toluenesulfonic acid, MIDA = N-methyliminodiacetic acid).
Fourth–generation approach: SNAr reaction with a methyl ester serving as a stable benzyl
alcohol precursor
At this point, it became clear that it would be essential to run the SNAr reaction with a fully
deprotected aniline bearing a benzyl alcohol precursor stable under acidic conditions – a methyl
ester would fulfill this requirement. Therefore, the remaining quantities of benzyl alcohol 30
from the second-generation route were oxidized in a two-step protocol. The aldehyle 32 was
accessed via Dess-Martin oxidation[15] in wet DCM – the same compound that was encountered
by chance during the attempted PMB-deprotection with DDQ shown in Scheme 13. A Pinnick
oxidation[16] using NaClO2 provided acid 37. Methylation was carried out with MeI and K2CO3
in acetone yielding methyl ester 38. After some experimentation it was found that TFA rather
than 4M HCl is best suited to cleave the Boc group. However, the Boc-deprotected compound
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39 was obtained only in a moderate 65% yield. Hydrogenation of the PMB group proceeded
smoothly producing the free aniline 40. With the methyl ester aniline 40 in hand, the crucial
SNAr reaction was performed using PTSA as catalyst generating di-amino pyrimidine 41 in very
good yield. Finally, reduction of the methyl ester in 41 to the benzyl alcohol [D7]-R935 was
carried out with DIBAL-H[17] in DCM as solvent. Interestingly, in order to achieve good
conversion, it was crucial to use a 1M DIBAL-H solution in DCM rather than in THF. The
target compound [D7]-R935 was obtained in 74% yield in form of a white solid (Scheme 15).
Scheme 15 Synthesis of methyl ester 41, SNAr reaction and late-stage reduction. Reagents and
conditions: (a) DMP (1.5 eq), wet DCM, RT, 4.5 h, 79%; (b) NaClO2 (10 eq), NaH2PO4 (10 eq),
tBuOH/2-methyl-2-butene (5:1), H2O, 0 ºC to RT, 3 h, 98%; (c) MeI (8.0 eq), K2CO3 (5.0 eq),
acetone, 90 ºC, 3 h, 96%; (d) TFA (6.0 eq), DCM, 30 ºC, ON, 65%; (e) H2 balloon, Pd/C (10 wt
%), MeOH, RT, 6 h, 80%; (f) 4 (0.75 eq), PTSA•H2O (0.9 eq), iPrOH, 90 ºC, 15 h, 91%; (g)
DIBAL-H (3.5 eq), DCM, 0 ºC to RT, 6 h, 74%. (DMP = Dess–Martin periodinane, TFA =
trifluoroacetic acid, DIBAL-H = diisobutylaluminium hydride).
13

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SUMMARY AND CONCLUSION
To support advanced DMPK studies of the JAK1/3 inhibitor R545, we developed synthetic
routes to the isotopically labeled version [D7]-R545 and its oxidative metabolite [D7]-R935. The
deuterium labeled compounds were required as internal reference standards for bioanalytical
mass spectrometry used to elucidate the metabolic fate of R545.
Firstly, four deuterium atoms were introduced by synthesizing a common intermediate, the
“[D4]-mono-SNAr product”; the deuterium source was commercially available [D4]-thymine (1).
Secondly, three more deuterium atoms were added via a [D3]-methoxy aniline.
While the [D7]-R545 synthesis was accomplished fairly quickly with standard chemistry, it took
considerable experimentation to prepare the oxidative metabolite [D7]-R935. To install the
required regiochemistry in the tetra-substituted [D3]-labeled bromo anilines (compounds 19 and
27) we employed an aryne reaction followed by a newly discovered protecting group-directed
regioselective bromination. After the desired right-hand side regiochemistry had been
established, the bromo aniline 27 was vinylated followed by an oxidation sequence to give
methyl ester aniline 40. Reaction with the [D4]-mono-SNAr product 4 delivered the late-stage
reduction precursor 41 which was converted to the target benzyl alcohol [D7]-R935. The above
described syntheses allowed us to obtain sufficient quantities (several hundred milligrams) of the
isotopically labeled material.
ACKNOWLEDGEMENTS
We are thankful to the Department of Analytical Chemistry (Van Nguyen, Duayne Tokushige,
and Dr. Mark Irving) of Rigel Pharmaceuticals for analytical support and numerous preparative
HPLC separations. The authors would also like to thank Ryan Kelley for proofreading the
manuscript.
REFERENCES
[1]
[2]
For Tofacitinib, see: (a) Zerbini, C. A.; Lomonte, A.B. Expert Rev. Clin. Immunol. 2012,
8, 319–331; de Lartigue, J. Drugs Today 2012, 48, 533–543; for Ruxolitinib, see: (b)
Vaddi, K.; Sarlis, N.J.; Gupta V. Expert Opin. Pharmacother. 2012, 13, 2397-2407; (b)
Med. Lett. Drugs Ther. 2012, 54, 27–28; (c) Ostojic, A.; Vrhovac, R.; Verstovsek, S.
Drugs Today 2011, 47, 817–827.
Deuse, T.; Hua, X.; Taylor, V.; Stubbendorff, M.; Baluom, M.; Chen, Y.; Park, G.;
Velden, J.; Streichert, T.; Reichenspurner, H.; Robbins, R.C.; Schrepfer, S.
Transplantation, 2012, 94, 695–702.
[3]
[4]
Schurgers, E.; Billiau, A.; Matthys, P. J Interferon Cytokine Res. 2011, 31, 917–926.
Li, H.; Heckrodt, T. J.; Chen, Y.; McMurtrie, D. J.; Taylor, V.; Singh, R.; Ding, P.; Yen,
R. PCT Int. Appl. (2012), WO 2012015972 A1 20120202.
[5]
[D4]-Thymine (1) (99.3 atom % D) was purchased from C/D/N Isotopes Inc., Pointe-
Claire, Quebec, Canada.
[6]
[7]
Aniline 3 is commercially available.
(a) Fráter, G.; Müller, U.; Günther, W. Tetrahedron, 1984, 40, 1269–1277; (b) Seebach,
D.; Wasmuth, D. Helv. Chim. Acta 1980, 63, 197–200.
[8]
(a) McOmie, J.F.W.; Watts, M. L.; West, D. E. Org. Synth. 1969, 49, 50–53.
14

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[9]
Elliott, E. L.; Bushell, S. M.; Cavero, M.; Tolan, B.; Kelly, T. R. Org. Lett. 2005, 7,
2449–2451. Note: The procedure described in the literature was modified in order to
avoid using liquid ammonia; it was replaced by solid NaNH2.
[10] Green, T.W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 4th Edition, Wiley,
New York, 2006, pp 825–827.
[11] Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457–2483.
[12] Hua, Z.; Yu, W.; Mei, Y.; Kang, Y.; Jin, Z. Org. Lett. 2004, 6, 3217–3219.
[13] Green, T.W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 4th Edition, Wiley,
New York, 2006, pp 123–130.
[14] Knapp, D. M.; Gillis, E. P.; Burke, M. D. J. Am. Chem. Soc. 2009, 131, 6961–6963.
[15] (a) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155–4156; (b) Boeckman, R. J. in
"Encyclopedia of Reagents for Organic Synthesis"; Paquette, L. A., Ed.; Wiley:
Chichester, UK, 1995, Vol. 7, pp 4982–4987; (c) Dess, D. B.; Martin, J. C. J. Am. Chem.
Soc. 1991, 113, 7277–7287; for the acceleration of the oxidation by water, see (d) Meyer,
S. D.; Schreiber, S. L. J. Org. Chem., 1994, 59, 7549–7552.
[16] Bal, B. S.; Childers Jr., W. E.; Pinnick, H. W. Tetrahedron, 1981, 37, 2091–2096.
