Design and synthesis of neuroprotective methylthiazoles and modification as NO-chimeras for neurodegenerative therapy

Article

pubs.acs.org/jmc

Design and Synthesis of Neuroprotective Methylthiazoles and

Modiﬁcation as NO-Chimeras for Neurodegenerative Therapy

Zhihui Qin,^†Jia Luo,^†Lawren VandeVrede,^†Ehsan Tavassoli,^†Mauro Fa’,^‡Andrew F. Teich,^‡

Ottavio Arancio,^‡and Gregory R. J. Thatcher*^,†

^†Department of Medicinal Chemistry and Pharmacognosy, University of Illinois College of Pharmacy, University of Illinois at

Chicago, 833 S. Wood Street, Chicago, Illinois 60612-7231, United States

^‡Department of Pathology and Cell Biology, The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia

University, 630 West 168th Street, New York, New York 10032, United States

S

* Supporting Information

ABSTRACT: Learning and memory deﬁcits in Alzheimer’s disease (AD) result from synaptic failure and neuronal loss, the latter

caused in part by excitotoxicity and oxidative stress. A therapeutic approach is described that uses NO-chimeras directed at

restoration of both synaptic function and neuroprotection. 4-Methylthiazole (MZ) derivatives were synthesized, based upon a

lead neuroprotective pharmacophore acting in part by GABA_Areceptor potentiation. MZ derivatives were assayed for protection

of primary neurons against oxygen−glucose deprivation and excitotoxicity. Selected neuroprotective derivatives were

incorporated into NO-chimera prodrugs, coined nomethiazoles. To provide proof of concept for the nomethiazole drug class,

selected examples were assayed for restoration of synaptic function in hippocampal slices from AD-transgenic mice, reversal of

cognitive deﬁcits, and brain bioavailability of the prodrug and its neuroprotective MZ metabolite. Taken together, the assay data

suggest that these chimeric nomethiazoles may be of use in treatment of multiple components of neurodegenerative disorders,

such as AD.

Scheme 1) potentiates the function of the inhibitory neuro-

transmitter GABA in the brain,⁶⁻⁸attenuating glutamate

induced excitotoxicity. CMZ has seen clinical use in a number

of indications,⁹⁻¹¹and study of compounds related to CMZ has

shown that anticonvulsant and hypnotic potency could be

INTRODUCTION

■

Alzheimer’s disease (AD) is an age-related neurodegenerative

disease manifested by progressive memory loss, decline in

language skills, and other cognitive impairments. The disorder

is reaching epidemic proportions, with a large human, social,

and economic burden.¹Currently approved treatments may

temporarily reduce the rate of cognitive decline in a limited

number of patients, without halting disease progression;

however, no treatment is considered disease-modifying.²

Diverse factors play a role in the pathogenesis of AD, notably

abnormal aggregation of amyloid β (Aβ) and tau protein,

glutamate-induced excitotoxicity, oxidative stress, and inﬂam-

mation.³Accordingly, a variety of therapeutic strategies

modulating the progression or prevention of AD are currently

being investigated.^4,5AD represents a major unmet medical

need.

Scheme 1. Design Concept for Novel Nomethiazole NO-

Chimeras Based upon Neuroprotective MZ Scaﬀolds

Excitotoxicity is a mechanism of neuronal dysfunction and

cell death characterized by excessive stimulation of glutamate

receptors, pathological elevation of intracellular calcium

concentration, and mitochondrial damage. The methylthiazole

(MZ) containing GABA-mimetic agent clomethiazole (CMZ,

Received: March 13, 2012

Published: July 10, 2012

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a

Scheme 2

a

Reagents and conditions: (a) Grignard reagent, THF, 0 °C to rt; (b) MsCl, NEt₃and then NaN₃, CH₃CN, rt; (c) alkyne, CuSO₄·5H₂O, sodium

ascorbate, t-BuOH/H₂O (1:1, v/v); (d) LiAlH₄, THF, reﬂux; (e) MsCl, NEt₃and then NaN₃, CH₃CN, reﬂux; (f) MOM-Br, K₂CO₃, THF; (g)

ethynyltrimethylsilane, CuI, Pd(Ph₃P)₂Cl₂, NEt₃, THF; (h) 3, CuSO₄·5H₂O, sodium ascorbate, K₂CO₃, MeOH−H₂O (2:1); (i) HCl/i-PrOH (1.5

M), 70 °C; (j) 4-nitrooxybutan-1-ol (for 18) or 1-acetoxy-4-hydroxybutane (for 17), Ph₃P, DIAD, 0 °C to rt, then K₂CO₃, MeOH (for 17).

dissociated and modulated separately.¹²⁻¹⁷Under the name

Zendra, CMZ was explored in clinical trials including phase III

trials as a neuroprotective drug for use in spinal cord injury and

ischemic stroke,¹⁸⁻²⁵and CMZ continues to be suggested as a

potential component of future combination therapies for

neuronal injury.²⁶CMZ has also been demonstrated to

counteract inﬂammation, lowering levels of proinﬂammatory

cytokines, including TNFα,^27,28and to rescue mitochondrial

function in brain tissues.²⁹Recent evidence, from both clinical

observations and mouse model studies, supports TNFα as a

therapeutic target in AD.³⁰⁻³²Likewise, restoration of

mictochondrial function is seen as a strategy for AD

therapy.^33,34Therefore, MZ derivatives, capable of eliciting

neuroprotection and attenuating neuroinﬂammation, are of

potential clinical utility in AD.

neuronal plasticity and neurogenesis.⁴⁴⁻⁴⁶Since activation of

CREB in the hippocampus is tightly coupled to NO/cGMP

signaling,^44,46−49restoration of normal NO/cGMP signaling in

the aging brain is essential to preserve both synaptic and

neuronal survival and function. NO-mimetic agents that

enhance CREB activity will likely be of beneﬁt in treatment

of AD.⁵⁰

We have previously reported a chimeric molecule with NO

and GABA-mimetic attributes, 1 (GT-1061), that contains an

MZ pharmacophore (Scheme 1).⁵⁰1 signiﬁcantly improved

learning and memory in rats with cognitive deﬁcits in the

cholinergic systems of the basal forebrain.³⁷A key objective of

the present study was to determine if 1 is a prototype of an

NO-chimera drug class. A positive observation would allow

further optimization and reﬁnement of this drug class for AD

and other CNS indications. We report herein (1) the

development of a small MZ library, (2) selection of MZ

derivatives based upon neuroprotective activity screened in

primary neurons, (3) synthesis of novel NO-chimeras, coined

nomethiazoles, (4) selection based upon chemical stability, (5)

assay for synaptic rescue in brain slices, (6) assay for restoration

An early event in AD is synaptic failure.³⁵Restoration of

synaptic function via activation of cAMP-responsive element

binding factor (CREB) is seen as a new target for treatment of

AD and other neurological disorders.³⁶⁻⁴³Activation of CREB

is known to enhance or restore long-term potentiation (LTP)

and the expression of corresponding gene products to enhance

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a

Scheme 3

a

Reagents and conditions: (a) NBS, CH₃CO₂H, dioxane−H₂O; (b) K₂CO₃, MeOH; (c) Grignard reagent, THF, 0 °C to rt; (d) NaN₃, CH₃CN/

H₂O (1:1, v/v), reﬂux; (e) NaH, DMSO, 90 °C, 5 h; (f) 3-ethynylpyridine, CuSO₄·5H₂O, sodium ascorbate, t-BuOH/H₂O (1:1, v/v); (g) fuming

HNO₃, Ac₂O, CH₂Cl₂, 0 °C; (h) 3-ethynylpyridine, toluene, reﬂux, 48 h; (i) 2-mercaptoethanol, Na₂CO₃, CH₃CN/H₂O (2:1); (j) 3-

pyridineboronic acid, (2-biphenyl)di-tert-butylphosphine, Pd(OAc)₂, KF, DMF, 120 °C.

of learning and memory in vivo, and (7) demonstration of brain

bioavailability and measurement of brain/plasma ratios. The

drug design approach is depicted in Scheme 1.

Synthesis of MZ analogues, retaining the 5-ethyl-4-

methylthiazole motif of both CMZ and 1, proceeded from 4-

methyl-5-vinylthiazole (19) to yield the alcohols 22 and 23

(Scheme 3). Epoxide intermediate 21 was added dropwise to

Grignard reagents to give 22a−c as major products. Although

the ultimate objective was to modify MZ derivatives as NO-

chimeras, compounds such as the indole derivative 24 served to

extend and explore the MZ structural space. Compound 24 also

represents an analogue of latrepirdine, a once-promising new

AD drug.⁵⁴Tetrahydrocarboline, the starting material for 24,

was made according to a published procedure.⁵⁵To incorporate

the newly discovered 3-(1H-1,2,3-triazole-4-yl)pyridine phar-

macophore into NO-chimera scaﬀolds, azido alcohol 25 was

obtained by treating epoxide 21 with sodium azide and reacting

with 3-ethylnylpyridine under standard click chemistry

conditions to give cycloaddition product 26. Compound 31,

a close structural analogue of 26, was synthesized from 22c

using Suzuki coupling to investigate the tolerance of neuro-

protection to triazole ring replacement in the novel

pharmacophore.

