Synthesis of thioridazine-VLA-4 antagonist hybrids using N-propargyl northioridazine enantiomers

Article abstract of DOI:10.1080/00397911.2020.1785503

Herein, we report the synthesis of thioridazine-VLA-4 hybrid epimers. These hybrid molecules were obtained by means of a click reaction involving N-propargyl northioridazine enantiomers and an azide containing VLA-4 antagonist. A synthesis of northioridaz

Full text of DOI:10.1080/00397911.2020.1785503

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Synthesis of thioridazine-VLA-4 antagonist hybrids
using N-propargyl northioridazine enantiomers
A. P. Martyn, A. C. Willis & M. J. Kelso
To cite this article: A. P. Martyn, A. C. Willis & M. J. Kelso (2020): Synthesis of thioridazine-VLA-4
antagonist hybrids using N-propargyl northioridazine enantiomers, Synthetic Communications, DOI:
10.1080/00397911.2020.1785503
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SYNTHETIC COMMUNICATIONSV
https://doi.org/10.1080/00397911.2020.1785503
Synthesis of thioridazine-VLA-4 antagonist hybrids using
N-propargyl northioridazine enantiomers
A. P. Martyna,b , A. C. Willisc, and M. J. Kelsoa,b
aMolecular Horizons and School of Chemistry & Molecular Bioscience, University of Wollongong (UOW),
b
Wollongong, NSW, Australia; Illawarra Health and Medical Research Institute, Wollongong, NSW,
c
Australia; Single Crystal X-ray Diffraction Unit, Research School of Chemistry, Australian National
University (ANU), Canberra, ACT, Australia
ABSTRACT
ARTICLE HISTORY
Received 28 April 2020
Herein, we report the synthesis of thioridazine-VLA-4 hybrid epimers.
These hybrid molecules were obtained by means of a click reaction
involving N-propargyl northioridazine enantiomers and an azide con-
taining VLA-4 antagonist. A synthesis of northioridazine enantiomers
from racemic thioridazine was developed and the absolute stereo-
chemistry was confirmed by X-ray crystallography. Access to N-
substituted thioridazine analogs may reveal that this phenothiazine
core has therapeutic potential in other disease avenues.
KEYWORDS
Click; northioridazine;
thioridazine; VLA-4
GRAPHICAL ABSTRACT
Introduction
Minimal residual disease (MRD) refers to the residual populations of malignant cells
that survive initial therapies.[1–3] This complication can result in disease recurrence and
significantly reduce the long-term survival rates of patients.[4,5] One therapeutic
approach to reduce these cell populations is to block the VLA-4/VCAM-1 protein-pro-
tein interaction[6] using competitive VLA-4 antagonists (e.g., compound 1, Fig. 1a).[7]
( )-Thioridazine (2) a discontinued anti-psychotic drug, was found to be a VLA-4
allosteric antagonist and HSPC mobilizer (Fig. 1b).[8] Additionally, it is able to
CONTACT A. P. Martyn
martynap@qut.edu.au
Molecular Horizons and School of Chemistry & Molecular
Bioscience, University of Wollongong (UOW), Wollongong, NSW, Australia.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2020 Taylor & Francis Group, LLC

2
A. P. MARTYN ET AL.
(a)
(b)
Figure 1. (a) Structure of potent competitive VLA-4 antagonist 1 (red);[7] (b) Structure of antipsychotic
and VLA-4 allosteric antagonist ( )-thioridazine 2 (blue).[8]
Figure 2. Structure of thioridazine-VLA-4 antagonist hybrid epimers (R)-3 and (S)-3. The thioridazine
chiral center (red circle) was used to annotate the stereochemistry of hybrid epimers. The hybrid
Incorporates the structural components of competitive VLA-4 antagonist 1 (red) and allosteric VLA-4
antagonist thioridazine 2 (blue) tethered by a 1,2,3-triazole linker.
selectively induce both apoptosis[9,10] and differentiation[11] in malignant cells. These
antagonists can mobilize malignant hematopoietic stem and progenitor cells (HSPCs)
from the bone marrow into peripheral blood, where they are more vulnerable to chemo-
therapy allowing them to be better cleared from the body during treatment
(Fig. 1).[12–14]
In a program aiming to investigate the biological activity of thioridazine-VLA-4
antagonist hybrids, we embarked on the synthesis of compound 3 (Fig. 2). All previous
studies investigating the biological effects of thioridazine on VLA-4 were conducted
using racemic mixtures. It is therefore unclear which thioridazine enantiomer (if not
both) is responsible for the effects. It is necessary to incorporate each thioridazine
enantiomer into the hybrid design to reveal any stereochemical effects caused by thiori-
dazine. Stereochemistry of the thioridazine chiral center was used to annotate the hybrid
epimers as (R)-3 and (S)-3.
Carrying racemic thioridazine through the hybrid synthesis would have created com-
plex diastereomeric product mixtures with unknown stereochemistry at the thioridazine