[17] Winterfeldt, E. Synthesis 1975, 617–630; Fernández-Bachiller, I. Synlett 2002, 179–180.
15

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EXPERIMENTAL
All moisture sensitive reactions were carried out under nitrogen. [D4]-Thymine (99.3 atom % D)
was purchased from C/D/N Isotopes, Canada. Anhydrous solvents were purchased from Aldrich
or EMD Millipore (DriSolv®). All other solvents were HPLC grade. Column chromatography
was performed with EMD Merck silica gel (0.040-0.63 µm, 240-400 mesh) under low pressure
of 5-10 psi. Flash chromatography was carried out on a Teledyne Isco CombiFlash Rf200
system with variable solvent gradients. TLC analysis was performed with E. Merck silica gel
60-F254 plates. NMR spectra were recorded on a Varian Mercury VX 300 MHz spectrometer.
NMR spectra measured in CDCl3 solutions were referenced to the residual CHCl3 signal (1H, δ =
7.26; 13C, δ = 77.0); spectra measured in DMSO-d6 were referenced to the residual DMSO signal
1
(1H, δ = 2.50; 13C, δ = 39.50). H and 13C shifts are given in ppm (s = singlet, d = doublet, t =
triplet, q = quadruplet, quin = quintet, m = multiplet, br s = broad signal). Coupling constants, J,
are given in Hz. Mass spectra were measured on a Waters Micromass ZQ or ZMD instrument.
Accurate mass measurements (HRMS) were obtained using an Acquity UPLC system with PDA,
ELSD and LCT Premier XE mass spectrometer (time-of-flight). Monitored wavelength was 254
nm. Mass analysis was performed in extended W-mode using leucine enkephalin as reference
lock mass, 556.2771 Da for positive ion and 554.2615 Da for negative ion. Samples were
initially prepared in methanol at 1 mg/ml and diluted as necessary to remove dead time artifacts.
Column: Acuity UPLC, BEH18, 1.7 ꢀm, 2.1x50 mm, solvents: A = water (w/ 0.05% formic
acid), B = acetonitrile (w/ 0.05% formic acid), flow rate: 0.735 ml/min, gradient: 95% A, 5% B
to 100% B in 4 minutes.
[D4]-2,4-Dichloro-5-methylpyrimidine (2)
[D4]-Thymine (1) (99.3 atom % D) (3.00 g, 23.1 mmol) was placed in a pressure vessel with 25
ml of phosphorus oxychloride (POCl3). The flask was sealed, and the white suspension was
heated at 130 °C for 3 hours. During heating the suspension became a clear yellow solution.
After cooling to room temperature, the reaction mixture was added very carefully dropwise to a
beaker filled with ice. The ice-water mixture was stirred for another 15 minutes and then
extracted with CHCl3 (3x50 ml). The organic layer was dried over MgSO4 and the solvent was
removed under reduced pressure to yield 3.81 g (99%) of the title compound 2.
13C NMR (75 MHz, CDCl3) δ 162.4, 159.5, 157.9, 128.7, 15.1; MS (ESI) (m/z): 167 [M+H]+;
HRMS (TOF ES+) m/z [M+H]+ calcd. for C5D4Cl2N2: 167.0077; found: 167.0081.
[D4]-5-((2-Chloro-5-methylpyrimidin-4-yl)amino)benzo[d]oxazol-2(3H)-one (4)
Aniline 3 (2.00 g, 13.3 mmol), 2,4-dichloro-pyrimidine (2), (2.34 g, 14.0 mmol) and NaHCO3
(1.12 g, 13.3 mmol) were dissolved in a 4:1 mixture of MeOH (80 ml) and H2O (20 ml). The
reaction mixture was stirred for one day at room temperature which resulted in a small amount of
product precipitate. To maximize the precipitation, the reaction flask was placed in the
refrigerator overnight. More precipitate was formed which was filtered off and air-dried. The
crude product was stirred in EtOH at 50 °C for 12 h, filtered and dried in high vacuum to yield
1.37 g of the desired [D4]-mono-SNAr product 4. Further crops were obtained from the mother
liquor bringing the total yield to 79% (2.94 g).
16

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1H NMR (300 MHz, DMSO-d6) δ 11.64 (s, 1H), 8.83 (s, 1H), 7.48 (s, 1H), 7.26 (s, 2H); 13C
NMR (75 MHz, DMSO-d6) δ 160.4, 156.8, 154.7, 139.8, 134.6, 130.2, 116.1, 114.1, 109.2,
104.6; MS (ESI) (m/z): 281 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C12H5D4ClN4O2:
281.0739; found: 281.0734.
[D3,13C]-Dimethyl 2-hydroxy-3-methylsuccinate (6)
Racemic dimethyl malate 5 (5.28 g, 32.6 mmol) was placed in a flask and degassed under
vacuum, then flushed with nitrogen. Dry THF was added (150 ml) and the solution was cooled
to -78 °C with a dry-ice bath. Lithium diisopropylamide (LDA, 2 M in
THF/heptane/ethylbenzene) (33.4 ml, 66.8 mmol) was slowly added in three portions via
syringe. The resulting thick suspension had a deep egg yolk color; stirring was continued at -78
°C for 30 minutes. [13CD3]-Iodomethane (Aldrich, 99.0 atom % 13C, 99.5 atom % D) (5.00 g,
34.2 mmol) was slowly added with a syringe. The reaction mixture was allowed to warm to
room temperature overnight and was then carefully quenched by the dropwise addition of sat.
NH4Cl solution. The resulting precipitate was dissolved by adding ca. 150 ml of 1 M HCl. The
mixture was then extracted with Et2O (1x 100 ml) and EtOAc (3x 100 ml); the combined organic
phases were dried over MgSO4 and solvents were removed under reduced pressure. The crude
oil was further purified by column chromatography eluting with hexane/EtOAc (7/3). TLC
analysis required staining to visualize product spots [H2SO4 (10%, 100 ml), phosphomolybdic
acid (4.5 g), Ce(IV)SO4) (0.1 g)]. The product 6 was obtained in form of a clear, pale yellow oil
(2.87 g, 49%). Rf (product) = 0.24 (SiO2, hexane/EtOAc 7/3).
1H NMR (300 MHz, DMSO-d6) δ 5.74 (d, J = 5.8 Hz, 1H), 4.26 – 4.15 (m, 1H), 3.64 (s, 3H),
3.58 (s, 3H), 2.89 – 2.77 (m, 1H), 2.83 (t, J = 5.0 Hz, 1H); 13C NMR (75 MHz, DMSO-d6) δ
172.9, 172.7, 72.0, 51.5, 43.3, 42.8, 11.8 (dp, J = 39.1, 19.5 Hz); MS (ESI) (m/z): 181 [M+H]+;
13
HRMS (TOF ES+) m/z [M+H]+ calcd. for C6 CH9D3O5: 181.0985; found: 181.0994.
[D3,13C,15N2]-5-Methylpyrimidine-2,4(1H,3H)-dione (7)
The methylated dimethyl malate 6 (2.67 g, 14.8 mmol) from the above reaction was combined in
a flask with [15N2]-urea (Aldrich, 98.0 atom % 15N) (918 mg, 14.8 mmol) and fuming H2SO4 (30
ml) was poured over the mixture. A strong heat tone and gas evolution was observed. The
reaction mixture was heated at 80 °C for 22 hours. Analysis by LCMS indicated that the
reaction had gone to completion and the desired thymine product had been formed. The reaction
mixture was poured on ice and then carefully neutralized with concentrated NaOH. Extraction
of the product with organic solvents (EtOAc, DCM, etc.) proved impracticable due to the low
solubility of thymine in organic solvents. It was then attempted to crystalize the labeled thymine
from the quenched reaction mixture, however, it was difficult to separate the product from the
Na2SO4 that was formed during neutralization. The thymine product 7 was not further purified
since a new route using commercially available [D4]-thymine was employed.