2. CMZ Pharmacology Underlying the Design and

Biossay of MZ Derivatives. Many papers have discussed the

biological activity of CMZ in clinical settings and in animal

models. Thiazole derivatives have long been known to manifest

sedative/hypnotic and anticonvulsant eﬀects that are indicative

of GABA-mimetic activity.^15,16The prototype NO-chimera 1

releases 5-(2-hydroxyethyl)-4-methylthiazole (HMZ) as pri-

mary metabolite, which was ﬁrst reported in 1956 to be an

anticonvulsant agent in rabbits.⁵⁶CMZ has been shown

extensively to be neuroprotective in animal models of transient

and global ischemia. CMZ potentiates GABA and muscimol

agonism at the GABA_Areceptor without evidence for

RESULTS AND DISCUSSION

■

1. Synthesis of MZ Derivatives. Copper(I)-catalyzed

Huisgen [3 + 2]-cycloaddition between an azide and a terminal

alkyne was used to aﬀord regioselectively 1,4-disubstituted-1H-

1,2,3-triazoles, based upon the mild conditions, reliable high

yield, and chemoselectivity of this “click” reaction.⁵¹⁻⁵³Azide 3

was synthesized in two steps and reacted with commercially

available alkynes, including alkyl (e.g., 1-hexyne, ethynyltrime-

thylsilane, propargylbenzene) and aryl (e.g., ethynylbenzene,

regioisomers of ethynylpyridine, 3-ethynylthiophene, and 5-

ethynyl-1-methyl-1H-imidazole), to generate a small library of

4-methyl-5-(phenyl(1H-1,2,3-triazol-1-yl)methyl)thiazole de-

rivatives (5a−i), and in addition, one ﬂuorinated analogue 8

was also prepared (Scheme 2). The goal was to increase

diversity of MZ derivatives and identify novel neuroprotective

scaﬀolds. Accordingly 5-(2-(1H-1,2,3-triazol-1-yl)ethyl)-4-

methylthiazole derivatives (11a−c) and two 5-benzyl-4-

methylthiazole derivatives were synthesized (6a,b). Of all the

click products, compound 5d was identiﬁed as an excellent

neuroprotectant using in vitro bioassays (discussed below),

whereas removal of the 3-pyridyl substitutent (5i), replacement

of the pyridine ring with phenyl (5b) or other heterocycles (5g,

5h), and changing the position of the nitrogen (5e, 5f) led to

reduced eﬃcacy. A similar structure−activity pattern was

observed for 11a−c. Thus, the 3-(1H-1,2,3-triazole-4-yl)-

pyridine fragment is a potential neuroprotective pharmaco-

phore and was used to develop further MZ derivatives.

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modulation of levels of GABA itself and opens neuronal Ca-

dependent chloride channels enhancing inhibitory neuro-

transmission. At concentrations 100-fold those required to

potentiate GABA function (30 μM), CMZ is a direct GABA_A

receptor agonist.⁵⁷⁻⁵⁹CMZ is not a ligand for the GABA_Bor

benzodiazepine receptors and potentiates the actions of glycine

on inhibitory neurotransmission.⁶⁻⁸

protective activity of CMZ, OGD in primary neurons was seen

as the preferred model to screen and to correlate structure of

MZ analogues with activity.

The simple 5-ethyl-4-methylthiazole derivatives (22, 23)

studied in primary neuron OGD retained neuroprotective

eﬃcacy comparable to that of CMZ (Table 1). In contrast,

The mechanism of action of CMZ appears not to be limited

to GABA_Apotentiation,^60,61since CMZ has been claimed to be

a p38-MAPK (mitogen activated protein kinase) pathway

inhibitor, although the exact protein target was not deﬁned.^68,69

In Alzheimer’s therapy, inhibition of p38-MAPK is seen as a

target, since, for example, Aβ-mediated inhibition of LTP

involves stimulation of the p38 MAPK cascade.^62,63Compound

4 (Scheme 2) is the lone analogue of CMZ for which

mechanistic data have been reported. In the gerbil model of

global ischemia, protection by R-4 and S-4 was equivalent to

CMZ;⁶however, R-4 was reported to be devoid of

anticonvulsant activity, suggested to be compatible with a

mechanism involving GABA reuptake inhibition.⁷

CMZ has a short half-life and is extensively metabolized.

Oxidative metabolism at carbon α to the thiazole ring leads to

α-hydroxylation and formation of metabolites from further

oxidation, including an α-chloro ketone and aldehyde.^64,65The

replacement of the secondary α-carbon by addition of a group

X (Scheme 1) was justiﬁed to prevent metabolic oxidation to

potentially electrophilic metabolites. The primary α-hydroxyl

metabolite of CMZ was also shown to have sedative activity,

providing further reason to limit metabolism at this position.

The rationale for exploration of the structural space represented

by Z (Scheme 1) using aromatic and heterocyclic groups was

based upon the reported biological data on MZ derivative 4

that clearly show central biological actions including neuro-

protection. In the absence of an identiﬁed CMZ binding site

associated with a GABA_Asubunit, iterative extension of the

structural space was carried out by varying the structure and

isomerism, represented by n, X, and Z in Scheme 1.

Table 1. Relative Percentage Neuroprotection of Primary

Neuronal Cultures in Response to OGD after Treatment

with MZ Derivatives

a

% normalized to CMZ after

b

OGD

5-ethyl-4-methylthiazoles

4-methyl-5-(phenyl-(1H-1,2,3-tria-

zol-1-yl)methyl)thiazoles

HMZ

CMZ

10

14.11 8.5

5a

5b

5c

5d

5e

5f

26.8 5.2

57.6 6.7

88.3 6.9

108 3.6

50.9 6.7

90.2 2.2

62.0 3.6

93.9 1.2

58.1 4.2

87.2 3.5

104 1.7

c

100

ns

22a

22b

23a

23b

24

99.6 1.4

98.9 3.0

102 1.5

101 3.9

15.4 5.9

ns

5g

5h

5i

25

31

25.0 7.3

8

16

(4-methylthiazol-5-yl)(phenyl)

methanols

5-(2-(1H-1,2,3-triazol-1-yl)ethyl)-4-

methylthiazoles

2

94.0 2.9

36.8 6.4

25.0 4.6

110.2 12.2

114.1 4.6

11a

11b

11c

51.5 11.8

53.9 6.8

99.6 2.5

6a

6b

38

41

2-(4-methylthiazol-5-yl)-1H-1,2,3-

triazol-1-ylethanols

26

30

107.3 2.7

92.9 7.3

a

% = (drug − DMSO)/(CMZ − DMSO). ns = not studied. Values are

b

presented as the mean SEM (n = 6). Readings from MTT assay at

The selection of a bioassay for iterative design and synthesis

of MZ derivatives was not immediately apparent. Despite the

wealth of reports on CMZ activity in vivo, relatively few

publications have described in vitro assays that could be

adapted for screening of novel MZ analogues and derivatives

for use as neuroprotectants. In SK-N-MC neuroblastoma cells,

CMZ was reported to inhibit the glutamate-induced activation

of AP-1 mediated by NMDA receptor activation.^66,67

Immortalized cell cultures (e.g., SK-N-MC and the SH-SY5Y

human neuroblastoma cell lines) would provide a facile system

for cell-based drug screening; however, in SH-SY5Y cell

cultures subject to excitotoxic insults including glutamate,

NMDA, and oxygen−glucose deprivation (OGD), CMZ did

not produce robust and reproducible neuroprotection (data not

shown). Therefore, rat primary cortical neurons were studied as

a more complete neuronal model. Excitotoxic cell death from

treatment with NMDA (100 μM) was inhibited reproducibly

by CMZ treatment and by a number of MZ derivatives and

analogues (Supporting Information, Table S1).

24 h normalized to vehicle (0% survival) and CMZ (100% survival).

Comparison, before normalization, with vehicle treated cells

demonstrated a mean increase in cell survival of approximately 30%

caused by CMZ treatment.

c

substitution of the phenyl ring in (4-methylthiazol-5-yl)-

(phenyl)methanol derivatives (2 versus 6a and 6b in Table

1) signiﬁcantly reduced eﬃcacy. Further, O-alkylation of 2 led

to improved neuroprotection (2 versus 38 and 41 in Scheme

4). Compound 26 (Scheme 3) incorporated a 3-(1H-1,2,3-

triazole-4-yl)pyridine pharmacophore within the MZ scaﬀold.

Both 26 and its regioisomer 30 were protective against OGD,

whereas replacement of the triazole substitutent in 26 (26 vs

31) or substitution of 3-pyridyl in 5d with 2-pyridyl (5e) led to

signiﬁcant loss of eﬃcacy. The results indicate that both triazole

and 3-pyridyl moieties are critical structural elements to the

neuroprotective 3-(1H-1,2,3-triazole-4-yl)pyridine fragment.

Mindful of the reports of GABAergic and GABA-

independent mechanisms of action for CMZ, the mechanism

of neuroprotection of four MZ derivatives was probed using the

GABA_Areceptor channel blocker picrotoxin (100 μM) that has

been used extensively for selective antagonism of GABA_A

mediated signaling.⁶⁸⁻⁷¹Lower concentrations of picrotoxin

deliver only partial blockade,^72,73and some studies employ

higher concentrations.^74,75Muscimol, a potent, selective

NMDA excitotoxicity represents only one aspect of neuro-

toxicity, whereas OGD provides a much more complete model

simulating ischemia followed by reperfusion inducing ATP

depletion, glutamate release, NMDA receptor overstimulation,

mitochondrial dysfunction, ROS generation, apoptosis, and

proinﬂammatory cytokine release. Given the evidence that

multiple pathways underlie the anti-inﬂammatory and neuro-

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a

Scheme 4

a

Reagents and conditions: (a) fuming HNO₃, Ac₂O, CH₂Cl₂, 0 °C; (b) TEMPO, PhI(OAc)₂, DCM; (c) NaClO₂, NaH₂PO₄, 2-methyl-2-butene, t-

BuOH; (d) 35 (for 36, 44) or 1-methylcyclopropane-1-carboxylic acid (for 37), EDCI, DIPEA, HOBT, DCM; (e) NaH, CH₃I, DMF; (f) bis(2-

bromomethyl) ether, NaH, DMF; (g) AgNO₃, CH₃CN, reﬂux; (h) LiAlH₄, THF, reﬂux; (i) triphosgene, AcOEt, reﬂux; (j) 34 (for 42) or 4-

nitrooxybutan-1-ol (for 43), NEt₃, THF.

