Multiple-step, one-pot synthesis of 2-substituted-3-phosphono-1-thia-4-aza-2-cyclohexene-5-carboxylates and their corresponding ethyl esters

Article abstract of DOI:10.1016/j.bmcl.2018.01.052

The multiple-step, one-pot procedure for a series of 2-substituted-3-phosphono-1-thia-4-aza-2-cyclohexene-5-carboxylates, analogues of the natural, sulfur amino acid metabolite lanthionine ketimine (LK), its 5-ethyl ester (LKE) and 2-substituted LKEs is described. Initiating the synthesis with the Michaelis-Arbuzov preparation of α-ketophosphonates allows for a wide range of functional variation at the 2-position of the products. Nine new compounds were synthesized with overall yields range from 40 to 62%. In addition, the newly prepared 2-isopropyl-LK-P, 2-n-hexyl-LKE-P and 2-ethyl-LKE were shown to stimulate autophagy in cultured cells better than that of the parent compound, LKE.

Full text of DOI:10.1016/j.bmcl.2018.01.052

Bioorganic & Medicinal Chemistry Letters 28 (2018) 562–565
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Multiple-step, one-pot synthesis of 2-substituted-3-phosphono-1-thia-
4-aza-2-cyclohexene-5-carboxylates and their corresponding ethyl
esters
Dunxin Shen a, Kenneth Hensley b, Travis T. Denton a,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Spokane, WA 99201, United States
b Arkansas College of Osteopathic Medicine, Fort Smith, AR 72916, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The multiple-step, one-pot procedure for a series of 2-substituted-3-phosphono-1-thia-4-aza-2-cyclo-
hexene-5-carboxylates, analogues of the natural, sulfur amino acid metabolite lanthionine ketimine
(LK), its 5-ethyl ester (LKE) and 2-substituted LKEs is described. Initiating the synthesis with the
Received 10 October 2017
Revised 22 January 2018
Accepted 25 January 2018
Available online 1 February 2018
Michaelis-Arbuzov preparation of
a-ketophosphonates allows for a wide range of functional variation
at the 2-position of the products. Nine new compounds were synthesized with overall yields range from
40 to 62%. In addition, the newly prepared 2-isopropyl-LK-P, 2-n-hexyl-LKE-P and 2-ethyl-LKE were
shown to stimulate autophagy in cultured cells better than that of the parent compound, LKE.
Ó 2018 Elsevier Ltd. All rights reserved.
Keywords:
Autophagy
Lanthionine ketimine
Neurodegenerative diseases
ALS
Alzheimer’s
Cysteine
Phosphonate
Arbuzov
Lanthionine ketimine (LK, (5R)-3, 5-dicarboxy-1-thia-4-aza-2-
cyclohexene) is a natural compound first described as an alterna-
tive product of the mammalian transsulfuration pathway arising
from the metabolism of homocysteine, in which cystathionine beta
synthase (CbS) and glutamine transaminase K (GTK) are integral
components.1,2 Although the specific target of LK remains elusive,
LK, and its more cell membrane permeable analogue, lanthionine
ketimine ethyl ester (LKE), have been shown to possess multiple
neuroprotective, anti-neuroinflammatory and neurotrophic activi-
ties.2–11 Chief amongst the biological actions of LKE is its ability to
promote autophagy in cells and in vivo.12,13 LKE penetrates the
blood-brain barrier and is more potent than LK in cell culture
assays, possibly due to increased lipophilicity, inherent to the
esterified 5-carboxylate group.7,12,13 LKE increases lifespan and
slows decline of motor function in the SOD1G93A mouse model of
amyotrophic lateral sclerosis (ALS),14 decreases production of
native Ab(1-40) in SH-SY5Y cells,5 decreases amyloid burden inside
neurons and in plaques,5 decreases protein phosphorylated-tau
accumulation,5 decreases microglial activation9 and slows cogni-
tive decline in the 3ÂTg-AD mouse model of Alzheimer’s disease
(AD).5,7 Besides neurodegenerative diseases, preclinical rodent
studies indicate stroke and glioma as additional disease targets of
LKE and its analogues.6,15 Although the exact molecular target of
LK(E) has not yet been identified, past studies have identified
collapsin response mediator protein-2 (CRMP2), an adaptor protein
involved in neurite outgrowth via interaction with tubulin dimers,
cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3b
(GSK-3b), synaptic plasticity, vesicular trafficking and autophagy
regulation.16,17 The effects of lanthionine ketimine may result from
the compound’s ability to engage CRMP2 pathways to alter local-
ization of the protein mTOR (mammalian target of rapamycin)
and, thus, promote beneficial autophagy.2,12
Due to the necessity for new compounds in the fight against
neurological disorders such as ALS, AD and Parkinson’s disease,
we set out to prepare new analogues of LK and LKE. To do so, mod-
ifications were made to the preexisting synthetic strategy to incor-
porate substituents on the 2-position of LK(E)s (Scheme 1). The
synthesis of 2-substituted-LKEs begins by the bromination of
a-ketocarboxylic acids, 1, by treatment with bromine for one hour
in refluxing dichloromethane. After removal of the solvent and
excess bromine, the brominated acid is reacted with an aqueous
solution of cysteine ethyl ester hydrochloride (3) to afford the
2-substituted LKEs (4). An important feature of LKE as a drug
⇑
Corresponding author.
E-mail address: travis.denton@wsu.edu (T.T. Denton).
https://doi.org/10.1016/j.bmcl.2018.01.052
0960-894X/Ó 2018 Elsevier Ltd. All rights reserved.