MS (ESI) (m/z): 133 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C413CH3D3O2 N2:
15
133.0670; found: 133.0679.
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[D3]-1,3-Dibromo-4-methoxy-5,6-dimethyl-2-nitrobenzene (9)
1,3-Dibromo-4-methoxy-5,6-dimethyl-2-nitrobenzene (8) (8.00 g, 23.6 mmol) (the synthesis of
this compound is described in PCT Int. Appl. (2012), WO 2012/015972 A1, page 143) was
dissolved in dry DCM (300 ml). BBr3 (1 M in THF) (30.6 ml, 30.6 mmol) was added via
syringe and the reaction mixture was heated at 45 °C for two days. The reaction was carefully
quenched by the addition of water, the phases were separated, and the aqueous layer was
extracted with DCM. The combined organic layers were dried over MgSO4 and the crude
product was purified via CombiFlash eluting with hexanes/EtOAc (gradient 0 to 30%). The
phenol (4.95 g, 65%) was obtained in form of a yellow solid.
MS (ESI) (m/z): 326 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C8H7Br2NO3: 323.8871;
found: 323.8889.
The phenol (4.95 g, 15.2 mmol) was dissolved in MeCN (200 ml); K2CO3 (3.15 g, 22.8 mmol)
and CD3I (3.15 g, 22.8 mmol) were added. The reaction mixture was stirred overnight at a
temperature of 40 °C. Half of the MeCN was evaporated and the K2CO3 was filtered off, then
silica gel was added, and the mixture was concentrated in vacuo. Chromatography eluting with
hexanes/ethyl acetate (isocratic, 30/1) afforded the desired product 9 (3.21 g, 62%) as a
yellowish solid.
MS (ESI) (m/z): 343 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C9H6D3Br2NO3:
340.9216; found: 340.9208.
[D3]-3-Methoxy-4,5-dimethylaniline•HBr (10)
The nitro compound 9 (3.21 g, 9.38 mmol) from the above reaction was placed in a
hydrogenation bottle; Pd/C (320 mg, 10 wt. % loading, dry basis, Degussa type E101 NE/W) and
methanol (150 ml) were added. The bottle was degassed and flushed with hydrogen (3x). After
shaking in a Parr apparatus at 80 psi hydrogen pressure for 21 hours, the catalyst was filtered off
using a glass frit charged with celite. Solvents were evaporated under reduced pressure and the
aniline 10 was obtained as a yellow solid (2.13 g, 97% yield) in form of the HBr salt.
1H NMR (300 MHz, DMSO-d6) δ 10.06 (s, 3H), 6.85 (s, 1H), 6.78 (s, 1H), 2.20 (s, 3H), 2.02 (s,
3H); 13C NMR (75 MHz, DMSO-d6) δ 157.5, 138.6, 129.0, 124.1, 116.3, 103.5, 39.5, 19.7, 11.2;
MS (ESI) (m/z): 155 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C9H10D3NO: 155.1264;
found: 155.1255.
[D7]-5-((2-((3-Methoxy-4,5-dimethylphenyl)amino)-5-methylpyrimidin-4-
yl)amino)benzo[d]oxazol-2(3H)-one ([D7]-R545)
The mono-SNAr product 4 (251 mg, 0.894 mmol) was placed in a 40 ml vial and aniline 10 (315
mg, 1.34 mmol) was added. Isopropanol (15 ml) and TFA (0.2 ml, 2.61 mmol) were added and
the mixture was heated at 100 °C overnight. The reaction mixture was concentrated under
reduced pressure and further purified using column chromatography eluting with DCM/MeOH
(2 M NH3) (gradient 0 to 10%). Combining the clean fractions furnished 296 mg (83% yield) of
[D7]-R545 in form of an off-white solid.
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1H NMR (300 MHz, DMSO-d6) δ 11.80 (s, 1H), 10.24 (s, 1H), 9.82 (s, 1H), 7.42 – 7.14 (m, 3H),
6.84 (s, 1H), 6.72 (s, 1H), 1.99 (s, 6H); 13C NMR (75 MHz, DMSO) δ 161.7, 157.1, 154.5,
150.8, 141.3, 137.4, 134.7, 133.0, 130.4, 120.3, 119.0, 114.5, 109.4, 107.6, 107.0, 105.0, 101.7,
19.6, 11.1; MS (ESI) (m/z): 399 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for
C21H14D7N5O3: 399.2155; found: 399.2129.
[D3]-1-Chloro-2-methoxy-4-methylbenzene (12)
2-Chloro-5-methylphenol (11) (40.0 g, 281 mmol), [D3]-iodomethane (48.8 g, 337 mmol) and
K2CO3 (50.4 g, 365 mmol) were dissolved in acetone (250 ml). The reaction mixture was
refluxed overnight, the salts were filtered off and the solvents were removed in vacuo. The crude
oil was purified using column chromatography eluting with hexanes/ethyl acetate (isocratic,
30/1). The yield of the methylated phenol 12 was 91% (40.8 g). The product was a pale-yellow
liquid.
1H NMR (300 MHz, DMSO-d6) δ 7.25 (d, J = 8.0 Hz, 1H), 6.94 (s, 1H), 6.73 (d, J = 8.0 Hz, 1H),
2.29 (s, 3H); 13C NMR (75 MHz, DMSO) δ 154.2, 138.1, 129.3, 121.7, 118.0, 113.4, 20.8; MS
(ESI) (m/z): 160 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C8H6D3ClO: 160.0609;
found: 160.0621.
[D3]-3-Methoxy-5-methylaniline (13)
Chloride 12 (3.00 g, 18.8 mmol), NaNH2 (2.21 g, 56.4 mmol) and dry THF were combined in a
pressure vessel. The mixture was heated at 60 °C for 16 hours. The red-brownish suspension
was then quenched by the addition of H2O (3 ml). More water was added, and the aqueous layer
was extracted with ethyl acetate (3x 50 ml). After drying over MgSO4, the crude product was
further purified by CombiFlash chromatography eluting first with CHCl3/MeOH (iscocratic, 1%)
then with hexanes/ethyl acetate (gradient 0 to 20%). After combining the clean fractions, 456
mg (17% yield) of the desired aniline 13 were obtained.
1H NMR (300 MHz, CDCl3) δ 6.16 (s, 1H), 6.14 (s, 1H), 6.08 (s, 1H), 3.51 (s, 2H), 2.24 (s, 3H);
MS (ESI) (m/z): 141 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C8H8D3NO: 141.1107;
found: 141.1123.
[D3]-2-Bromo-3-methoxy-5-methylaniline (14)
Aniline 13 (3.51 g, 24.7 mmol) was dissolved in MeCN (100 ml) and cooled to ca. -5 °C using a
NaCl-ice bath. NBS (4.39 g, 24.7 mmol) was added in portions and the reaction was allowed to
warm to room temperature over the course of two hours. The reaction mixture was adsorbed on
silica gel and further purified using CombiFlash chromatography eluting with hexanes/ethyl
acetate (gradient 0 to 20%). Combining the clean fractions afforded 2.31 grams (43%) of the
title compound 14 as a white solid.
19

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1H NMR (300 MHz, DMSO-d6) δ 6.15 (s, 2H), 5.17 (s, 2H), 2.18 (s, 3H); 13C NMR (75 MHz,
DMSO) δ 155.9, 148.7, 138.1, 108.4, 98.2, 96.3, 22.9; MS (ESI) (m/z): 219/221 [M+H]+; HRMS
(TOF ES+) m/z [M+H]+ calcd. for C8H7D3BrNO: 219.0209; found: 219.0214.