GABA_Areceptor agonist, was studied for comparison. Instead

of OGD, the simpler excitotoxic insult delivered by glutamate

(100 μM) was used to induce neuronal loss, measured at 24 h

(Figure 1). As anticipated, muscimol produced eﬀective

neuroprotection against glutamate toxicity, which was lost

and reversed on blockade of the GABA_Achloride channel.

Neuroprotection produced by 22a mirrored that by muscimol,

indicating a GABAergic neuroprotective mechanism. In

contrast, neuroprotection by 11c was signiﬁcantly attenuated

by GABA_Areceptor blockade but was not ablated or reversed,

indicating a neuroprotective mechanism only partially depend-

ent on the GABA_Areceptor. MZ derivatives 26 and 41

demonstrated intermediate neuroprotective mechanisms. On

the basis of these collected data, a neuroprotective

pharmacophore was speculated (Figure 1). Full elucidation of

the individual GABA-dependent and GABA-independent

pharmacophores requires extended structure−activity studies;

however, the objective was achieved: to develop novel MZ

derivatives to be functionalized as NO-chimeras.

3. Synthesis of NO-Chimeras. MZ derivatives (e.g.,

compounds 5d, 22, 23, and 26, Table 1) were selected for

further study based upon eﬃcacy for neuroprotection against

OGD-induced cell death and the feasibility of structural

modiﬁcation to give nitrate NO-chimeras. Compound 5d is

as an excellent neuroprotectant against OGD and NMDA

induced cytotoxicity. Compound 16 (Scheme 2) is an analogue

of 5d providing a hydroxyl group on the pyridine ring and

allowing modiﬁcation as an NO-chimera. A TMS protected

alkyne was introduced into the pyridine derivative 13 via a

Sonogoshira reaction, yielding 14, which underwent direct

reaction with azide 3 in a basic methanolic solution, as adapted

from the literature.⁷⁶The MOM protecting group of 15 was

removed, and NO-chimera 18 was prepared by coupling of 16

with 4-nitrooxybutan-1-ol under Mitsunobu conditions. Sim-

ilarly, 17, the denitration product of 18, was made by using 4-

acetyloxybutan-1-ol as reactant followed by deacetylation. Both

16 and 17 are potential metabolites of the NO-chimera 18;

notably 16 retained neuroprotective eﬃcacy comparable to that

of CMZ (Table 1).

Two general strategies were used for synthesis of nitrate NO-

chimeras. The ﬁrst used simple electrophilic or nucleophilic

nitration procedures to generate nitrate esters from alcohol or

halide intermediates, respectively. For instance, NO-chimeras

32a,b and 33 were prepared from hydroxyl MZ precursors

22a,b and 23a by the treatment of a mixture of acetic anhydride

and fuming nitric acid (Scheme 3). However, this method failed

to directly convert 26 to NO-chimera 28; therefore, nitrate 27

was prepared from intermediate 25 and then reacted with 3-

ethynylpyridine in the presence of ascorbate and CuSO₄. The

expected instability of the nitrate moiety under these reductive

conditions led to a low yield of 26 as major and 28 as minor

product. However, a mixture of 25 and alkyne reﬂuxed in

toluene successfully gave both regioisomers 28 and 29 by

thermal cycloaddition. The nitrate group of 29 was removed

using thiol under basic conditions to yield MZ derivative 30

(Scheme 3).

The second strategy of synthesizing NO-chimeras was to

incorporate a labile linker group that would distance the nitrate

from the MZ core in an approach more akin to that used in

hybrid nitrates such as NO-NSAIDs. Esters of cyclopropane

carboxylic acid have been reported to possess substantially

increased stability toward both acid- and base-catalyzed

hydrolysis.⁷⁷Inspired by this observation, 1-nitrooxymethylcy-

clopropanecarboxylic acid (35, Scheme 4) was prepared by

mononitration and sequential oxidation. Acid 35 was coupled

with 2 to yield NO-chimera 36 which was expected to have

enhanced stability to esterase hydrolysis and therefore

theoretically predicted to have enhanced brain bioavailability.

An alternative use of this strategy led to design and synthesis of

the carbamate prodrug 42, using the isocyanate precursor

obtained from 4 reacting with mononitrate 34, again predicted

to have further enhanced stability. A similar procedure was used

for coupling of 4-nitrooxybutan-1-ol to yield the carbamate 43.

Preparation of the amide-linked prodrug 44 and compound 37

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Figure 1. Inhibition by picrotoxin of neuroprotection elicited by MZ derivatives in primary neuronal cultures subject to excitotoxic insult. Primary

cortical cultures (10−11 DIV) were subjected to 24 h of glutamate (Glut, 100 μM) toxicity with co-treatment of muscimol (Musc) or MZ derivatives

(50 μM). Picrotoxin (Picro, 100 μM) was added, where indicated, 1 h before glutamate treatment to provide blockade of GABA_Asignaling. Survival

was measured with the MTT assay and normalized to vehicle treated controls. Data show the mean and SEM from at least six individual experiments

analyzed by ANOVA with post hoc Dunnett’s MCT compared to nonglutamate treated vehicle control or compared to the eﬃcacy of picrotoxin in

blocking each drug treatment eﬀect with a two-tailed t test: ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ns, not signiﬁcant.

Figure 2. Representative kinetic traces for hydrolysis of 36 and 37 (10 μM) in phosphate buﬀer (100 mM, pH 7.4). Incubations were conducted at

room temperature and monitored by HPLC with UV detection at 254 nm and ﬁt to a pseudo-ﬁrst-order rate equation. A putative mechanism is

shown for rate acceleration by anchimeric stabilization of the anionic transition state by the nitrate group acting as intramolecular Lewis acid.

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allowed comparison with the ester-linked prodrug 36. Aliphatic

nitrate 40 was synthesized from 2 by O-alkylation with

dibromide and subsequent nucleophilic substitution using

AgNO₃. The ethylene glycol linker was incorporated to

increase aqueous solubility.

4. Stability of NO-Chimeras. It was anticipated that future

animal studies would require simulation of an extended release

formulation, potentially by use of drug in buﬀered drinking

water; therefore, the stability of nitrates was tested in phosphate

buﬀer (100 mM, pH 7.4) using HPLC−UV or LC−MS

monitoring. Since the nitrate group itself is stable in acid and

mild alkaline solution, the observed stability over 5 days at

room temperature of 28, 29, 32a,b, 33, and 40 was expected;

the carbamate 43 and amide 44 were also stable. However, to

our surprise, hydrolysis of 36 was rapid, rather than being

retarded by the hindered α-cyclopropane group, with a half-life

of only about 30 min (Figure 2). The lability of the

nitrooxymethylcyclopropanecarboxylate was unexpected, and

therefore, comparison was made with the stability of 37 as an

analogue lacking only the nitrate group. The observed half-life

of about 380 min for ester hydrolysis conﬁrms that the nitrate

group contributes to the instability of 36. We have previously

reported the phenomenon of accelerated hydrolysis caused by

an adjacent nitrate group ascribed to intramolecular Lewis acid

catalysis⁷⁸and propose a similar mechanism for 36 enhanced

by a Thorpe−Ingold eﬀect in which the developing negative

charge is stabilized by the nitrate moiety through a cyclic

transition state (Figure 2). Although more stable than 36,

carbamate 42 also decomposed in phosphate buﬀer with a half-

life of approximately 3000 min to yield at least four UV-

detectable products that from LC−MS analysis included two

denitrated products (m/z = 333) (Supporting Information,

Figure S1).

5. Restoration of Synaptic Function by NO-Chimeras.

An early event in AD is synaptic failure leading to deﬁcits in

learning and memory.³⁵The importance of synaptic alterations

in AD has also been conﬁrmed by studies in transgenic mouse

models of AD⁷⁹as well as on LTP, a widely studied cellular

model of learning and memory.⁸⁰Previous work has reported

that synaptic transmission and cognition are altered in double

transgenic APP/PS1 mice expressing both the human APP

mutation (K670M:N671L) and the human PS1 mutation

(M146L).^81,82In hippocampal slices obtained from APP/PS1

and WT littermates between 4 and 5 months of age,

reproducible LTP responses in the CA1 are observed, with a

reduction of LTP in APP/PS1 hippocampal slices, as measured

by ﬁeld excitatory postsynaptic potential (fEPSP). Aβ-induced

inhibition of LTP has been observed to be reversed by NO-

donors via a mechanism shown to be mediated by the NO/

cGMP signaling pathway.⁴⁷Basal synaptic transmission was

determined by measuring the slope of fEPSP at increasing

stimulus intensities in slices from 4 to 5 month old APP/PS1

mice for NO-chimeras 32a and 42 (100 μM) perfused for 5

min before inducing LTP through a θ-burst tetanic stimulation

of the Schaeﬀer collateral pathway (Figure 3).⁸¹Potentiation

was observed for 32a, although data for 42 did not reach

signiﬁcance (P < 0.1). The requirement for reductive

bioactivation of nitrates to NO and the instability of 42

discussed above are likely causes of the observed diﬀerences.