D. Shen et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 562–565
563
water to serve as a reductant to neutralize any remaining bromine
from the previous reaction. The addition of the reducing agent both
protected the reaction from becoming dark in color and ultimately
increased the overall yield of the precipitated products. The final,
solid materials were collected by centrifugation, triturated with
ethyl ether and isolated by vacuum filtration.
Scheme 1. Synthetic sequence for the preparation of 2-substituted-LKEs.
Using these procedures, two 2-substituted LKEs and seven 2-
substututed LK(E)-Ps were prepared (Table 1) and their structures
were confirmed by 1H, 13C and 31P NMR and liquid chromatogra-
phy tandem UV spectrophotometry high-resolution mass spec-
trometry (LC/UV/HRMS).
Considering the existence of imine-enamine tautomerism, the
final product could be in either imine or enamine form. The enam-
ine form was confirmed by one- and two-dimensional NMR analy-
sis. The 1H and 13C NMR spectra for 2-n-hexyl-LKE-P are shown in
Fig. 1.
candidate is its relatively small size and possession of functional
groups that can be further modified, while remaining within the
accepted parameters of the rule of five for druglikeness.18 We rea-
soned that amongst the potential modes of target engagement,
electrostatic interaction of the 3-carboxylate group has a high
probability of importance, which could possibly be further
enhanced by increasing the charge density or the hydrogen bond-
ing opportunities near this corner of the molecule. Thus, we
designed a synthetic sequence to prepare 3-phosphonate ana-
logues of LK(E), namely lanthionine ketimine (ester) phosphonates
(LK(E)-Ps, Scheme 2). The synthesis of 2-substituted-LK(E)-Ps
Proton assignments were confirmed by the COSY cross peaks
between methine proton 1 and the diastereotopic methylene pro-
tons 3 and 4 (Supplementary Fig. 1). The smaller, vicinal coupling
constant for peak 3 (3.3 Hz) indicates the cis (staggered) orienta-
tion of protons 1 and 3 whereas the larger, vicinal coupling con-
stant (6.3 Hz) indicates the trans (antiperiplanar) orientation of
protons 1 and 4. The 13C NMR peak assignments for 2-n-hexyl-
LKE-P are shown in Fig. 1, B. Carbons 1, 2 and 3 were confirmed
to be proton-free by DEPT analysis (Supplementary Fig. 2. The
chemical shift of carbon 1 is 170.5 ppm, indicative of a carbonyl
carbon. The remaining two, proton-free, alkene (enamine) carbons
give rise to the doublet at 127.0 ppm with a coupling constant (J =
192.5 Hz) significantly larger than the doublet at 110.3 ppm (J =
17.5 Hz) resulting in the assignments made. Carbons 4–11 were
also confirmed by DEPT and HSQC analysis (Supplementary infor-
mation). According to the peak assignment of 2-n-hexyl-LKE-P,
all other synthesized LK(E)-Ps were characterized accordingly.
Upon analysis of 2-benzyl-LK-P and 2-ethyl-LKE, some intrigu-
ing features were revealed in the 1H NMR spectra. The two methy-
lene (benzyl) protons in 2-benzyl-LK-P are split into a quartet. This
peak should be, theoretically, a simple singlet integrating to two
protons. This quartet was confirmed to represent the benzyl pro-
tons by HSQC analysis (Supplementary Fig. 4). Correspondingly,
the methylene protons in 2-ethyl-LKE, which should be split into
a quartet, are split into two multiplets, each integrating to a single
proton. The two multiplets were confirmed to be the methylene
protons of the 2-ethyl group by HSQC analysis (Supplementary
Fig. 5). We hypothesize that the benzyl and ethyl groups have
restricted rotation about the CH2-alkene (enamine) bond, thus gen-
erating two distinguishable rotamers at room temperature,
although this does not explain the multiplicity of the benzyl pro-
begins with the preparation of dimethyl
(DMAKPs, 6), utilizing standard Michaelis-Arbuzov (MA) reaction
conditions. -Ketophosphonate diesters are extremely reactive
a-ketophosphonates
a
towards nucleophiles, therefore, to avoid any unnecessary side
reactions, the remaining transformations were all performed in
the same pot. Accordingly, following the MA reaction, the methyl
chloride by-product and excess trimethyl phosphite were removed
by simple rotary evaporation. The crude dimethyl a-ketophospho-
nates (6) were dissolved in excess trimethylsilyl bromide (TMS-Br)
and heated to 100 °C, using microwave irradiation, for ten minutes
to afford the intermediate bis-TMS esters (7). After removal of the
excess TMS-Br and methyl bromide, intermediates 7 were bromi-
nated by treatment of the crude reaction mixture with bromine
in refluxing dichloromethane for one hour. After removal of the
solvent and excess bromine, the crude intermediates were treated
with water for three minutes to hydrolyze the bis-TMS esters to
the diacid form before reacting with cysteine ethyl ester
hydrochloride (3) or cysteine hydrochloride (9) to afford the LKE-
Ps or LK-Ps, respectively. Most products precipitated from the final,
aqueous reaction system as white to off-white solids.
During conditions screening, the reaction mixtures would often
turn very dark after the bromination step of the sequence. It was
suspected that the sulfur containing intermediates were being oxi-
dized, and ultimately polymerized, by the bromine that was not
completely removed from the previous reaction. In an effort to
eliminate this problem, in the final step, where the brominated
intermediate is reacted with the amino acid, a 5% aqueous sodium
bisulfite solution was used as the reaction solvent in place of pure
Scheme 2. Synthetic sequence for the preparation of 2-substituted-LK(E)-Ps.