[D3]-3-Methoxy-5-methyl-N-tritylaniline (15)
Chloride 12 (6.30 g, 39.5 mmol) was dissolved in dry THF and cooled to -78 °C using an
acetone/dry-ice bath. NaNH2 (4.61 g, 118 mmol) was added and the clear, slightly milky
solution was stirred for 3 hours at -78 °C. Then, triphenylmethylamine (20.5 g, 78.9 mmol) was
added and the reaction mixture was allowed to warm to room temperature overnight. Analysis
by TLC showed a product spot along with various by-products. The brown-reddish suspension
was then carefully quenched by the dropwise addition of H2O (exothermic, gas evolution). More
water was added, and the aqueous layer was extracted with ethyl ether (3x 100 ml). After drying
over Na2SO4 the crude product was further purified by CombiFlash chromatography eluting with
hexanes/ethyl acetate (gradient 0 to 5%). Pooling the clean fractions resulted in 763 mg (5%
yield) of the desired aniline 15 in the form of a beige solid. The low yield might be due to
precipitation of product on the column.
1H NMR (300 MHz, CDCl3) δ 7.42 (t, J = 2.0 Hz, 2H), 7.41 – 7.37 (m, 4H), 7.35 – 7.29 (m, 4H),
7.29 – 7.19 (m, 5H), 6.01 (s, 1H), 5.96 (s, 1H), 5.67 (t, J = 2.2 Hz, 1H), 5.04 (s, 1H), 2.13 (s,
3H); 13C NMR (75 MHz, CDCl3) δ 159.6, 147.4, 145.4, 138.7, 129.2, 127.8, 126.7, 109.7, 104.2,
99.1, 71.3, 21.6; MS (ESI) (m/z): 383 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for
C27H22D3NO: 383.2200; found: 383.2168.
Trityl-aniline 15 (64 mg, 0.167 mmol) was dissolved in CHCl3 (5 ml) and cooled to 0 °C. NBS
(30 mg, 0.167 mmol) was added and the solution was stirred for 5 hours at ice-bath temperature.
The reaction mixture was allowed to warm to room temperature overnight and then worked up.
Analysis by LCMS and NMR revealed that ~ 2:1 mixture of the two bromo-regioisomers had
formed. An attempted separation by flash chromatography was unsuccessful.
1H NMR (300 MHz, CDCl3) selected peaks (major isomer) δ 6.10 (s, 1H), 5.64 (s, 1H), 2.58 (s,
3H); (minor isomer) δ 6.03 (s, 1H), 5.57 (s, 1H), 2.33 (s, 3H); MS (ESI) (m/z): 461/463 [M+H]+.
[D3]-N-Benzyl-3-methoxy-5-methylaniline (17)
Chloride 12 (31.5 g, 197 mmol) was dissolved in dry THF (500 ml) and cooled to -78 °C with an
acetone/dry-ice bath. Under a protective stream of nitrogen, NaNH2 (23.1 g, 592 mmol) was
added and the pale yellow-beige suspension was stirred for 1 hour at -78 °C. Benzylamine (43
ml, 395 mmol) was added and after 2 hours the reaction mixture was allowed to warm to room
temperature overnight. Stirring was continued for an additional 2 days in which the mixture
turned into a dark violet solution. The reaction was then carefully quenched by the dropwise
addition of H2O (exothermic, gas evolution). More water was added, and the aqueous layer was
extracted with ethyl ether (3x 200 ml). The organic layers were filtered through Na2SO4 and
solvents were evaporated under reduced pressure. The crude product was further purified by
column chromatography eluting with hexanes/ethyl acetate (20/1, isocratic). Combining the
clean fractions resulted in 19.7 g (43% yield) of the desired benzyl aniline 17.
20

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1H NMR (300 MHz, DMSO-d6) δ 7.41 – 7.16 (m, 5H), 6.16 (t, J = 6.0 Hz, 1H), 6.05 (s, 1H),
5.95 (s, 2H), 4.24 (d, J = 6.1 Hz, 2H), 2.12 (s, 3H); 13C NMR (75 MHz, DMSO) δ 160.2, 149.8,
140.4, 138.7, 128.2, 127.1, 126.5, 106.2, 102.2, 95.5, 46.5, 21.6; MS (ESI) (m/z): 231 [M+H]+;
HRMS (TOF ES+) m/z [M+H]+ calcd. for C15H14D3NO: 231.1574; found: 231.1473.
[D3]-tert-Butyl benzyl(3-methoxy-5-methylphenyl)carbamate (18)
Benzyl aniline 17 (10.5 g, 45.6 mM) was dissolved in anhydrous THF (200 ml). Et3N (9.50 ml,
68.4 mmol) and Boc2O (24.9 g, 114 mmol) were added via syringe. The resulting clear yellow
solution was stirred at 60 °C overnight. Analysis by TLC and LCMS showed complete
conversion of the starting material. Silica gel was added to the reaction mixture and solvents
were removed under reduced pressure. Further purification of the crude product by CombiFlash
chromatography eluting with hexanes/ethyl acetate (gradient 0 to 8%) furnished 11.1 g (74%
yield) of the Boc-protected aniline 18 in form of a clear oil. Rf (starting material) = 0.40; Rf
(product) = 0.36 (SiO2, hexanes/EtOAc 10/1).
1H NMR (300 MHz, CDCl3) δ 7.34 – 7.20 (m, 5H), 6.61 (s, 1H), 6.53 (s, 1H), 6.51 (s, 1H), 4.80
(s, 2H), 2.27 (s, 3H), 1.43 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 159.5, 154.7, 143.7, 139.3,
138.7, 128.3, 127.3, 126.9, 119.6, 112.3, 109.5, 80.4, 54.0, 28.3, 21.5; MS (ESI) (m/z): 331
[M+H]+; HRMS (TOF ES+) m/z [M-tBu+H]+ calcd. for C16H14D3NO3: 275.1472; found:
275.1474.
[D3]-tert-butyl benzyl(4-bromo-3-methoxy-5-methylphenyl)carbamate (19)
Benzyl Boc-aniline 18 (6.61 g, 20.0 mmol) was dissolved in dry DCM (200 ml) and NBS (3.73
g, 21.0 mmol) was added. The clear, pale yellow solution was stirred at room temperature for
one day. Silica gel was added to the reaction mixture and the solvent was evaporated in vacuo.
Purification by CombiFlash chromatography eluting with hexanes/ethyl acetate (gradient 0 to
8%) provided 7.39 g (90% yield) of a light yellow, highly viscous oil (19) which crystalized
upon standing. The regiochemistry of the bromination was confirmed by NOESY experiments
(observed NOEs are shown as arrows in Scheme 9). Rf (starting material) = 0.30; Rf (product) =
0.28 (SiO2, hexanes/EtOAc 10/1).
1H NMR (300 MHz, CDCl3) δ 7.37 – 7.20 (m, 5H), 6.71 (s, 1H), 6.51 (s, 1H), 4.81 (s, 2H), 2.35
(s, 3H), 1.45 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 155.7, 154.4, 142.1, 139.3, 138.4, 128.4,
127.4, 127.1, 120.7, 111.3, 108.3, 80.7, 53.9, 28.2, 23.2; MS (ESI) (m/z): 353/355 [M-tBu+H]+;
HRMS (TOF ES+) m/z [M-tBu +H]+ calcd. for C16H13D3BrNO3: 235.0577; found: 235.0575.
[D3]-tert-butyl benzyl(3-methoxy-5-methyl-4-vinylphenyl)carbamate (20)
Aryl bromide 19 (4.69 g, 11.5 mmol), vinylboronic acid pinacol ester (3.83 g, 24.9 mmol),
PdCl2(PPh3)2 (1.01 g, 1.43 mmol) and Na2CO3 (2 M in H2O, 15 ml, 30 mmol) were mixed with
DME/EtOH/H2O (ratio: 7/3/2, 80 ml). The mixture was degased and flushed with N2 (3x). The
egg yolk yellow suspension was then heated at 110 °C for 5 hours. Water (200 ml) was added to
the now homogenous pale-yellow solution and the aqueous phase was extracted with EtOAc (2x
21

ACCEPTED MANUSCRIPT
100 ml) and Et2O (1x 100 ml). The combined organic layers were filtered through MgSO4 and
silica gel was added. Solvents were removed under reduced pressure and the crude product was
further purified by CombiFlash chromatography eluting with hexanes/EtOAc (gradient 0 to 7%).