The potentiation of the actions of the inhibitory neuro-

transmitter GABA is not expected to enhance LTP, and

accordingly, CMZ did not restore LTP (data not shown).

Figure 3. Restoration of CA1-LTP impairment in 3-month-old APP/

PS1 mouse hippocampal slices. (A) NO-chimera 32a rescued the LTP

impairment in APP/PS1 mice (F_(1,16)= 4.129, p = 0.05, compared with

vehicle; 50−80 min analysis); (B) NO-chimera 42 showed a tendency

toward reversal of LTP impairment without reaching signiﬁcance

(F_(1,12)= 2.537, p = 0.13, compared with vehicle).

6. Restoration of Cognition by NO-Chimeras. The step-

through passive avoidance (STPA) task was used to measure

the reversal of a stimulus-response memory deﬁcit induced by

the amnesic agent scopolamine that blocks muscarinic

acetylcholine receptor activation. Since muscarinic acetylcho-

line receptor dysfunction is relevant to AD, scopolamine-

induced amnesia remains a useful model for screening of drugs

for procognitive actions.⁸³The STPA task tests learned

aversion memory, since mice have a natural preference for

dark areas over brightly lit and open areas. Following a period

of habituation, the animal is placed in the light compartment of

the apparatus and inevitably crosses to the dark compartment

of the apparatus, at which time an electric shock is delivered.

The animals rapidly learn to avoid moving into the dark

compartment during the training phase, despite the admin-

istration of the amnesic agent. Retention of this newly learned

behavior can be tested on subsequent days to assess the

strength of the aversive memory by measuring the latency to

enter the dark area. In both training and testing phases, the task

is halted after 300 s if the animal does not enter the dark side.

STPA has a signiﬁcant advantage for drug screening because

the potential locomotor eﬀects of drugs are very unlikely to

confound the observations. First, all animals must show a

uniform learning response to training regardless of drug

treatment, which initially entails movement between the

compartments until the task is learned. Second, animals are

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tested 24 and/or 48 h after a single drug treatment, and since

most of the drugs, including all NO-chimeras that we have

studied to date, have cleared the system long before memory is

tested, the STPA task is primarily a measure of memory

consolidation.

Scopolamine was administered 30 min and drugs 20 min

before training (both ip). Drugs were administered at

concentrations equimolar to 1 (1 mg/kg, i.e., 4.45 μmol/kg)

to provide comparison to previous work.⁸⁴Administration of

scopolamine and vehicle resulted in a latency of less than 100 s

for mice to enter the dark side of the apparatus when tested 24

h later, demonstrating an inability to consolidate memory. In

contrast, treatment with scopolamine and all novel NO-

chimeras studied (28, 29, 32a, 32b, 40, 42, 43) signiﬁcantly

restored memory at 24 h (Figure 4).

predictive of oral bioavailability by comparison with data for

1.³⁷

CONCLUSIONS

■

Synaptic failure is an early event in both AD and mild cognitive

impairment (MCI), which causes impaired cognition and

memory.¹Disease progression involves neurodegeneration of

cortical and hippocampal neurons and loss of hippocampal

volume. A drug that targets synaptic failure in addition to

providing neuroprotection is a rational approach to AD

therapy. Abnormally assembled Aβ and tau proteins are

associated with both synaptic and neuronal dysfunction and

loss.³Aβ has been shown to down-regulate signaling via the

NO/cGMP/CREB pathway; therefore, enhancement of NO/

cGMP signaling and activation of CREB may provide a novel

approach for the treatment of AD.^{39−42,47,85}Gene products

associated with CREB activation may provide neuroprotection;

however, prevention of neuronal loss can also be achieved by

selective pharmacological modulation of GABA_Areceptors, a

mechanism that has been shown to provide neuroprotection

against Aβ mediated toxicity.⁸⁶⁻⁸⁹

Herein, we have elaborated on the concept of an NO-

chimera, incorporating GABA-mimetic and NO-mimetic

mechanisms, by developing and derivatizing novel MZ

scaﬀolds. These chimeric agents represent a novel drug class

coined nomethiazoles. The milestones for this study were (1)

development of the MZ pharmacophore through synthesis of

novel derivatives, (2) assay of neuroprotection in primary

neurons, (3) synthesis of nomethiazoles by derivatization of

novel, neuroprotective MZ scaﬀolds, possessing (4) satisfactory

stability. Proof-of-concept for nomethiazoles was achieved by

demonstration of (5) synaptic rescue using LTP measurements

ex vivo, (6) restoration of learning and memory in vivo, and (7)

brain bioavailability of both NO-chimeras and their MZ

metabolites. It was conﬁrmed that 1 is a prototype of a novel

drug class that possesses a combination of GABA-mimetic and

NO-mimetic activities, thus providing both neuroprotection

and neurorestoration. Since neurodegenerative diseases such as

AD are multifactorial, the development of drugs designed to

target more than one mechanism of action is likely to be a

promising therapeutic approach.

Figure 4. Procognitive eﬀects of NO-chimeras evaluated in the

scopolamine-induced memory deﬁcit model using the STPA task.

Scopolamine was administered 30 min and drug was administered 20

min prior to the start of training, and memory was tested 24 h after

training and drug treatment. NO-chimeras 28, 29, 32a, 32b, 42, 43

(∗∗∗, p < 0.01, compared with vehicle), and 40 (∗∗, p < 0.05,

compared with vehicle) signiﬁcantly reversed the scopolamine induced

memory deﬁcit. Data were obtained from at least ﬁve mice per group.

7. Brain Bioavailability Assessed by LC−MS/MS.

Although the procognitive eﬀects of the NO-chimeras are

indicative of brain bioavailability, LC−MS/MS measurements

were used to conﬁrm blood−brain barrier penetration.

Furthermore, a second objective of these assays was to measure

the concentrations of MZ metabolites in the brain after NO-

chimera delivery, since the neuroprotective properties of the

MZ metabolite are an important aspect of the drug design.

Figure 5 shows LC−MS/MS chromatograms for multiple

reaction monitoring (MRM) after administration of two NO-

chimeras and their MZ metabolites, representing two subclasses

of NO-chimera with diﬀerent physicochemical properties. The

NO-chimera 32a showed good brain bioavailability, and its 5-

ethyl-4-methylthiazole metabolite 22a was also observed in the

brain of mice 20 min after administration. In contrast, the

bioavailability of NO-chimera 28 was low at this time point,

whereas the 2-(4-methylthiazol-5-yl)-1H-1,2,3-triazol-1-yletha-

nol metabolite 26 was readily detected. Calculated parameters

suggest that 26 (tPSA = 73 Å²; ClogP < 0) should not

penetrate the blood−brain barrier, whereas 22a (tPSA = 33 Å²;

ClogP = 1.56) has more favorable properties; however, both

MZ derivatives were observed in mouse brains after direct ip

administration or after administration of the NO-chimera

prodrug. The preliminary replicate analyses after administration

of 26 and 28 suggest that the nitrate chimera prodrug elevates

brain levels of 26, relative to direct administration of 26 itself

(Table 2). These data, obtained after ip administration, are

EXPERIMENTAL SECTION

■

1

Synthesis. H and ¹³C NMR spectra were obtained with Bruker

Ultrashield 400 MHz and Advance 300 MHz spectrometers. Chemical

shifts are reported as δ values in parts per million (ppm) relative to

tetramethylsilane (TMS) for all recorded NMR spectra. All reagents

and solvents were obtained commercially from Acros, Aldrich, and

Fluka and were used without puriﬁcation. Low-resolution mass

spectrometry (LRMS) was conducted on an Agilent 6300 ion-trap

LC−MS instrument. High resolution mass spectral data were collected

using a Shimadzu QTOF 6500 instrument. All compounds submitted

for biological testing were found to be >95% pure by analytical HPLC.

Purity and/or low resolution mass spectral (ESI or APCI) analysis was

measured using an Agilent Zorbax Bonus C18, 3.5 μm, 150 mm × 3.0

mm column on either a Shimadzu UFLC or Agilent 1100 series HPLC

instrument in tandem with an Agilent 6310 ion trap LC−MS

instrument. HPLC parameters were the following: 0.1% FA in 10%

CH₃OH (solvent A); 0.1% FA in CH₃CN (solvent B); ﬂow rate 0.5

mL/min; gradient, t = 0 min, 10% A; t = 1 min, 10% A; t = 18 min,

80% A; t = 20 min, 80% A; t = 21 min, 95% A.

1-(4-Methyl-5-thiazolyl)-1-phenylmethanol (2). 4-Methyl-5-

thiazolecarboxaldehyde (1.0 g, 7.8 mmol) was dissolved in THF (40

mL) and cooled in an ice bath, and phenylmagnesium bromide (1 M

in THF, 8.5 mL) was added dropwise. The reaction mixture was

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Figure 5. MRM LC−MS/MS chromatograms of extracted mouse brain samples 20 min after ip delivery of each drug at a dosage that was equimolar

with 1 (4.5 μmol/kg): (A) 26 with 8 used as internal standard; (B) 28 with 8 used as internal standard; (C) 22a with 22b used as internal standard;

(D) 32a with 22b used as internal standard.

NMR (acetone-d₆, 100 MHz): δ 151.57, 148.90, 145.14, 137.96,

129.09, 128.20, 126.92, 69.66, 15.54.