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3
(a)
(b)
Figure 3. Structures of key intermediates required for the synthesis of hybrids (R)-3 and (S)-3. (a) pro-
tected azido-VLA-4 antagonist 4 (red); (b) (R)- or (S)-N-propargyl northioridazine 5 (blue).
portion. The use of optically pure thioridazine ensured that the stereochemistry of the
final hybrids was unambiguous at all three stereocenters.
Results and discussion
Synthetic strategy toward thioridazine-VLA-4 antagonist hybrids
Click chemistry was chosen to connect the thioridazine and VLA-4 antagonist moieties
due to its inherent reliability. The click reagents were designed such that the azide was
substituted on the benzene sulfonamide group of the protected VLA-4 antagonist (4)
and the terminal alkyne was incorporated into N-propargyl northioridazine enantiomers
(R)-5 and (S)-5 (Fig. 3). The thioridazine and VLA-4 antagonist precursors were to be
prepared in parallel and then combined using click chemistry to assemble the methyl
ester protected hybrid scaffold. Subsequent methyl ester hydrolysis would result in the
generation of the hybrid epimers (R)-3 and (S)-3.
Synthesis of the protected azido-VLA-4 antagonist
This class of VLA-4 antagonist has been thoroughly explored and reported in the litera-
ture.[7,15] The protected VLA-4 antagonist (4) contains two major constituents; an N-
sulfonylated proline and a phenylalanine modified with 3,5-dichloroisonicotinamide.
The preparation of the L-proline portion of the antagonist (4) is achieved using Scheme
1. Compound 7 was prepared through the N-sulfonylation of L-proline-OMe.HCl with
4-bromo benzenesulfonyl chloride. Use of organic bases, such as Et3N and DIPEA were
explored, producing 7 in 30% and 35% yield, respectively. Using neat pyridine as the
solvent produced 7 in an improved yield of 61%. Premixing the sulfonyl chloride with
pyridine prior to the addition of L-proline-OMe.HCl accelerated the reaction and led to
isolation of 7 in 83% yield.
Conversion of aryl bromide 7 to the aryl amine (8) was achieved using the conditions
reported by Markiewicz et al.[16] A solution of the aryl bromide (7), sodium azide,
copper(I) iodide and N,N0-dimethylethylenediamine (DMEDA) in anhydrous DMSO
was heated to 100 ꢀC for 3 h. Under these conditions, the aryl amine (8) was isolated in
81% yield after purification by column chromatography. The aryl azide (9) was prepared
in 75% yield by diazotization of 8 followed by the addition of aqueous sodium azide.[17]