The pooled, clean fractions resulted in 3.38 g (83% yield) of the desired product 20 in the form
of a greenish oil that started to crystalize upon standing.
1H NMR (300 MHz, CDCl3) δ 7.36 – 7.21 (m, 5H), 6.80 – 6.66 (m, 1H), 6.66 (s, 1H), 6.53 (s,
1H), 5.65 (d, J = 18.0 Hz, 1H), 5.49 (d, J = 11.8 Hz, 1H), 4.83 (s, 2H), 2.31 (s, 3H), 1.46 (s, 9H);
13C NMR (75 MHz, CDCl3) δ 157.6, 154.7, 142.0, 138.9, 137.4, 130.8, 130.7, 128.4, 128.2,
127.4, 127.0, 123.6, 120.6, 119.1, 107.3, 80.5, 54.0, 28.3, 21.0; MS (ESI) (m/z): 357 [M+H]+;
HRMS (TOF ES+) m/z [M-tBu +H]+ calcd. for C18H16D3NO3: 301.1631; found: 301.1652.
[D3]-tert-butyl benzyl(4-formyl-3-methoxy-5-methylphenyl)carbamate (22)
Vinyl phenyl compound 20 (3.19g, 8.93 mmol) was dissolved in dioxane (170 ml) and 2,6-
lutidine (2.08 ml, 17.8 mmol) was added. An aqueous OsO4 solution (4 wt %, Aldrich) (5 ml,
0.786 mmol) was transferred into the flask via syringe; the reaction mixture turned red-blackish
within minutes and was stirred at room temperature for 20 minutes. In a separate flask, NaIO4
(7.64 g, 35.7 mmol) was dissolved in hot water (60 ml) using sonication. The NaIO4 solution
was added to the reaction resulting in the formation of a voluminous grey-beige precipitate.
Stirring was continued for 90 min; analysis by TLC indicated that the reaction had been
completed. Water (~200 ml) was added and the mixture was made slightly acidic with 0.5 M
HCl to drive the lutidine into the aqueous phase. The mixture was extracted with Et2O (3x 200
ml) and the pooled organic layers were filtered through MgSO4. After removing the solvents
under reduced pressure, a blackish oil (22) remained (3.01 g, 94%).
1H NMR (300 MHz, CDCl3) δ 10.52 (s, 1H), 7.38 – 7.18 (m, 5H), 6.72 (s, 1H), 6.66 (s, 1H), 4.87
(s, 2H), 2.51 (s, 3H), 1.44 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 191.2, 163.4, 154.0, 148.2,
142.6, 138.3, 128.5, 128.5, 127.2, 126.9, 120.4, 120.4, 106.5, 81.4, 53.5, 28.2, 21.8; MS (ESI)
(m/z): 359 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C21H22D3NO4: 359.2047; found:
359.2040.
[D3]-3-Methoxy-4,5-dimethylaniline (25)
For analytical data see compound 10 (HBr-salt).
[D3]-3-methoxy-N-(4-methoxybenzyl)-5-methylaniline (26)
Chloride 12 (41.4 g, 259 mmol) was placed in flask and dissolved in dry THF (500 ml). The
solution was degased, flushed with nitrogen (3x) and subsequently cooled to -78 °C using an
acetone/dry-ice bath. NaNH2 (30.3 g, 778 mmol) was added under a protective stream of
nitrogen and the grey-white suspension was stirred for 1 hour at -78 °C. (4-
methoxyphenyl)methanamine (71.1 g, 518 mmol) was added. After 10 minutes the dry-ice bath
was removed, and the reaction mixture was allowed to warm to room temperature overnight.
The mixture had turned into a dark violet-purple solution and stirring was continued for one
more day. Analysis by TLC showed the desired product along with many side product spots.
22

ACCEPTED MANUSCRIPT
The reaction was then carefully quenched by the dropwise addition of H2O (exothermic, NH3
formation, strong heat tone, some ice-bath cooling required). More water (~400 ml) was added
and the aqueous layer was extracted with ethyl ether (3x 300 ml) and ethyl acetate (1x 200 ml).
The pooled organic phases were filtered through Na2SO4 and solvents were evaporated under
reduced pressure. The amber crude oil was further purified by column chromatography eluting
with hexanes/ethyl acetate (10/1, isocratic). Combining the clean fractions resulted in 22.8 g
(34% yield) of the desired PMB-aniline 26 in the form of a pale-yellow oil.
1H NMR (300 MHz, CDCl3) δ 7.35 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.6 Hz, 2H), 6.21 (s, 1H),
6.16 (s, 1H), 6.10 (s, 1H), 4.28 (s, 2H), 4.00 (s br, 1H), 3.86 (s, 3H), 2.33 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 160.6, 158.7, 149.3, 139.8, 131.3, 128.6, 113.8, 106.6, 103.5, 95.9, 55.1, 47.6,
21.7; MS (ESI) (m/z): 261 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C16H16D3NO2:
261.1679; found: 261.1635.
[D3]-tert-Butyl (4-bromo-3-methoxy-5-methylphenyl)(4-methoxybenzyl)carbamate (27)
PMB aniline 26 (12.6 g, 48.4 mmol) was placed in a pressure vessel and dissolved in dry THF
(300 ml). Et3N (13.4 ml, 96.8 mmol) and Boc2O (21.1 g, 96.8 mmol) were added via syringe. A
catalytic amount of DMAP (110 mg) was also added. The clear pale-yellow solution was stirred
at 85 °C overnight. Water (300 ml) was added and the mixture was stirred at 50 °C for one hour.
The mixture was extracted with Et2O (3x 200 ml) and dried over Na2SO4. Silica gel was added
to the pooled organic fractions and solvents were removed under reduced pressure. Further
purification of the crude product by CombiFlash chromatography eluting with DCM (neat)
delivered 11.5 g (66% yield) of the Boc-protected aniline in the form of a clear oil. Rf (starting
material) = 0.49; Rf (product) = 0.36 (SiO2, DCM).
1H NMR (300 MHz, CDCl3) δ 7.17 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 6.59 (s, 1H),
6.54 (s, 1H), 6.48 (s, 1H), 4.74 (s, 2H), 3.78 (s, 3H), 2.27 (s, 3H), 1.45 (s, 9H); 13C NMR (75
MHz, CDCl3) δ 159.5, 158.6, 154.6, 143.6, 139.2, 130.8, 128.7, 119.8, 113.6, 112.2, 109.7, 80.2,
55.1, 53.3, 28.2, 21.5; MS (ESI) (m/z): 361 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for
C21H24D3NO4: 361.2204; found: 361.2221.
The PMB Boc-aniline (13.6 g, 37.6 mmol) from the above reaction was dissolved in dry DCM
(250 ml) and NBS (7.03 g, 39.5 mmol) was added. The clear, pale yellowish solution was stirred
at room temperature for one day. Since the reaction was not quite complete, MeCN (300 ml)
was added and the mixture was stirred for one more day at room temperature. Finally, the
mixture was heated at 40 °C for one hour to complete the conversion. Silica gel was added to the
reaction mixture and the solvents were evaporated in vacuo. Purification by CombiFlash
chromatography eluting with DCM (neat) gave 13.8 g (83%) of the desired bromide 27 in the
form of a pale orange, highly viscous oil. The regiochemistry of the bromination was confirmed
by NOESY experiments (observed NOEs are shown as arrows in Scheme 11). Rf (starting
material) = 0.48; Rf (product) = 0.53 (SiO2, DCM).