Table 2. Analysis of Selected MZ Derivatives and NO-

Chimeras in Mice Brain Tissue

5-(Azidophenylmethyl)-4-methylthiazole (3). MsCl (2.9 mL)

was added to a solution of 2 (6.2 g) and NEt₃(6.0 mL) in anhydrous

acetonitrile (70 mL). Reaction was monitored by TLC until the

starting material was consumed. NaN₃(6.1 g) was added, and the

mixture was stirred at room temperature overnight. Most of the

acetonitrile was removed under reduced pressure. The residue was

diluted with ethyl acetate and washed with water. The organic phase

was separated and concentrated, and the crude product was puriﬁed by

MRM

transition of

analytes

injected

internal standard and brain concn

a

drug (ip) analyte

MRM transition

(ng/mL)

28

333 → 288

288 → 147

265 → 220

220 → 189

8, 352 → 147

22b, 238 → 207

2.1 0.6

76.6 10.2

56.4 5.5

9.5 0.6

9.5 1.1

32.9 3.7

28

26

32a

22a

32a

22a

1

column chromatography (hexane/ethyl acetate, 2:1, 75%). H NMR

(acetone-d₆, 400 MHz): δ 8.87 (s, 1H), 7.34−7.47 (m, 5H), 6.41 (s,

1H), 2.45 (s, 3H). ¹³C NMR (acetone-d₆, 100 MHz): δ 152.86,

151.16, 140.13, 131.97, 129.67, 129.24, 127.62, 61.78, 15.55. HRMS

calcd for C₁₁H₁₀N₄S [M + H]⁺231.0704, found 231.0713.

a

Four mice were used for the injection of each drug. Values are

presented as the mean SEM (n = 4).

α-Phenyl-(4-methyl-5-thiazolyl)methanamine (4). LiAlH₄

(130 mg, 3.5 mmol) was added to a solution of 3 (320 mg, 1.39

mmol) in anhydrous THF (10 mL). The mixture was reﬂuxed for 1 h

and quenched with 0.1 N NaOH. The residue was diluted with water

and extracted using ethyl acetate (2 × 25 mL). Organic solutions were

combined and concentrated, and the crude product was puriﬁed by

column chromatography (DCM/MeOH, 15:1, 230 mg, 82%). ¹³C

warmed to room temperature and stirred overnight. The reaction was

quenched with aqueous NH₄Cl, and extraction was with ethyl acetate

(2 × 100 mL). Organic solutions were combined and concentrated,

and the crude product was puriﬁed by column chromatography

1

(hexanes/ethyl acetate, 1:1, 1.4 g, 87%). H NMR (acetone-d₆, 400

MHz): δ 8.81 (s, 1H), 7.45(d, 2H, J = 7.2 Hz) 7.24−7.36 (m, 3H),

6.16 (d, 1H, J = 3.6 Hz), 5.34 (d, 1H, J = 3.6 Hz), 2.38 (s, 3H). ¹³C

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NMR (CD₃CN, 100 MHz): δ 151.62, 148.35, 145.96, 139.76, 129.52,

128.23, 127.51, 53.69, 15.61. HRMS calcd for C₁₁H₁₂N₂S [M + H]⁺

205.0799, found 205.0807.

4-(1-Methyl-1H-imidazol-5-yl)-1-[(4-methyl-5-thiazolyl)-

phenylmethyl]-1H-1,2,3-triazole (5h). 5-Ethynyl-1-methyl-1H-imi-

dazole was reacted with 3 to give 5h. ¹H NMR (CDCl₃, 400 MHz): δ

8.75 (s, 1H), 7.70(s, 1H), 7.50 (bs, 2H), 7.39−7.40 (m, 3H), 7.31(s,

1H), 7.17−7.19 (m, 2H), 3.93 (s, 3H), 2.44 (s, 3H). ¹³C NMR

(CDCl₃, 100 MHz): δ 152.50, 151.87, 137.66, 129.04, 128.99, 128.90,

126.52, 120.21, 60.89, 15.22. HRMS calcd for C₁₇H₁₆N₆S [M + H]⁺

337.1235, found 337.1235.

Compound 5. Standard Click Chemistry Procedure. Azide (1

equiv) and alkyne (1 equiv) were dissolved in t-BuOH and water

(1:1). Sodium ascorbate (0.2 equiv) and CuSO₄·5H₂O (0.1 equiv)

were added. The reaction mixture was stirred overnight, diluted with

ethyl acetate, and washed with water. The organic phase was separated

and concentrated, and the crude product was puriﬁed by column

chromatography (DCM/MeOH, 20:1, yield 75−90%). For com-

pounds 5i and 11a, the solvent was changed to a mixture of MeOH

and H₂O (2:1, v/v). K₂CO₃(1 equiv) was also added in addition to

other reactants.

4-Butyl-1-[(4-methyl-5-thiazolyl)phenylmethyl]-1H-1,2,3-tri-

azole (5a). 1-Hexyne was reacted with 3 to give 5a. ¹H NMR

(acetone-d₆, 400 MHz): δ 8.92 (s,1H), 7.83 (s, 1H), 7.48 (s, 1H),

7.35−7.41(m, 3H), 7.27−7.28 (m, 2H), 2.68 (t, 2H, J = 8.0 Hz), 2.36

(s, 3H), 1.58−1.66 (m, 2H), 1.29−1.38 (m, 2H), 0.88 (t, 3H, J = 7.6

Hz). ¹³C NMR (acetone-d₆, 100 MHz): δ 153.50, 152.51, 148.66,

140.45, 131.10, 129.67, 129.34, 127.72, 122.08, 61.08, 32.23, 25.86,

22.79, 15.40, 14.02. HRMS calcd for C₁₇H₂₀N₄S [M + H]⁺313.1481,

found 313.1468.

1-[(4-Methyl-5-thiazolyl)phenylmethyl]-4-phenyl-1H-1,2,3-

triazole (5b). Ethynylbenzene was reacted with 3 to give 5b. ¹H

NMR (acetone-d₆, 400 MHz): δ 9.02 (s, 1H), 8.48 (s, 1H), 7.92−7.94

(m, 2H), 7.58 (s, 1H), 7.30−7.45 (m, 8H), 2.41 (s, 3H). ¹³C NMR

(acetone-d₆, 100 MHz): δ 147.98, 140.23, 131.82, 129.77, 129.55,

129.48, 128.75, 127.74, 126.29, 121.31, 61.47, 15.47. HRMS calcd for

C₁₉H₁₆N₄S [M + H]⁺333.1174, found 333.1186.

1-[(4-Methyl-5-thiazolyl)phenylmethyl]-4-(phenylmethyl)-

1H-1,2,3-triazole (5c). Propargylbenzene was reacted with 3 to give

5c. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.98 (s, 1H), 7.97 (s, 1H), 7.53

(s, 1H), 7.17−7.41 (m, 10H), 4.01 (s, 2H), 2.31 (s, 3H). ¹³C NMR

(CDCl₃, 100 MHz): δ 153.82, 150.92, 146.33, 139.46, 139.36, 129.81,

128.99, 128.57, 128.52, 126.81, 126.29, 122.78, 59.37, 31.19, 15.05.

HRMS calcd for C₂₀H₁₈N₄S [M + H]⁺347.1330, found 347.1344.

3-[1-[(4-Methyl-5-thiazolyl)phenylmethyl]-1H-1,2,3-triazol-

4-yl]pyridine (5d). 3-Ethynylpyridine was reacted with 3 to give 5d.

¹H NMR (DMSO-d₆, 400 MHz): δ 9.10 (bs, 1H), 9.03 (s, 1H), 8.83

(s, 1H), 8.54 (bs,1H), 8.25 (d, 1H, J = 8.0 Hz), 7.67 (s, 1H), 7.29−

7.49 (m, 6H), 2.45 (s, 3H). ¹³C NMR (DMSO-d₆, 100 MHz): δ

153.98, 151.42, 149.12, 146.58, 143.77, 139.09, 132.71, 129.48, 129.08,

128.76, 126.87, 126.41, 124.08, 122.18, 59.87, 15.12. HRMS calcd for

C₁₈H₁₅N₅S [M + H]⁺334.1126, found 334.1118.

2-[1-[(4-Methyl-5-thiazolyl)phenylmethyl]-1H-1,2,3-triazol-

4-yl]pyridine (5e). 2-Ethynylpyridine was reacted with 3 to give 5e.

¹H NMR (acetone-d₆, 400 MHz): δ 8.95 (s, 1H), 8.55 (d, 1H, J = 4.0

Hz), 8.51 (s, 1H), 8.15 (d, 1H, J = 8.0 Hz), 7.85 (triple doublet, 1H, J

= 8.0, 1.6 Hz), 7.64 (s, 1H), 7.21−7.45 (m, 6H), 2.43 (s, 3H). ¹³C

NMR (acetone-d₆, 100 MHz): δ 153.73, 152.61, 151.51, 150.42,

148.90, 140.03, 137.59, 130.37, 129.79, 129.51, 127.74, 123.69, 123.32,

120.34, 61.43, 15.43. HRMS calcd for C₁₈H₁₅N₅S [M + H]⁺334.1126,

found 334.1129.

4-[1-[(4-Methyl-5-thiazolyl)phenylmethyl]-1H-1,2,3-triazol-

4-yl]pyridine (5f). 4-Ethynylpyridine was reacted with 3 to give 5f.