4
A. P. MARTYN ET AL.
Scheme 1. Synthesis of intermediate 10. Reagents and conditions: (a) 4-bromobenzene sulfonyl chlor-
ide, pyridine, 0 ꢀC-rt, 16 h (83%); (b) NaN3, CuI (100 mol%), DMEDA (150 mol%), DMSO, 100 ꢀC, 3 h
(81%); (c) conc. HCl, NaNO2, EtOAc, 0 ꢀC-rt, 1 h; (d) NaN3, rt, 3 h, 75%; (e) 1 M LiOH, THF:H2O (3:1), rt,
1 h, quant.
Scheme 2. Synthesis of protected azido-VLA-4 antagonist 4 (red). Reagents and conditions: (a) SOCl2,
DMF (cat.), CH2Cl2, reflux; (b) N-Boc-Phe(p-NH2)-OMe, N-methyl morpholine, CH2Cl2, 0 ꢀC-rt, 94% over
two steps (lit. 82%); (c) TFA:TIPS:H2O (95:2.5:2.5), 30 min, rt, quant; (d) HATU, DIPEA, DMF, rt,
16 h, 69%.
Hydrolysis with 1 M LiOH in 3:1 THF:H2O then afforded carboxylic acid 10 in quanti-
tative yield.
Compound 6 was prepared and purified as per the literature conditions.[18] These
conditions were also reported when this class of VLA-4 antagonists were produced with
an ethyl ester.[15] The acid chloride of 3,5-dichloroisonicotinic acid was freshly made
using thionyl chloride, where it was immediately coupled with N-Boc-Phe(p-NH2)-OMe
affording compound 6 in 94% yield over two steps (Scheme 2). The characterization
data collected matched those reported within the patent. The N-Boc protecting group of
6 was deprotected using a cocktail of trifluoroacetic acid (TFA), triisopropylsilane
(TIPS) and water (95:2.5:2.5). The resulting amine (as a TFA salt) was used immediately
in a coupling reaction with compound 10 to generate the protected azido-VLA-4 antag-
onist 4 (Scheme 2).
Three coupling reagents in two solvents were explored for the preparation of com-
pound 4 (Table 1). Competing amide coupling with TFA was avoided by pre-activating
the carboxylic acid (10) with the coupling reagent prior to its addition. The pre-activa-
tion process took approximately 5–10 min and was characterized by the formation of an
intense yellow solution. Pre-activation was performed in all experiments by slowly add-
ing an excess of N,N-diisopropylethylamine (DIPEA) to a solution of the acid (10) with
the coupling reagent in either dichloromethane (CH2Cl2) or dimethylformamide
(DMF). Each reaction was initiated by transferring this solution via cannula to a flask

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Table 1. Coupling conditions explored to afford the protected azido-VLA-4
antagonist (4).
Entry
Coupling reagent
Solvent
Yield 4 (%)
1
2
3
4
5
6
HBTU
HATU
PyBOP
HBTU
HATU
PyBOP
CH2Cl2
CH2Cl2
CH2Cl2
DMF
DMF
DMF
20
62
67
44
69
68
containing the amine 6.TFA salt. These reactions were stirred at room temperature
overnight (16 h), upon completion they were purified by column chromatography.
Initial experiments aimed to explore the efficiency of the three coupling reagents in
CH2Cl2. Use of N,N,N0,N0-Tetramethyl-O-(1H-benzotriazol-1-yl)uranium (HBTU) pro-
duced a complex mixture of products and afforded compound 4 in 20% yield (Entry 1).
A significant improvement was observed when using 1-[Bis(dimethylamino)methylene]-
1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), which
increased the isolated yield to 62% (Entry 2). Use of Benzotriazole-1-yl-oxy-tris-pyrroli-
dino-phosphonium hexafluorophosphate (PyBOP) was found to generate 4 in compar-
able yields to HATU (67%, Entry 3). Switching the solvent to DMF improved HBTU
effectiveness, generating compound 4 in 44% yield (Entry 4). Although yields were simi-
lar with HATU (69%, Entry 5) and PyBOP (68%, Entry 6), HATU was preferred as it
produced cleaner reaction mixtures allowing for easier purification by silica gel column
chromatography. Over 1 g of the protected VLA-4 antagonist 4 was isolated as a result
of these experiments.
Synthesis of N-propargyl northioridazine enantiomers from ( )-thioridazine
The synthetic strategy toward preparing the N-propargyl northioridazine enantiomers is
outlined in Scheme 3. Choi and coworkers reported that racemic thioridazine can be
converted into two isolable diastereomers ((R)-11 and (S)-11) using a menthol carba-
mate chiral auxiliary.[19] It was envisaged that hydrolysis of these menthyl carbamates
would produce northioridazine (12) enantiomers (Route A). Alternatively, these could
be obtained through N-demethylation of each thioridazine enantiomer (Route B). N-
Alkylation of the northioridazine enantiomers ((R)-12 and (S)-12) with propargyl brom-
ide would produce the N-propargyl northioridazine enantiomers ((R)-5 and (S)-5)
required to assemble the hybrid epimers ((R)-3 and (S)-3).
Using the method developed by Choi and coworkers, the two diastereomeric menthyl
carbamates were formed by reacting commercially available racemic thioridazine. HCl
with (–)-(1 R)-menthol chloroformate in the presence of DIPEA in CH2Cl2.[19] Two
products consistent with thioridazine diastereomers (one less polar and one more polar
by TLC analysis) formed overnight and were isolated by silica gel chromatography. For
ease of reference, F1 refers to the diastereomer that was less polar according to TLC
analysis, and F2 refers to the more polar diastereomer (Scheme 3, F1: Rf ¼ 0.77, F2: Rf
¼ 0.54; EtOAc:Pet. Spirit, 20:80). The reaction was performed multiple times on 5 g