564
D. Shen et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 562–565
Table 1
2-substituted LKEs and LK(E)-Ps prepared and yield.
With the synthesis of 2-alkyl-LK(E)s and 2-alkyl-LK(E)-Ps estab-
lished, an initial biological evaluation was performed. 2-ethyl-LKE,
2-isopropyl-LK-P and 2-n-hexyl-LKE-P were selected to test their
effect on autophagy enhancement in vitro, marked by an increase
in LC3-II (the phosphatidylethanolamine-appended microtubule
associated protein 1, light chain 3). Using this assay, the increased
LC3-II signal may be due to the late-stage blockade of autophagy
clearance. To account for this, bafilomycin-A1 (baf), a vacuolar
ATPase inhibitor, was included to fully block autophagy clearance
and, therefore, any increase in signal, above this induced baseline,
indicates stimulation of autophagy, not blockage of autophagoso-
mal clearance.19 RG2 glioma cells were treated with LK, 2-iso-
propyl LK-P, 2-ethyl-LKE and 2-n-hexyl LKE-P and 50 nM
bafilomycin-A1 and analyzed for stimulation of autophagy (Figs. 2
and 3).
Name
Yield (%)
2-methyl-LKE
2-ethyl-LKE
40%
47%
61%
55%
58%
58%
62%
51%
57%
2-isopropyl-LK-P
2-n-butyl-LK-P
2-n-butyl-LKE-P
2-n-hexyl-LK-P
2-n-hexyl-LKE-P
2-phenyl-LK-P
2-benzyl-LK-P
tons of 2-benzyl-LK-P. In the case of 2-benzyl-LK-P rotamers, the
region of the spectrum corresponding to the benzyl protons should
contain two singlets that, together, would integrated to two pro-
tons Yet, these protons are, as indicated, split into a quartet. In
regards to 2-ethyl-LKE, this phenomenon is also observed. In the-
ory, these protons should be split into a single quartet, yet, the
splitting pattern is a complicated doublet of multiplets. These mul-
tiplets, together, integrate to the proper value of two protons and,
if rotamers are the purpose for this complicated spitting pattern,
the multiplets should coalesce at higher temperature as the rota-
tional barrier is overcome. As indicated by 1H NMR analysis (Sup-
plementary Fig. 6), when increasing the temperature of the sample
from room temperature to 35 °C, the signals began to coalesce,
endorsing the likelihood of this assessment. Although this is only
a partial explanation for this phenomenon, all corresponding
NMR analyses support the structures assigned. In future rounds
of synthesis, as the library of compounds is increased, this phe-
nomenon will be examined in further detail.
The results indicate a number of biological consequences
including 1) the cellular permeability of the new compounds and
2) the successful stimulation of autophagy in the presence of bafi-
lomycin. The increase in autophagy activity of 2-isopropyl-LK-P
was greater than LK at 10
100 M. This biphasic relationship may indicate that, due to the
high potency of the new compounds, 100 M may be above the
lM but showed less of an effect at
l
l
optimum concentration for autophagy stimulation and in the range
of being toxic to the cells. This is also indicated in the testing of 2-
ethyl-LKE and 2-n-hexyl-LKE-P (Fig. 3). In these preliminary exper-
iments, it is assumed that the optimum concentration for autop-
hagy stimulation of 2-n-hexyl-LKE-P is between 1 and 10
lM
while that of 2-ethyl-LKE is above 10 M. With the preliminary
l
potencies estimated, future analyses will be performed on these,
and future analogues, with additional doses, in replicate experi-
ments, to specifically determine the concentrations at which the
compounds most effectively stimulate autophagy.
Fig. 1. 1H (Panel A, Left) and 13C (Panel B, Right) NMR spectra of 2-n-hexyl-LKE-P.
Fig. 2. Autophagy enhancement of LK and 2-isopropyl-LK-P.