¹H NMR (CDCl₃, 400 MHz): δ 8.75 (s, 1H), 8.61 (d, 2H, J = 4.8 Hz),

1-[(4-Methyl-5-thiazolyl)phenylmethyl]-1H-1,2,3-triazole

(5i). Ethynyltrimethylsilane was reacted with 3 in the presence of

1

CuSO₄, sodium ascorbate, and K₂CO₃to give 5i. H NMR (acetone-

d₆, 400 MHz): δ 8.90 (s, 1H), 8.06 (d, 1H, J = 0.8 Hz), 7.75 (s, 1H, J =

0.8 Hz), 7.57 (s, 1H), 7.26−7.43 (m, 5H), 2.37 (s, 3H). ¹³C NMR

(acetone-d₆, 100 MHz): δ 153.57, 152.41, 140.39, 134.11, 130.73,

129.73, 129.42, 127.71, 125.00, 61.00, 15.35. HRMS calcd for

C₁₃H₁₂N₄S [M + H]⁺257.0861, found 257.0873.

1-(4-Methyl-5-thiazolyl)-1-(4-ﬂuorophenyl)methanol (6a). 4-

Fluorophenylmagnesium bromide was reacted with 1 to give 6a

according to the method described for compound 2. ¹H NMR

(CDCl₃, 400 MHz): δ 8.49 (s, 1H), 7.33−7.37 (m, 2H), 7.03 (t, 2H, J

= 8.4 Hz), 6.04 (s, 1H), 2.32 (s, 3H). HRMS calcd for C₁₁H₁₀FNOS

[M + H]⁺224.0545, found 224.0544.

1-(4-Methyl-5-thiazolyl)-1-(4-chlorophenyl)methanol (6b). 4-

Chlorophenylmagnesium bromide was reacted with 1 to give 6b

according to the method described for compound 2. ¹H NMR

(CDCl₃, 400 MHz): δ 8.56 (s, 3H), 7.33 (bs, 4H), 6.07 (s, 1H), 2.38

(s, 3H). HRMS calcd for C₁₁H₁₀ClNOS [M + H]⁺240.0250, found

240.0245.

5-[Azido(4-ﬂuorophenyl)methyl]-4-methylthiazole (7). 6a

was used to prepare 7 according to the method described for

compound 3. ¹H NMR (CDCl₃, 400 MHz): δ 8.69 (s, 1H), 7.27−7.34

(m, 2H), 7.07 (t, 2H, J = 8.4 Hz), 5.98 (s, 1H), 2.43 (s, 3H). ¹³C NMR

(CDCl₃, 100 MHz): δ 163.82, 161.35, 151.85, 150.28, 134.43, 134.40,

130.92, 128.60, 128.52, 115.98, 115.77, 60.74, 15.49.

3-[1-[(4-Fluorophenyl)(4-methyl-5-thiazolyl)methyl]-1H-

1,2,3-triazol-4-yl]pyridine (8). 3-Ethylnylpyridine was reacted with

1

7 to give 8 using a method described for compound 5. H NMR

(CDCl₃, 400 MHz): δ 8.99 (s, 1H), 8.77 (s, 1H), 8.56 (d, 1H, J = 3.6

Hz), 8.18−8.21 (m, 1H), 7.88 (s, 1H), 7.34−7.37 (m, 2H), 7.17−7.20

(m, 2H), 7.06−7.11 (m, 2H), 2.44 (s, 3H). ¹³C NMR (CDCl₃, 100

MHz): δ 163.97, 161.49, 152.59, 152.09, 149.33, 146.89, 144.89,

113.71, 113.68, 132.89, 128.79, 128.61, 128.53,126.16, 123.60, 119.27,

116.26, 116.04, 60.42, 15.30. . HRMS calcd for C₁₈H₁₄FN₅S [M + H]⁺

352.1032, found 352.1040.

5-(2-Azidoethyl)-4-methylthiazole (10). MsCl (2.1 g, 18.1

mmol) was added to a solution of 9 and DIPEA (3.7 mL, 22.3

mmol) in anhydrous acetonitrile (100 mL). Reaction was monitored

by TLC until the starting material was consumed. NaN₃(2.7 g, 41.9

mmol) was added, and the mixture was reﬂuxed for 3 h. Most of the

acetonitrile was removed under reduced pressure. The residue was

diluted with ethyl acetate and washed with water. The organic phase

was separated and concentrated, and the crude product was puriﬁed by

column chromatography. Product was obtained as a slightly yellow oil.

¹H NMR (acetone-d₆, 400 MHz): δ 8.74 (s, 1H), 3.59 (t, 2H, J = 6.8

Hz), 3.09 (t, 2H, J = 6.8 Hz), 2.38 (s, 3H). ¹³C NMR (acetone-d₆, 100

MHz): δ 150.75, 128.19, 52.72, 26.54, 14.96.

Compound 11. 11a−c were synthesized using the method

described for compound 5.

8.01 (s, 1H), 7.71 (d, 2H, J = 5.6 Hz), 7.34−7.40 (m, 4H), 7.18−7.20

(m, 2H), 2.42 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 152.49,

151.94, 150.09, 145.12, 137.55, 137.39, 129.02, 128.99, 128.81, 126.46,

120.65, 119.73, 60.96, 15.18. HRMS calcd for C₁₈H₁₅N₅S [M + H]⁺

334.1126, found 334.1119.

1-[(4-Methyl-5-thiazolyl)phenylmethyl]-4-(3-thienyl)-1H-

1,2,3-triazole (5g). 3-Ethynylthiophene was reacted with 3 to give

5g. ¹H NMR (CDCl₃, 400 MHz): δ 8.74 (s, 1H), 7.69 (d, 1H, J = 2.0

Hz), 7.60 (s, 1H), 7.36−7.44 (m, 5H), 7.30 (s, 1H), 7.15−7.16 (m,

2H), 2.43 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 152.43, 152.16,

144.17, 138.07, 131.47, 129.34, 129.17, 129.08, 126.70, 126.40, 125.73,

121.42, 118.68, 60.91, 15.42. HRMS calcd for C₁₇H₁₄N₄S₂[M + H]⁺

339.0738, found 339.0754.

1-[2-(4-Methyl-5-thiazolyl)ethyl]-1H-1,2,3-triazole (11a).

Ethynyltrimethylsilane was reacted with 10 in the presence of

CuSO₄, sodium ascorbate, and K₂CO₃to give 11a. ¹H NMR

(acetone-d₆, 400 MHz): δ 8.70 (s, 1H), 7.83 (s, 1H), 7.62 (s, 1H),

4.68 (t, 2H, J = 6.8 Hz), 3.45 (t, 2H, J = 6.8 Hz), 2.18 (s, 3H). ¹³C

NMR (acetone-d₆, 100 MHz): δ 151.12, 150.93, 133.81, 127.25,

125.13, 51.14, 27.68, 14.59. HRMS calcd for C₈H₁₀N₄S [M + H]⁺

195.0704, found 195.0711.

1-[2-(4-Methyl-5-thiazolyl)ethyl]-4-phenyl-1H-1,2,3-triazole

1

(11b). Ethynylbenzene was reacted with 10 to give 11b. H NMR

(CDCl₃, 400 MHz): δ 8.60 (s, 1H), 7.76−7.78 (m, 2H), 7.51 (s, 1H),

7.39−7.43 (m, 2H), 7.31−7.35 (m, 1H), 4.60 (t, 2H, J = 6.8 Hz), 3.44

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(t, 2H, J = 6.8 Hz), 2.25 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ

150.73, 150.32, 147.78, 130.32, 128.80, 128.20, 125.84, 125.69, 119.94,

51.06, 27.34, 14.58. HRMS calcd for C₁₄H₁₄N₄S [M + H]⁺271.1017,

found 271.1015.

3-[1-[2-(4-Methyl-5-thiazolyl)ethyl]-1H-1,2,3-triazol-4-yl]-

pyridine (11c). 3-Ethynylpyridine was reacted with 10 to give 11c.

¹H NMR (CDCl₃, 400 MHz): δ 8.95 (s, 1H), 8.61 (s, 1H), 8.56 (d,

1H, J = 3.6 Hz), 8.13−8.15 (m, 1H), 7.70 (s, 1H), 7.34−7.37 (m, 1H),

4.64 (t, 2H, J = 6.8 Hz), 3.47 (t, 2H, J = 6.8 Hz), 2.26 (s, 3H). ¹³C

NMR (CDCl₃, 100 MHz): δ 150.82, 150.40, 149.33, 147.01, 144.73,

132.98, 126.49, 125.66, 123.74, 120.35, 51.23, 27.31, 14.61. HRMS

calcd for C₁₃H₁₃N₅S [M + H]⁺272.0970, found 272.0965.

3-Bromo-5-(methoxymethoxy)pyridine (13). 3-Bromo-5-hy-

droxypyridine (1.0 g, 5.7 mmol) and MOM-Br (0.64 mL, 6.9

mmol) were dissolved in THF(10 mL). Anhydrous K₂CO₃(1.5 g, 10.9

mmol) was added, and the reaction mixture was stirred overnight.

After ﬁltration and concentration, the product was puriﬁed by column

chromatography (SiO₂, hexane/ethyl acetate, 3:1, 1.1 g, 88%). ¹H

NMR (CDCl₃, 400 MHz): δ 8.34 (s, 1H), 8.33 (s, 1H), 7.56 (t, 1H, J

= 2.4 Hz), 5.19 (s, 2H), 3.49 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ

153.70, 143.96, 137.81, 125.72, 120.18, 94.66, 56.27.