6
A. P. MARTYN ET AL.
scales, producing the pure diastereomers in consistent yields (F1-11 38% and F2-
11 43%).
The absolute stereochemistry of the two thioridazine menthyl carbamates (F1 and F2)
was unknown because Choi and coworkers did not report optical rotation data. The thi-
oridazine-based target compounds made herein after removing the chiral auxiliary
would, therefore, have unknown stereochemistry if the thioridazine stereochemistry was
not first established. The specific rotation of thioridazine enantiomers was most recently
reported by Antonsen et al. as [a]25 ¼ þ19 and [a]25 ¼ ꢁ21 for (R) and (S)-thiorida-
D
D
zine, respectively.[20] Reduction of each thioridazine diastereomer (F1 and F2) to the
constituent thioridazine enantiomers would establish the thioridazine stereochemistry
(by comparison to the reported specific rotation) and by inference, F1 and F2
stereochemistry. Hydrolysis of each diastereomer (denoted (R)-11 or (S)-11)) would
then produce a northioridazine enantiomer ((R)-12 or (S)-12) with known stereochem-
istry. N-substituted thioridazine analogs with known stereochemistry could then be pre-
pared, such as (R)-5 and (S)-5.
The two diastereomers F1-11 and F2-11 were reduced in parallel using LiAlH4 in dry
THF at reflux to generate the two thioridazine enantiomers. These conditions produced the
thioridazine enantiomers from F1 and F2 in 62 and 67% yield, respectively. For unambigu-
ous optical rotation measurements, the enantiomers were further purified by preparative
RP-HPLC, and their measurements performed immediately after lyophilization.
Previous investigations had shown that (ꢁ) optical rotation corresponds to (S)-thiori-
dazine and (þ) optical rotation corresponds to (R)-thioridazine.[20,21] Optical rotations
of the thioridazine enantiomers prepared here indicated that F1-11 provided (þ)-(R)-
thioridazine and F2-11 provided (-)-(S)-thioridazine. Through inference, F1-11 has (R)
chirality and F2-11 has (S). The two carbamate diastereomers are hereafter labeled (R)-
11 and (S-11, respectively.
With absolute stereochemistry assigned to the menthyl thioridazine diastereomers,
preparation of northioridazine enantiomers ((R)-12 and (S)-12) was targeted via
hydrolysis from the corresponding carbamate (Scheme 3, Route A). Hydrolysis using
various nucleophilic bases (e.g., LiOH, KOH, NaOMe, and LiOMe) failed to yield any
northioridazine 12. An extensive set of conditions probing acidic hydrolysis were also
investigated, however, TFA was the only reagent to produce any northioridazine (12).
It was observed that addition of any acid immediately formed a deep-blue solution,
presumably due to protonation of the phenothiazine nitrogen and generation of charge-
delocalized species. Several noteworthy conditions are highlighted in Table 2 where (R)-
11 was used as a standard throughout.
The use of neat TFA at room temperature showed no evidence of hydrolysis; how-
ever, upon heating the reaction mixture to reflux (R)-northioridazine (12) was generated
in 8% yield (Entry 1). Bender and coworkers explored conditions for menthyl carbamate
hydrolysis on related substrates, and reported that cleavage was difficult.[22] Their best
method involved treating the menthyl carbamates with TFA and heating the mixture at
110 ꢀC in a sealed tube for 6 h. Under these conditions, secondary amines were report-
edly generated in modest to high yields. Subjecting the thioridazine menthyl carbamate
(R)-11 to these conditions resulted in a complex mixture of side products. Despite a
considerable amount of starting material remaining (R)-northioridazine (12) was