D. Shen et al. / Bioorganic & Medicinal Chemistry Letters 28 (2018) 562–565
565
Fig. 3. Autophagy enhancement of 2-ethyl LKE and 2-n-hexyl-LKE-P.
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the compounds ability to permeate into living cells to elicit their
biological response. In an effort to define a comparable experimen-
tal value for cell-membrane permeating ability, LKE, 2-ethyl-LKE,
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indicate. In other words, if cellular membrane permeability for
the phosphonates is lower than that of LKE, when comparing LKE
to a phosphonate at any given extracellular concentration, the
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and, since the phosphonate activates autophagy better than LKE
at this lower concentration, it is more potent than LKE.
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flux in cell culture systems. Future directions include the synthesis
of more lipophilic forms of the LK(E)-Ps as well as the preparation
of phosphonate-ester pro-drug forms in an effort to increase the
cell-permeability and overall efficacy of the compounds. Target
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Acknowledgments
The authors would like to thank Kateryna Kostenkova and
Mahamudul Haque for their technical assistance in the chemistry
lab. We would like to thank the National Institute of Neurological
Disorders and Stroke (1 R15 NS093594-01A1) for financial support
of this research. The Health Sciences & Services Authority of Spo-
kane County is acknowledged for their financial support of the
NMR and Mass Spectrometry core facilities at WSU Spokane.
17. Myllykoski M, Baumann A, Hensley K, Kursula P. Collapsin response mediator
protein 2: high-resolution crystal structure sheds light on small-molecule
binding, post-translational modifications, and conformational flexibility. Amino
Acids. 2017;49:747–759.
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computational approaches to estimate solubility and permeability in drug
discovery and development settings. Adv Drug Deliv Rev. 2001;46:3–26.
19. Klionsky DJ, Abdalla FC, Abeliovich H, et al. Guidelines for the use and
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.01.052.
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