Solvent was removed, and the residue was dissolved in ethyl acetate

and washed with water. The crude product was puriﬁed by column

chromatography (DCM/MeOH, 15:1, 67 mg, 28%). ¹H NMR

(CDCl₃, 400 MHz): δ 8.76 (s,1H), 8.50 (d, 1H, J = 0.8 Hz), 8.24

(d, 1H, J = 2.4 Hz), 7.84 (s,1H), 7.77 (t, 1H, J = 1.6 Hz), 7.39−7.41

(m, 3H), 7.33 (s,1H), 7.17−7.19 (m, 2H), 4.10 (t, 2H, J = 6.4 Hz),

3.73 (t, 2H, J = 6.4 Hz), 2.44 (s, 3H), 1.89−1.94 (m, 2H), 1.74−1.79

(m, 2H). ¹³C NMR (CDCl₃, 100 MHz): δ 155.30, 152.60, 152.09,

144.66, 138.82, 138.13, 137.74, 129.19, 129.16, 129.06, 126.84, 126.64,

119.58, 117.38, 68.19, 62.01, 61.11, 29.07, 25.55, 15.35. HRMS calcd

for C₂₂H₂₃N₅O₂S [M + H]⁺422.1645, found 422.1657.

4-[[5-[1-[(4-Methyl-5-thiazolyl)phenylmethyl]-1H-1,2,3-tria-

zol-4-yl]-3-pyridinyl]oxybutane]-1-nitrate (18). 1-Nitrooxy-4-hy-

droxybutane (25 mg, 0.19 mmol), 16 (25 mg, 0.07 mmol), and PPh₃

(37 mg, 0.14 mmol) were dissolved in THF (2 mL), and the ﬂask was

ﬁlled with argon. The solution was cooled in an ice bath, and DIAD

(40 μL) was added using a syringe. The mixture was stirred overnight

and quenched with water. Solvent was removed, and crude product

was puriﬁed by puriﬁed by column chromatography (DCM/MeOH,

1

20:1, 67%). H NMR (CDCl₃, 400 MHz): δ 8.77 (s, 1H), 8.53 (s,

1H), 8.26 (s, 1H), 7.81 (s, 1H), 7.80−7.79 (m, 1H), 7.40−7.42 (m,

3H), 7.33 (s, 1H), 7.17−7.19 (m, 2H), 4.55 (t, 2H, J = 6.0 Hz), 4.12

(t, 2H, J = 6.0 Hz), 2.45 (t, 3H), 1.95−1.97 (m, 4H). ¹³C NMR

(CDCl₃, 100 MHz): δ155.14, 152.65, 152.24, 144.67, 139.20, 138.14,

137.81, 129.29, 129.26, 129.09, 126.98, 126.72, 119.54, 117.39, 72.70,

67.47, 61.21, 25.47, 23.71, 15.45. HRMS calcd for C₂₂H₂₂N₆O₄S [M +

H]⁺467.1501, found 467.1488.

α-(Bromomethyl)-4-methyl-5-thiazolemethanol (20). 4-

Methyl-5-vinylthiazole (900 mg, 7.2 mmol) was dissolved in a mixture

of dioxane (2.5 mL), H₂O (5 mL) and acetic acid (430 mg). NBS (1.4

g, 7.9 mmol) was added in three portions. The reaction mixture was

stirred at room temperature for 1 h and then diluted with ethyl acetate

(50 mL). The organic phase was separated and concentrated, and the

product was puriﬁed by column chromatography (SiO₂, hexane/ethyl

acetate, 1.5:1, 900 mg, 52%). ¹H NMR (CDCl₃, 400 MHz): δ 8.64 (s,

1H), 5.22 (t, 1H, J = 6.8 Hz), 3.57 (d, 2H, J = 6.8 Hz), 2.42 (s, 3H).

¹³C NMR (CDCl₃, 100 MHz): δ 151.53, 149.48, 132.10, 67.63, 38.44,

3-(Methoxymethoxy)-5-[2-(trimethylsilyl)ethynyl]pyridine

(14). Compound 13 (1.0 g, 4.6 mmol), CuI (44 mg, 0.23 mmol), and

Pd(Ph₃P)₂Cl₂(162 mg, 0.23 mmol) were placed in a ﬂask and ﬁlled

with argon. THF (10 mL) was added using a syringe followed by NEt₃

(3.0 mL, 21 mmol) and ethynyltrimethylsilane (0.7 mL, 5.1 mmol).

The reaction mixture was stirred overnight at room temperature. Most

of the solvent was removed, and the residue was puriﬁed by column

1

chromatography (SiO₂, hexane/ethyl acetate, 5:1, 0.85 g, 81%). H

NMR (CDCl₃, 400 MHz): δ 8.32−8.35 (m, 2H), 7.43−7.44 (m, 2H),

5.19 (s, 2H), 3.48 (s, 3H), 0.26 (s, 9H). ¹³C NMR (CDCl₃, 100

MHz): δ 152.65, 145.97, 139.21, 125.18, 120.38, 101.15, 98.03, 94.53,

56.17.

3-(Methoxymethoxy)-5-[1-[(4-methyl-5-thiazolyl)-

phenylmethyl]-1H-1,2,3-triazol-4-yl]pyridine (15). Azide 3 (783

mg, 3.4 mmol) and alkyne 14 (0.8 g, 3.4 mmol) were dissolved in a

mixture of MeOH and water (2:1, 5 mL). K₂CO₃(484 mg, 3.5 mmol),

CuSO₄·5H₂O (170 mg, 0.68 mmol), and sodium ascorbate (272 mg,

0.68 mmol) were added, and the reaction mixture was stirred at room

temperature overnight. Most of the solvent was removed under

reduced pressure. The residue was dissolved in DCM and washed with

water. The organic phase was separated and concentrated, and the

crude product was puriﬁed by column chromatography (DCM/

15.39.

4-Methyl-5-(2-oxiranyl)thiazole (21). Bromohydrin 20 (650 mg,

2.94 mmol) was dissolved in MeOH (10 mL). K₂CO₃(1.2 g, 8.8

mmol) was added, and the reaction mixture was stirred at room

temperature for 1 h. Solvent was removed under reduced pressure, and

the residue was diluted with hexane and ethyl acetate (50 mL, 2:1 v/

v). After ﬁltration and concentration, the product was obtained with

enough purity for the next step reaction. ¹H NMR (CDCl₃, 400

MHz): δ 8.63 (s, 1H), 4.10 (t, 1H, J = 3.6 Hz), 3.22−3.24 (m, 1H),

2.91−2.93 (m, 1H), 2.53 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ

151.90, 150.69, 128.97, 51.25, 46.80, 14.95.

1

MeOH, 20:1, 1.15 g, 86%). H NMR (CDCl₃, 400 MHz): δ 8.75 (s,

1H), 8.65 (s, 1H), 8.37 (s, 1H), 7.92 (s, 1H), 7.90 (s, 1H), 7.35−7.39

(m, 4H), 7.18−7.20 (m, 2H), 5.24 (s, 2H), 3.48 (s, 3H), 2.43 (s, 3H).

¹³C NMR (CDCl₃, 100 MHz): δ 153.40, 152.50, 151.92, 144.42,

140.07, 139.05, 137.69, 129.01, 128.96, 128.92, 126.79, 126.51, 119.70,

119.48, 94.31, 60.93, 56.03, 15.20.

β-Phenyl-4-methyl-5-thiazoleethanol (22a) and α-(Phenyl-

methyl)-4-methyl-5-thiazolemethanol (23a). Crude epoxide 21

(1.0 g, 7.1 mmol) was dissolved in anhydrous THF (10 mL) and

added dropwise to a phenylmagnisum bromide solution (1 M in THF,

10 mL) at 4 °C. The reaction mixture was stirred overnight at room

temperature, quenched with aqueous NH₄Cl, and extracted with ethyl

acetate (2 × 50 mL). The organic phase was separated and

concentrated. 22a and 23a were separated and puriﬁed by column

chromatography (SiO₂, hexane/ethyl acetate 1:1). 22a (major product,

5-[1-[(4-Methyl-5-thiazolyl)phenylmethyl]-1H-1,2,3-triazol-

4-yl]-3-pyridinol (16). Click product 15 (230 mg, 0.58 mmol) was

dissolved in HCl/i-PrOH solution (1.5M, 5.0 mL) and heated at 70

°C for 1 h. The solvent was removed. The residue was dissolved in

AcOEt and washed with aqueous NaHCO₃. The organic phase was

separated and concentrated, and the crude product was puriﬁed by

column chromatography (DCM/MeOH, 15:1, 170 mg, 83%). ¹H

NMR (acetone-d₆, 400 MHz): δ 9.11 (bs, 1H), 8.93 (s, 1H), 8.61 (s,

1H), 8.59 (s, 1H), 8.17 (d, 1H, J = 2.8 Hz), 7.76 (t, 1H, J = 2.4 Hz),

7.60 (s, 1H), 7.35−7.46 (m, 5H), 2.41 (s, 3H). ¹³C NMR (acetone-d₆,

100 MHz): δ154.63, 153.77, 152.72, 145.09, 140.14, 139.11, 138.41,

130.41, 129.83, 129.56, 128.41, 127.78, 122.09, 119.22, 61.50, 15.43.

HRMS calcd for C₁₈H₁₅N₅OS [M + H]⁺350.1076, found 350.1068.

4-[5-[1-[(4-Methyl-5-thiazolyl)phenylmethyl]-1H-1,2,3-tria-

zol-4-yl]-3-pyridinyl]oxy-1-butanol (17). 1-Acetoxy-4-hydroxybu-

tane (150 mg, 1.2 mmol), 16 (200 mg, 0.57 mmol), and PPh₃(227

mg) were dissolved in THF (5 mL), and the ﬂask was ﬁlled with

argon. The solution was cooled in an ice bath, and DIAD (230 μL, 1.1

mmol) was added using a syringe. The mixture was stirred overnight

and quenched with water. After workup, the crude product was

dissolved in methanol (10 mL) and treated with K₂CO₃(140 mg).