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Scheme 3. Resolution of ( )-thioridazine and synthesis of N-propargyl northioridazine enantiomers
(R)-5 and (S)-5. Reagents and conditions: (a) (ꢁ)-menthyl chloroformate, DIPEA, CH2Cl2, rt, 18 h; (b)
chromatographic separation, F1-11 (38%) and F2-11 (43%); (c) LiAlH4, THF, reflux, 8 h (R)-2 (73%) and
(S)-2 (68%); (d) TFA:TIPS:H2O (95:2.5:2.5), sealed tube, 110 ꢀC, 3 d (R)-12 (36%) and (S)-12 (31%); (e) 1-
chloroethyl chloroformate, DIPEA, CH2Cl2, rt, 16 h; (f) MeOH, reflux, 4 h (R)-12 (65%) and (S)-12 (71%)
over two steps; (g) 4-nitrobenzene sulfonyl chloride, DIPEA, CH2Cl2, rt, 18 h, 56%; (h) 60% NaH, DMF,
rt; (i) 80% propargyl bromide, rt, 16 h (R)-5 (51%) and (S)-5 (58%) over two steps.
Table 2. Reaction conditions and isolated yields for hydrolysis of (R)-11 to (R)-northioridazine (12)
using route A.
Entry
Solvent
TFA
TFA
TFA
TFA
Temperature
Time
Yield (%)
1
reflux
18 h
6 h
2 d
1 h
3 d
8
20
22
62
36
2a
3a
4b
5a
110 ꢀC
110 ꢀꢀC
ꢂ
110 C
110 ꢀC
TFA:TIPS:H2O
aReaction performed in a sealed tube.
bReaction performed in microwave reactor at 200 MHz.
isolated in 20% yield (Entry 2). A similar result was found if the reaction was left to
continue for 2 d at 110 ꢀC (Entry 3).
Use of a microwave reactor was explored by dissolving the menthyl carbamate in
neat TFA with heating at 200 MHz for one hour. The reaction mixture was significantly
cleaner as observed by TLC analysis. After purification by column chromatography (R)-
northioridazine (12) was isolated in 62% yield (Entry 4). It was observed that the rubber
septum of the microwave vial began protruding outwards during the reaction due to an
internal pressure. A proposed mechanism suggests that TFA promoted cleavage and
was releasing carbon dioxide in situ, therefore, generating the pressure (Fig. 4). This
limited the scale in which the reaction could be performed safely in the microwave.
Using a mixture of TFA:TIPS:H2O (95:2.5:2.5) with the sealed tube method improved
the yield to 36% and produced a cleaner reaction mixture (Entry 5). Our requirement

8
A. P. MARTYN ET AL.
Figure 4. Proposed mechanism for TFA-promoted removal of menthyl carbamate (R)-11 to provide
(R)-12.
to produce larger quantities of the northioridazine enantiomers meant that a more effi-
cient route was required.
An alternative approach for obtaining optically pure northioridazine enantiomers
(12) involved the N-demethylation of thioridazine enantiomers (R)-2 and (S)-2 (Scheme
3, Route B). Reduction of carbamates (R)-11 and (S)-11 to enantiomerically pure thiori-
dazine enantiomers was previously optimized and produced useful quantities of material
for N-demethylation experiments. Despite introducing another synthetic step, this
avoided the challenges faced with hydrolysis.
Preliminary experiments used racemic free base thioridazine to study reaction behav-
ior. After the formation of the 1-chloroethyl thioridazine carbamate, the crude residue
was dissolved into methanol and heated to reflux to generate ( )-northioridazine (11).
Isolation of northioridazine (11) was challenging by column chromatography as
unreacted thioridazine co-eluted in several solvent systems. Purification was simplified
by adsorbing the crude 1-chloroethyl carbamate onto a plug of silica and washing exces-
sively with chloroform. This resulted in the isolation of the thioridazine 1-chloroethyl
carbamate without any trace of residual thioridazine. After heating a solution of the
pure 1-chloroethyl carbamate in methanol to reflux, racemic northioridazine (12) was
isolated in 70% yield without further purification. These conditions were then applied
to both thioridazine enantiomers ((R)-2 and (S)-2) to produce the northioridazine enan-
tiomers ((R)-12 and (S)-12) in 65 and 71% yields, respectively.
Specific rotation data for northioridazine has, to the best of our knowledge, not
previously been reported. Each enantiomer was purified by preparative HPLC and
their purities confirmed by analytical RP-HPLC. A representative chromatogram for
(S)-12 is shown in Figure S1. Accordingly, the measured optical rotations indicated
that (R)-northioridazine and (S)-northioridazine showed (ꢁ) and (þ) rotations,
respectively.
Absolute stereochemistry of the thioridazine enantiomers was inferred from data
reported in the literature.[20,21] Confirmation of the stereochemistry was pursued using