1H NMR (300 MHz, CDCl3) δ 7.14 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 8.7 Hz, 2H), 6.66 (s, 1H),
6.47 (s, 1H), 4.72 (s, 2H), 3.78 (s, 3H), 2.34 (s, 3H), 1.44 (s, 9H); 13C NMR (75 MHz, CDCl3) δ
158.7, 155.7, 154.4, 142.1, 139.3, 130.4, 128.9, 121.0, 113.7, 111.3, 108.5, 80.6, 55.1, 53.3, 28.3,
23

ACCEPTED MANUSCRIPT
23.2; MS (ESI) (m/z): 439/441 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for
C21H23D3BrNO4: 439.1309/441.1291; found: 439.1309/441.1297.
[D3]-tert-Butyl (3-methoxy-5-methyl-4-vinylphenyl)(4-methoxybenzyl)carbamate (28)
A pressure vessel was charged with aryl bromide 27 (8.50 g, 19.3 mmol), vinyl boronic acid
pinacol ester (7.28 ml, 42.5 mmol), PdCl2(PPh3)2 (2.00 g, 2.90 mmol) and Na2CO3 (2 M in H2O,
6.15 g in 10 ml, 58.0 mmol) were mixed with DME/EtOH/H2O (ratio: 7/3/2, 200 ml). The
mixture was degassed and flushed with N2 (3x). The flask was sealed and the pale-yellow
suspension was heated to 110 °C. The mixture turned into clear solution after about 20 minutes;
heating was continued for 5 hours. After completion of the reaction was confirmed by TLC and
LCMS (Rf/Rt are very similar), the crude mixture was poured into a separation funnel; water
(150 ml) was added and the mixture was extracted with EtOAc (2x 100 ml) and Et2O (1x 100
ml). The pooled organic phases were filtered through MgSO4 and silica gel was added. Solvents
were removed under reduced pressure and the crude product was further purified by CombiFlash
chromatography eluting with hexanes/EtOAc (gradient 0 to 15%). The combined clean fractions
furnished 6.41 g (86% yield) of the desired product 28 as a clear, highly viscous oil with a
blackish tint. Rf (starting material) = 0.37; Rf (product) = 0.39 (SiO2, hexanes/EtOAc 8/2).
1H NMR (300 MHz, CDCl3) δ 7.19 (d, J = 8.8 Hz, 2H), 6.84 (d, J = 8.8 Hz, 2H), 6.73 (dd, J =
18.0, 11.8 Hz, 1H), 6.64 (s, 1H), 6.52 (s, 1H), 5.65 (dd, J = 18.0, 2.3 Hz, 1H), 5.48 (dd, J = 11.8,
2.3 Hz, 1H), 4.76 (s, 2H), 3.76 (s, 3H), 2.31 (s, 3H), 1.47 (s, 9H); 13C NMR (75 MHz, CDCl3) δ
158.5, 157.4, 154.5, 141.7, 137.2, 130.7, 130.5, 128.7, 123.4, 120.6, 118.9, 113.5, 107.3, 80.2,
54.9, 53.2, 28.2, 20.8; MS (ESI) (m/z): 387 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for
C23H26D3NO4: 387.2360; found: 387.2352.
[D3]-tert-Butyl (4-(hydroxymethyl)-3-methoxy-5-methylphenyl)(4-
methoxybenzyl)carbamate (30)
2,6-Lutidine (2.06 ml, 17.7 mmol) and vinyl phenyl compound 28 (3.43g, 8.87 mmol) were
combined in a flask and dissolved in dioxane (170 ml). An aqueous OsO4 solution (4 wt %,
Aldrich) (5.61 ml, 0.881 mmol) was added via syringe. The reaction mixture turned blackish
within minutes, stirring was continued for 20 minutes at room temperature. In a separate flask,
NaIO4 (7.59 g, 35.5 mmol) was dissolved in hot water (50 ml) using sonication. The NaIO4
solution was added to the reaction mixture which in turn resulted in the immediate formation of a
voluminous grey-beige precipitate. Stirring was continued for one hour; analysis by TLC
indicated that the reaction had been completed. Water (~100 ml) was added and the mixture was
made slightly acidic using 0.5M HCl to drive the lutidine into the aqueous phase. The mixture
was extracted with Et2O (3x 150 ml) and the pooled organic layers were filtered through Na2SO4.
Removing the solvents under reduced pressure produced a green-blackish oil/liquid. This crude
aldehyde was taken into the next reaction without further purification. Rf (Ar-vinyl) = 0.51; Rf
(Ar-CHO) = 0.32 (SiO2, hexanes/EtOAc 8/2); MS (ESI) (m/z): 389 [M+H]+.
The crude oil from the above reaction was dissolved in reagent alcohol (EtOH, iPrOH 95/5, 120
ml) and NaBH4 (671 mg, 17.7 mmol) was added in portions. The mixture was stirred overnight
at room temperature. The reaction was quenched with water (10 ml) and solvents were removed
in vacuo. The crude product was adsorbed on silica gel and purified by CombiFlash
24

ACCEPTED MANUSCRIPT
chromatography eluting with hexanes/EtOAc (gradient 0 to 30%). Combining the clean fraction
provided 1.43 g (41% over two steps) of the desired benzyl alcohol 30 in form of a brown oil. Rf
(Ar-CHO) = 0.32; Rf (Ar-CH2OH) = 0.08 (SiO2, hexanes/EtOAc 8/2).
1H NMR (300 MHz, CDCl3) δ 7.16 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.4 Hz, 2H), 6.60 (s, 1H),
6.49 (s, 1H), 4.72 (s, 2H), 4.67 (s, 2H), 3.78 (s, 3H), 2.31 (s, 3H), 2.20 (s br, 1H), 1.44 (s, 9H);
13C NMR (75 MHz, CDCl3) δ 158.6, 157.9, 154.6, 142.8, 137.8, 130.8, 128.8, 124.8, 120.8,
113.6, 107.4, 80.4, 57.0, 55.1, 53.3, 28.3, 19.2; MS (ESI) (m/z): 391 [M+H]+; HRMS (TOF ES+)
m/z [M+H]+ calcd. for C22H26D3NO5: 391.2309; found: 391.2301.
[D3]-tert-Butyl (3-methoxy-4,5-dimethylphenyl)carbamate (31)
Benzyl alcohol 30 (368 mg, 0.942 mmol) was dissolved in isopropanol (50 ml) and Pd/C (5% wt.
% loading, dry basis) (100 mg, 46.9 µmol) was added. The flask was degassed and flushed with
H2 (3x). The mixture was stirred under hydrogen (balloon) at room temperature overnight. The
mixture was then filtered through celite and adsorbed onto silica gel. Purification by
CombiFlash eluting with hexanes/EtOAc (gradient 0 to 8%) resulted in isolation of methoxy-4-
methylbenzene and the Boc-protected aniline 31 (219 mg, 91%). Rf (starting material) = 0.24; Rf
(methoxy-4-methylbenzene) = 0.66; Rf (Boc-protected aniline 31) = 0.71 (SiO2, hexanes/EtOAc
7/3).
1H NMR (300 MHz, CDCl3) δ 6.97 (s, 1H), 6.64 (s, 1H), 6.50 (s, 1H), 2.22 (s, 3H), 2.08 (s, 3H),
1.53 (s, 9H); MS (ESI) (m/z): 253 [M-H]+.