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Figure 5. X-ray crystal structure of (R)-nosyl-northioridazine (13) (CCDC 1982915†). Anisotropic dis-
placement ellipsoids show 30% probability levels. Hydrogen atoms are drawn as circles with
small radii.
X-ray crystallographic analysis. The menthyl carbamates were amorphous solids and
could not be used. Both thioridazine (2) and northioridazine (12) are oils at room
temperature and their hydrochloride salts were too hygroscopic to produce crystals
suitable for analysis. Novel N-substituted northioridazine derivatives were thus pur-
sued in attempt to generate crystals suitable for X-ray studies. N-Sulfonylation
of (R)-northioridazine (12) with 4-nitrobenzene sulfonyl chloride produced com-
pound 13 in 56% yield after column chromatography (Scheme 3). Fortunately, crys-
tals of compound 13 formed within pure fractions collected from purification
(EtOAc:Pet. Spirit, 1:5). The crystal structure of (R)-nosyl-northioridazine (13) was
obtained and its ORTEP diagram is shown in Figure 5. X-ray diffraction data are
provided in the experimental section. This data unambiguously confirmed the
expected stereochemistry at C-17 as having (R) stereochemistry. This evidence con-
firms the stereochemistry assignments of compounds made from racemic thioridazine
are correct.
Synthesis of compound 13 supports that northioridazine (12) is a suitable substrate
for creating enantiomerically pure N-substituted thioridazine analogs. Synthesis of the
target N-propargyl northioridazine enantiomers ((R)-5 and (S)-5) was then prepared via
N-alkylation of northioridazine enantiomers ((R)-12 or (S)-12) with propargyl bromide
(Scheme 3). Several bases and solvent systems were investigated, however, the use of
60% sodium hydride in DMF generated the highest yields of (R)-5 and (S)-5 in 58%
and 51% yields, respectively. Each enantiomer was further purified by preparative HPLC
and their purities assessed by analytical RP-HPLC. A representative chromatogram for
(S)-5 is shown in Figure S2.

10
A. P. MARTYN ET AL.
Scheme 4. Synthesis of thioridazine-VLA-4 hybrids via a click reaction of (R)- or (S)-N-propargyl north-
ioridazine 5 with azido-VLA-4 antagonist (4 or 15). Reagents and conditions: (a) Cu(OAc)2.H2O
t
(40 mol%), sodium ascorbate (80 mol%), BuOH:H2O (1:1), 33 ꢀC, 18 h (R)-14 64%, (R)-3 24%, (S)-3 20%;
(b) 1 M LiOH, THF:H2O (3:1), rt, 1 h, quant; (c) 1 M LiOH, THF:H2O (3:1), rt, 1 h, decomp.
Synthesis of thioridazine-VLA-4 antagonist hybrid epimers
The click reaction was investigated using (R)-N-propargyl northioridazine (5), where it
and the protected azido-VLA-4 antagonist (4) were dissolved in tert-butanol/water (1:1)
with copper acetate and sodium ascorbate. The reaction mixture was heated and moni-
tored by TLC analysis (Scheme 4). After 2 h, a yellow precipitate had formed and TLC
analysis indicated complete consumption of (R)-N-propargyl northioridazine (5). The
crude residue was purified by silica gel column chromatography (100% EtOAc) where
the desired hybrid methyl ester (R)-14 was obtained in 64% yield. Hydrolysis of the
methyl ester to the target carboxylic acid ((R)-3) with 1 M LiOH, unexpectedly resulted
in a complex mixture of products when observed by TLC analysis. As an alternative,
methyl ester hydrolysis was performed on the protected VLA-4 antagonist (4), which
resulted in quantitative cleavage to the carboxylic acid (15). The carboxylic acid (15)
did not require further purification, as it showed excellent purity when assessed with
analytical RP-HPLC (Fig. S3). The VLA-4 antagonist (15) was freshly prepared and
immediately used in the click reaction with (S)-N-propargyl-northioridazine (5) using
previously described conditions.
As the retention times of the two click reagents were known, this reaction was moni-
tored using analytical RP-HPLC (see Fig. S5). It was determined that the reaction could
be pushed to completion by stirring for 58 h with more catalyst added (same quantities)
on the 50th h. After lyophilization, the crude material was purified by preparative RP-
HPLC, affording (S)-3 in 20% yield showing excellent purity when assed by analytical
RP-HPLC (Fig. S4). The same conditions and monitoring process were used to
prepare the (R)-3 epimer, which resulted in an isolated yield of 24% after preparative
RP-HPLC.

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11
Experimental
General
Reagents and solvents were used without further purification unless otherwise stated.
CH2Cl2, DMF, toluene, and diethyl ether (Et2O) were used directly from a PureSolv
Solvent purification system. Tetrahydrofuran was purified by drying over KOH before
distillation from sodium benzophenone ketyl. Low-resolution electrospray ionization
(ESI) mass spectra were obtained using a Shimadzu LCMS-2010EV spectrometer. ESI
High-Resolution Mass Spectra (HRMS) were obtained on a Xevo QToF mass spectrom-
eter with perfluorokerosene as internal standard. Fourier-Transform Infrared (FTIR)
spectra were obtained using a Shimadzu IRAffinity-1 spectrometer with a MIRacle 10
1
accessory. H, 13C and two-dimensional (2D) NMR experiments were performed using
a Varian Inova 500 MHz or Varian Premium Shielded 500 MHz spectrometer at 25 ꢀC.
Chemical shifts are reported as d (ppm) relative to internal TMS or other solvent where
stated. The abbreviations s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, dd ¼ doublet of
doublets, m ¼ multiplet, and br s ¼ broad singlet are used throughout. Analytical reverse
phase HPLC (RP-HPLC) was performed using a Waters 600 Multi-solvent Delivery
System with a Waters 2996 Photodiode Array Detector and Grace Vision HT C18 HL
column (3 m, 150 ꢃ 4.6 mm) at 1 mL minꢁ1. Preparative RP-HPLC was performed with
a Waters PrepLC System and Waters 2489 UV/visible detector operating at 214 and
254 nm. Separation was achieved with a SunFire Prep C18 OBD column (5 m,
100 ꢃ 30 mm) at a flow rate of 15 mL.minꢁ1. Compounds were eluted with linear gra-
dients as specified. For analytical and preparative RP-HPLC solvent A consisted of
100% H2O with 0.1% TFA and solvent B consisted of 100% CH3CN with 0.1% TFA. All
RP-HPLC was conducted with Waters Empower software. X-ray crystallography data
collection: CrysAlis PRO;[23] cell refinement: CrysAlis PRO[23]; data reduction: CrysAlis
Pro;[23] program used to solve structure: SIR92;[24] program used to refine struc-
ture: CRYSTALS.[25]
Procedure for synthesis of N-propargyl northioridazine enantiomers (5)
To a stirring solution of (R)-northioridazine 12 (156 mg, 392 mmol) in dry DMF (4 mL)
was slowly added 60% NaH in mineral oil (17.2 mg, 432 mmol). This solution was then
allowed to stir at room temperature for 2 h under argon. A solution containing 80%
propargyl bromine in toluene (97 mL, 785 mmol) was then added and the mixture was
left to stir overnight. Upon completion, the reaction mixture was concentrated in vacuo
the residue was dissolved in EtOAc (20 mL) and placed into a separating funnel. The
organic layer was washed with water (3 ꢃ 20 mL) and brine (1 ꢃ 20 mL), dried over
MgSO4, and concentrated in vacuo. The crude residue was purified by silica gel column
chromatography (EtOAc:Pet. Spirit, 0:100–40:60) to give 5 as a yellow oil (89 mg, 58%).
[a]25 ¼ þ11.6 (c ¼ 1.3, MeOH). Rf ¼ 0.60 (EtOAc:Pet. Spirit, 40:60). FTIR: neat
D
(cmꢁ1) 3292.6, 2923.3, 1456.3, 748.4. HR-MS (ESI) m/z ¼ 395.1593 [M þ H]þ, 395.1616
1
calcd for C23H27N2S2. H and 13C NMR data were as for (S)-5 below.
Method of preparation as for (R)-5. From (S)-12 (156 mg, 0.438 mmol) (S)-5 (88 mg,
1
51%) was obtained. [a]25 ¼ ꢁ11.0 (c ¼ 0.5, MeOH). H NMR (500 MHz, CDCl3): d
D