[D3]-tert-Butyl (4-formyl-3-methoxy-5-methylphenyl)(4-methoxybenzyl)carbamate (32)
Oxidation via DDQ
Benzyl alcohol 30 (56 mg, 0.143 mmol) was dissolved in DCM (4 ml). DDQ (97 mg, 0.431
mmol) was suspended in H2O (1 ml) and the orange suspension was added to the reaction
mixture. The two-phase mixture turned dark-red and was stirred vigorously overnight at room
temperature. Analysis by TLC showed a spot to spot reaction. The mixture was then filtered
through a Büchner funnel (medium frit) to remove excess DDQ. More water (3 ml) was added,
the organic phase was separated and passed through a plug of MgSO4. Silica gel was added and
solvents were removed under reduced pressure. Further purification by CombiFlash
chromatography (gradient 0 to 20%) yielded 47 mg (85%) of the aldehyde 32. Rf (starting
material) = 0.18; Rf (aldehyde) = 0.32 (SiO2, hexanes/EtOAc 7/3).
1H NMR (300 MHz, CDCl3) δ 10.46 (d, J = 1.9 Hz, 1H), 7.11 (d, J = 6.6 Hz, 2H), 6.78 (d, J =
6.5 Hz, 2H), 6.68 (s, 1H), 6.62 (s, 1H), 4.77 (s, 2H), 3.68 (s, 3H), 2.46 (s, 3H), 1.40 (s, 9H); 13C
NMR (75 MHz, CDCl3) δ 209.0, 190.6, 163.1, 158.5, 153.7, 147.9, 142.0, 130.0, 128.0, 120.4,
120.0, 113.6, 106.5, 80.9, 54.8, 52.5, 27.9, 21.4; MS (ESI) (m/z): 389 [M+H]+; HRMS (TOF
ES+) m/z [M+H]+ calcd. for C22H24D3NO5: 389.2153; found: 389.2155.
Oxidation via DMP
Benzyl alcohol 30 (760 mg, 1.95 mmol) was dissolved in wet DCM (50 ml, water[15d] seems to
improve the reaction rate). Solid Dess–Martin periodinane (DMP) (825 mg, 2.92 mmol) was
added and the resulting grey-brownish suspension was stirred in an open flask at room
25

ACCEPTED MANUSCRIPT
temperature for 4.5 hours. Sat. aq. NaHCO3 (50 ml) was added and the mixture was partitioned
between organic and aqueous phase. After further extraction of the aqueous layer with DCM (3x
50 ml) the combined organics were dried (MgSO4) and purified by CombiFlash chromatography
on silica gel eluting with hexanes/EtOAc (gradient 0 to 20%). The aldehyde 32 was obtained as
a clear, pale-yellow oil (598 mg, 79%).
Analytical data was identical to the data obtained for the compound above using the DDQ
oxidation procedure.
[D7]-5-((2-(benzyl(4-bromo-3-methoxy-5-methylphenyl)amino)-5-methylpyrimidin-4-
yl)amino)benzo[d]oxazol-2(3H)-one (34)
Chloro-pyrimidine 4 (1.02 g, 3.63 mmol), aniline 19 (2.23 g, 5.45 mmol) and PTSA•H2O (1.04
g, 5.45 mmol) were placed in a pressure vessel and isopropanol (80 ml) was added. The flask
was sealed and the off-white/peach-colored suspension was heated at 100 °C for 21 hours.
During this time the reaction mixture became a clear, light reddish solution. After cooling to
room temperature, 2 M methanolic ammonia (3 ml) was added to neutralize the mixture. The
crude product was adsorbed on silica gel by removing solvents under reduced pressure.
CombiFlash chromatography eluting with DCM/MeOH-NH3 (2 M) (gradient 0 to 8%) gave 983
mg (49%) of compound 34 in the form of an off-white solid. Rf (starting material 4) = 0.33; Rf
(product 34) = 0.54; Rf (aniline 19) = 0.95 (SiO2, DCM/MeOH-NH3 (2 M) 9/1).
1H NMR (300 MHz, DMSO-d6) δ 11.48 (s, 1H), 8.23 (s, 1H), 7.40 – 7.08 (m, 6H), 6.83 (d, J =
9.5 Hz, 2H), 6.74 (d, J = 8.8 Hz, 1H), 5.19 (s, 2H), 3.34 (s, 1H), 2.24 (s, 3H); 13C NMR (75
MHz, DMSO-d6) δ 184.3, 159.7, 158.7, 155.3, 154.7, 144.4, 139.2, 138.6, 138.2, 136.0, 129.9,
128.1, 127.0, 126.5, 121.2, 114.7, 109.2, 108.9, 107.9, 105.1, 103.3, 52.6, 22.8; MS (ESI) (m/z):
553/555 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C27H17D7BrN5O3: 553.1573; found:
553.1569.
[D7]-5-((2-(Benzyl(3-methoxy-5-methyl-4-vinylphenyl)amino)-5-methylpyrimidin-4-
yl)amino)benzo[d]oxazol-2(3H)-one (35)
Bromide 34 (567 mg, 1.02 mmol), vinyl-MIDA (225 mg, 1.22 mM), Pd(OAc)2 (11.5 mg, 5 mol
%), SPhos (42 mg, 10 mol %) and K3PO4 (1.63 g, 7.68 mmol) were combined in a pressure
vessel and a 5:1 mixture of dioxane/water (108 ml) was added. The mixture was degassed and
flushed with argon (3x). The clear, light brown reaction mixture was then heated at 60 °C for 23
hours. Analysis by TLC and HPLC indicated that only traces of product had formed. More
vinyl-MIDA (230 mg), Pd(OAc)2 (50 mg), SPhos (200 mg) were added and the mixture was
heated at 100 °C for an additional 24 hours. TLC and HPLC showed roughly 50% conversion
and the reaction was worked up at this point: Silica gel was added and solvents were evaporated
in vacuo. Purification by CombiFlash eluting with DCM/MeOH-NH3 (2 M) (gradient 0 to 8%)
furnished 240 mg (47%) of compound 35 as beige solid. Rf (starting material) = 0.38; Rf
(product) = 0.42 (SiO2, DCM/MeOH-NH3 (2 M) 9/1).
1H NMR (300 MHz, DMSO-d6) δ 11.46 (s, 1H), 8.19 (s, 1H), 7.38 (dd, J = 8.8, 2.3 Hz, 1H), 7.31
– 7.13 (m, 6H), 6.80 – 6.64 (m, 4H), 5.65 (dd, J = 17.9, 2.4 Hz, 1H), 5.43 (dd, J = 11.9, 2.4 Hz,
26

ACCEPTED MANUSCRIPT
1H), 5.19 (s, 2H), 2.22 (s, 3H); 13C NMR (75 MHz, DMSO-d6) δ 159.9, 158.7, 157.5, 154.7,
144.1, 139.5, 138.6, 136.6, 136.1, 130.7, 129.9, 128.1, 126.9, 126.4, 121.5, 120.9, 118.5, 114.6,
108.0, 104.9, 103.1, 52.7, 20.8; MS (ESI) (m/z): 501 [M+H]+; HRMS (TOF ES+) m/z [M+H]+
calcd. for C29H20D7N5O3: 501.2625; found: 501.2598.
[D7]-5-((2-(Benzyl(4-(hydroxymethyl)-3-methoxy-5-methylphenyl)amino)-5-
methylpyrimidin-4-yl)amino)benzo[d]oxazol-2(3H)-one (36)
The styrene derivative 35 (86.1 mg, 0.172 mmol) and 2,6-lutidine (~0.05 ml, 0.344 mmol) were
dissolved in dioxane (8 ml) to give a clear, yellow solution. An aqueous OsO4 solution (4 wt %,
Aldrich) (0.33 ml, 0.052 mmol) was added via syringe. The reaction mixture turned blackish
within minutes; stirring was continued for 10 minutes at room temperature. NaIO4 (147 mg,
0.688 mmol) was dissolved in hot water (2 ml) with the help of sonication. This solution was
added to the reaction mixture. Immediately, formation of a voluminous grey-beige precipitate
was observed. Stirring was continued for two hours; analysis by TLC & LCMS indicated that
the reaction had gone to completion. Silica gel was added to the crude reaction mixture and
solvents were evaporated under reduced pressure. The crude product was further purified using
CombiFlash chromatography eluting with DCM/MeOH-NH3 (2 M) (gradient 0 to 5%).