12
A. P. MARTYN ET AL.
1.56–1.68 (m, 2H), 1.75 (m, 4H), 1.81–1.89 (m, 1H), 2.17–2.27 (m, 1H), 2.46 (s, 3H),
2.52–2.66 (m, 2H), 2.79–2.81 (m, 1H), 3.27 (d, J ¼ 5.0, 1H), 3.69 (d, J ¼ 5.0 Hz, 1H),
3.82–3.91 (m, 1H), 3.92–4.01 (m, 1H), 6.79 ꢁ 6.81 (S, 1H), 6.82 (d, J ¼ 10 Hz, 1H), 6.88
(d, J ¼ 10 Hz, 1H), 6.91 (d, J ¼ 10 Hz, 1H), 7.04 (d, J ¼ 10 Hz, 1H), 7.10–7.16 (t,
J ¼ 15 Hz, 2H). N.B. The alkyne signal was not observed due to deuterium exchange.
13C NMR (126 MHz, CDCl3): d 16.5, 24.1, 25.5, 29.3, 30.4, 43.0, 44.1, 53.3, 57.4, 74.0,
76.5, 114.6, 115.8, 120.9, 122.3, 122.7, 125.4, 127.3, 127.5, 127.6, 137.7, 144.8, 146.0.
FTIR: neat (cmꢁ1) 3292.4, 2924.3, 1456.4, 748.4. HR-MS (ESI) m/z ¼ 395.1613
[M þ H]þ, 395.1616 calcd for C23H27N2S2.
Supporting information
1
Full experimental details, characterization data, HPLC traces, H, and 13C spectra can
be found in the supplementary information provided.
Conclusions
A practical synthesis of northioridazine enantiomers ((R)-12 and (S)-12) from racemic
thioridazine (2) was developed. The absolute stereochemistry of the northioridazine
enantiomers was confirmed through an X-ray crystallographic analysis of (R)-nosyl-
northioridazine (13). This methodology demonstrates that northioridazine (12) is a suit-
able substrate for creating enantiomerically pure N-substituted thioridazine analogs. The
aim of synthesizing two epimeric thioridazine-VLA-4 antagonist hybrids was success-
fully accomplished. The convergent synthetic strategy delivered the azido-VLA-4 antag-
onist (15) and N-propargyl northioridazine enantiomers ((R-5 and (S)-5). A click
reaction between these compounds then resulted in assembly of the target hybrids ((R)-
3 and (S)-3) which were purified by preparative RP-HPLC.
Acknowledgments
We would like to thank the Arrow Bone Marrow Transplant Foundation for their financial sup-
port with an Arrow HCC PhD Scholarship.
Disclosure statement
There are no conflicts of interest to declare.
Funding
This study was supported by the Arrow Bone Marrow Transplant Foundation for their financial
support with an Arrow HCC PhD Scholarship.
ORCID
A. P. Martyn
M. J. Kelso
http://orcid.org/0000-0001-7211-0477
http://orcid.org/0000-0001-7809-6637

R
SYNTHETIC COMMUNICATIONSV
13
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