Combining the clean fractions afforded 64.1 mg (74%) of the desired aldehyde as a yellowish
solid. Rf (starting material) = 0.34; Rf (product) = 0.24 (SiO2, DCM/MeOH-NH3 (2 M) 95/5).
1H NMR (300 MHz, CDCl3) δ 10.47 (s, 1H), 8.62 (s, 1H), 7.45 – 7.12 (m, 6H), 6.90 (s, 1H), 6.87
– 6.83 (m, 1H), 6.77 (dd, J = 2.0, 1.0 Hz, 2H), 6.52 (s, 1H), 5.29 (s, 2H), 2.46 (s, 3H); MS (ESI)
(m/z): 503 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C28H18D7N5O4: 503.2417; found:
503.2414.
The aldehyde (64.1 mg, 0.127 mmol) from the above reaction was dissolved in reagent alcohol
(5 ml) and NaBH4 (9.6 mg, 0.254 mmol) was added. The clear, pale yellow solution was stirred
at room temperature for two hours. A drop of water was added to quench the reaction and the
mixture was then adsorbed on silica gel. Further purification by flash chromatography eluting
with DCM/MeOH-NH3 (2 M) (gradient 0 to 10%) gave alcohol 36 in the form of a white
crystalline solid (55 mg, 86%). Rf (starting material) = 0.48; Rf (product) = 0.55 (SiO2,
DCM/MeOH-NH3 (2 M) 9/1).
1H NMR (300 MHz, CDCl3) δ 10.10 (s, 1H), 7.54 (d, J = 2.2 Hz, 1H), 7.37 – 7.16 (m, 5H), 6.94
(d, J = 8.5 Hz, 1H), 6.75 (d, J = 1.9 Hz, 1H), 6.61 (d, J = 2.0 Hz, 1H), 6.50 (dd, J = 8.6, 1.7 Hz,
1H), 6.32 (s, 1H), 5.22 (s, 2H), 4.88 (d, J = 2.0 Hz, 2H), 3.47 (s, 1H), 2.36 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 160.9, 158.4, 158.1, 156.0, 145.7, 139.2, 139.1, 138.7, 136.1, 130.1, 128.2,
127.7, 126.8, 123.4, 122.5, 112.0, 109.4, 109.0, 103.7, 101.6, 56.0, 53.4, 19.0; MS (ESI) (m/z):
505 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for C28H20D7N5O3: 505.2574; found:
505.2551.
[D3]-4-((tert-Butoxycarbonyl)(4-methoxybenzyl)amino)-2-methoxy-6-methylbenzoic acid
(37)
The aldehyde 32 (598 mg, 1.54 mmol) was dissolved in tBuOH (50 ml) and 2-methyl-2-butene
(β-Isoamylene) (10 ml) was added. The clear solution was cooled to 0 °C with an ice-bath. In a
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ACCEPTED MANUSCRIPT
separate flask, NaClO2 (80%, technical) (1.39 g, 15.4 mmol) and NaH2PO4 (1.85 g, 15.4 mmol)
were dissolved in H2O (30 ml). This solution was added dropwise to the reaction mixture which
turned into a milky suspension; it was allowed to warm to room temperature over three hours.
Sat. NH4Cl (50 ml) was added and the mixture was extracted with EtOAc (3x 50 ml). The
combined organic phases were passed through a plug of MgSO4 and solvents were removed
under reduced pressure. The desired acid 37 (611 mg, 98%) was obtained in the form of a pale
yellowish-milky viscous oil that started to crystallize upon standing. Rf (starting material) =
0.52; Rf (product) = 0.02 (SiO2, hexanes/EtOAc 7/3).
1H NMR (300 MHz, CDCl3) δ 7.45 (s, 1H), 7.09 (d, J = 8.2 Hz, 2H), 6.77 (d, J = 8.1 Hz, 2H),
6.58 (s, 1H), 6.52 (s, 1H), 4.69 (s, 2H), 3.68 (s, 3H), 2.27 (s, 3H), 1.39 (s, 9H); 13C NMR (75
MHz, CDCl3) δ 170.1, 158.4, 156.5, 154.2, 144.3, 137.4, 130.0, 128.5, 120.3, 113.5, 107.3, 80.6,
54.8, 52.9, 28.0, 19.6; MS (ESI) (m/z): 405 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for
C22H24D3NO6: 405.2102; found: 405.2090.
[D3]-Methyl 4-((tert-butoxycarbonyl)(4-methoxybenzyl)amino)-2-methoxy-6-
methylbenzoate (38)
Acid 37 (611 mg, 1.51 mmol) was placed in a pressure vessel and dissolved in acetone. K2CO3
(1.04 g, 7.55 mmol), Cs2CO3 (492 mg, 1.51 mmol) and methyl iodide (1.71 g, 12.1 mmol) were
added. The mixture was heated at 90 °C for two hours. Analysis by TLC showed complete
conversion and salts were filtered off. Silica gel was added to the solution and solvents were
removed under reduced pressure. Purification by CombiFlash chromatography eluting with
hexanes/EtOAc (gradient, 0 to 20%) furnished 606 mg (96%) of methyl ester 38 as a clear
viscous oil. Rf (starting material) = 0.01; Rf (product) = 0.19 (SiO2, hexanes/EtOAc 8/2).
1H NMR (300 MHz, CDCl3) δ 7.11 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 8.7 Hz, 2H), 6.57 (s, 1H),
6.49 (s, 1H), 4.70 (s, 2H), 3.82 (s, 3H), 3.71 (s, 3H), 2.18 (s, 3H), 1.40 (s, 9H); 13C NMR (75
MHz, CDCl3) δ 168.1, 158.5, 156.4, 154.1, 144.3, 136.4, 130.2, 128.6, 120.8, 120.0, 113.5,
107.3, 80.4, 54.8, 52.9, 51.8, 28.0, 19.1; MS (ESI) (m/z): 419 [M+H]+; HRMS (TOF ES+) m/z
[M+H]+ calcd. for C23H26D3NO6: 419.2258; found: 419.2272.
[D3]-Methyl 2-methoxy-4-((4-methoxybenzyl)amino)-6-methylbenzoate (39)
Boc-protected aniline 38 (439 mg, 1.05 mmol) was dissolved in dry DCM (20 ml) and TFA
(0.48 ml, 6.30 mmol) was added. The clear solution was heated at 30 °C overnight; the color
changed to yellow during heating. Sat. aq. NaHCO3 (20 ml) was poured into the mixture which
was subsequently extracted with DCM (3x 20 ml). The combined organic layers were dried over
MgSO4 and the crude product was adsorbed on silica gel. Purification by flash chromatography
eluting with hexanes/EtOAc (gradient 0 to 25%) yielded 281 mg (65%) of the desired compound
39. Rf (starting material) = 0.44; Rf (product) = 0.29 (SiO2, hexanes/EtOAc 7/3).
1H NMR (300 MHz, CDCl3) δ 7.26 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 6.04 (s, 1H),
5.99 (s, 1H), 4.25 (s, 2H), 4.20 (s br, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 2.23 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 168.9, 158.8, 158.7, 150.1, 138.6, 130.8, 128.6, 114.0, 112.3, 106.5, 93.5, 55.2,
51.7, 47.3, 20.2; MS (ESI) (m/z): 319 [M+H]+; HRMS (TOF ES+) m/z [M+H]+ calcd. for
C18H18D3NO4: 319.1734; found: 319.1733.
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