1

1.0 g, 65%), H NMR (CDCl₃, 400 MHz): δ 8.43 (s, 1H), 7.25−7.29

(m, 2H), 7.17−7.21 (m, 3H), 4.38 (t, 1H, J = 6.4 Hz), 3.95−4.05 (m,

2H), 3.87 (s, 1H, OH), 2.28 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ

150.11, 149.87, 140.13, 131.48, 128.92, 127.91, 127.33, 67.13, 46.27,

15.34. HRMS calcd for C₁₂H₁₃NOS [M + H]⁺220.0796, found

1

220.0795. 23a (minor product, 0.2 g, 13%), H NMR (CDCl₃, 400

MHz): δ 8.43 (s, 1H), 7.28−7.33 (m, 5H), 4.83 (t, 1H, J = 6.4 Hz),

3.29 (s, 1H), 3.10−3.19 (m, 2H), 2.17 (s, 3H). ¹³C NMR (CDCl₃, 100

MHz): δ 150.07, 149.89, 143.23, 128.42, 127.82, 126.95, 125.77, 74.36,

36.08, 14.63. HRMS calcd for C₁₂H₁₃NOS [M + H]⁺220.0796, found

220.0796.

β-(4-Fluorophenyl)-4-methyl-5-thiazoleethanol (22b) and α-

[(4-Fluorophenyl)methyl]-4-methyl-5-thiazolemethanol (23b).

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22b and 23b were synthesized using the procedure described above for

22a from epoxide 21 and 4-ﬂuorophenylmagnisum bromide. Products

were separated and puriﬁed by column chromatography (SiO₂,

hexane/ethyl acetate, 1:1). 22b (major product, 60%), ¹H NMR

(CDCl₃, 400 MHz): δ 8.45 (s, 1H), 7.16−7.19 (m, 2H), 6.30−6.97

(m, 2H), 4.37 (t, 1H, J = 6.4 Hz), 3.94−4.03 (m, 2H), 2.26 (s, 3H).

¹³C NMR (CDCl₃, 100 MHz): δ 162.66, 160.22, 150.07, 148.95,

136.24, 136.21, 131.98, 129.26, 129.18, 115.36, 115.14, 66.22, 45.16,

14.82. HRMS calcd for C₁₂H₁₂FNOS [M + H]⁺238.0702, found

238.0699. 23b (minor product, 14%), ¹H NMR (CDCl₃, 400 MHz): δ

8.39 (s, 1H), 7.21−7.24 (m, 2H), 6.96−6.99 (m, 2H), 4.81 (t, 1H, J =

6.4 Hz), 3.04−3.16 (m, 2H), 2.12 (s, 3H). ¹³C NMR (CDCl₃, 100

MHz): δ 163.50, 161.06, 150.25, 149.84, 139.14, 139.11, 127.51,

127.43, 126.89, 115.35, 115.14, 73.62, 36.19, 14.61. HRMS calcd for

C₁₂H₁₂FNOS [M + H]⁺238.0702, found 238.0709.

β-(4-Chlorophenyl)-4-methyl-5-thiazoleethanol (22c). Com-

pound 22c was synthesized using the procedure described above for

22a from epoxide 21 and 4-chlorophenylmagnisum bromide (1 M in

diethyl ether). Product was separated and puriﬁed by column

chromatography (SiO₂, hexane/ethyl acetate, 1:1, 79%). ¹H NMR

(CDCl₃, 400 MHz): δ 8.61 (s, 1H), 7.29 (d, 2H, J = 8.4 Hz), 7.18 (d,

2H, J = 8.4 Hz), 4.42 (t, 1H, J = 6.4 Hz), 4.03−4.07 (m, 2H), 2.35 (s,

3H). ¹³C NMR (CDCl₃, 100 MHz): δ 150.29, 149.95, 138.78, 133.13,

131.02, 129.26, 129.00, 66.86, 45.57, 15.32.

2,3,4,5-Tetrahydro-2,8-dimethyl-5-[2-(4-methyl-5-thiazolyl)-

ethyl]-1H-pyrido[4,3-b]indole (24). Caboline (500 mg, 2.5 mmol)

was dissolved in DMSO (5 mL). NaH (120 mg, 60%, 3 mmol) was

added, and the mixture was stirred for 5 min. 4-Methyl-5-vinylthiazole

(2.86 mL, 25 mmol) was added, and the reaction mixture was heated

at 90 °C for 5 h. Reaction was quenched with MeOH (0.5 mL), and

the solvent was evaporated under high vacuum. The residue was

dissolved in DCM (20 mL) and washed with water. The organic phase

was separated and concentrated, and the product was puriﬁed by

column chromatography (SiO₂, DCM/MeOH, 25:1, 0.1% HOAc, 350

mg, yellow solid, 36%). Compound 24 was obtained as an AcOH salt.

¹H NMR (acetone-d₆, 400 MHz): δ 8.67 (s, 1H), 7.18−7.22 (m, 2H),

μL) were added. The reaction mixture was stirred at 0−4 °C for 4 h.

After dilution with ethyl acetate (20 mL), the reaction mixture was

neutralized by saturated aqueous NaHCO₃. The organic phase was

separated and concentrated, and the crude product was puriﬁed by

column chromatography (SiO₂, hexane/ethyl acetate, 2.5:1, 350 mg,

1

71%). H NMR (CDCl₃, 400 MHz): δ 8.79 (s, 1H), 5.24 (t, 1H, J =

2.4 Hz), 4.58 (d, 2H, J = 6.0 Hz), 2.53 (s, 3H). ¹³C NMR (CDCl₃, 100

MHz): δ 152.61, 151.93, 125.14, 73.29, 55.79, 15.33.

β-(4-Methyl-5-thiazolyl)-[4-(3-pyridinyl)-1H-1,2,3-triazole-1-

ethane]-1-nitrate (28) and β-(4-Methyl-5-thiazolyl)-[5-(3-pyr-

idinyl)-1H-1,2,3-triazole-1-ethane]-1-nitrate (29). Compound 27

(260 mg, 1.1 mmol) and 3-ethynylpyridine (234 mg, 2.2 mmol) were

dissolved in toluene (5 mL). The reaction mixture was reﬂuxed for 48

h. Toluene was removed under reduced pressure, and the residue was

puriﬁed by column chromatography (SiO₂, DCM/MeOH, 20:1). 28

(165 mg, 44%), ¹H NMR (CDCl₃, 400 MHz): δ 8.98 (s, 1H), 8.82 (s,

1H), 8.59 (d, 1H, J = 3.2 Hz), 8.18 (d, 1H, J = 8.0 Hz), 7.91 (s, 1H),

7.37 (dd, 1H, J = 4.8 Hz, 8.0 Hz), 6.35−6.38 (m, 1H), 5.31−5.36 (m,

1H), 5.09−5.13 (m, 1H), 2.56 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz):

δ153.33, 153.10, 149.63, 147.04, 145.27, 133.11, 126.01, 123.77,

123.58, 119.70, 72.32, 55.41, 15.40. HRMS calcd for C₁₃H₁₂N₆O₃S [M

+ H]⁺333.0764, found 333.0751. 29 (120 mg, 32%), ¹H NMR

(CDCl₃, 400 MHz): δ 8.80 (dd, 1H, J = 4.8 Hz, 1.6 Hz), 8.78 (s, 1H),

8.59 (d, 1H, J = 1.6 Hz), 7.80 (s, 1H), 7.64−7.67 (m, 1H), 7.49−7.52

(m, 1H), 5.97−6.01 (m, 1H), 5.44−5.50 (m, 1H), 4.98−5.02 (m, 1H),

2.24 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 153.83, 151.55, 151.34,

149.41, 136.44, 135.22, 133.71, 124.74, 123.80, 122.30, 72.76, 52.95,

14.93. HRMS calcd for C₁₃H₁₂N₆O₃S [M + H]⁺333.0764, found

333.0776.

β-(4-Methyl-5-thiazolyl)-5-(3-pyridinyl)-1H-1,2,3-triazole-1-

ethanol (30). Nitrate 29 (100 mg, 0.3 mmol) was dissolved in

CH₃CN/H₂O (2:1, 5 mL). Na₂CO₃(300 mg, 3 mmol) and 2-

mercaptoethanol (70 μL, 1 mmol) were added. The reaction mixture

was stirred at room temperature until most of the starting material was

consumed. After dilution with DCM (20 mL), the solution was

washed with H₂O and concentrated. The crude product was puriﬁed

6.93 (d, 1H,J = 8.4 Hz), 4.28 (t, 2H, J = 6.4 Hz), 3.82 (s, 2H), 3.23 (t,

2H, J = 6.4 Hz), 2.92 (t, 2H, J = 6.0 Hz), 2.56−2.62 (m, 5H), 2.38 (s,

3H), 1.96 (s, 3H), 1.91 (s, 3H) . ¹³C NMR (acetone-d₆, 100 MHz): δ

172.71, 150.34,150.75, 135.69, 133.51, 128.69, 128.35, 126.83, 123.21,

118.16, 109.16, 106.11, 52.23, 51.42, 44.69, 44.22, 27.10, 21.84, 21.48,

20.89, 14.43. HRMS calcd for C₁₉H₂₃N₃S·CH₃CO₂H [M + H]⁺

326.1691, found 326.1687.

1

with column chromatography (DCM/MeOH, 20:1, 81%). H NMR

(CDCl₃, 400 MHz): δ 8.73 (d, 1H, J = 4.0 Hz), 8.66 (bs, 2H), 7.76−

7.77 (m, 1H), 7.72 (s, 1H), 7.45−7.49 (m, 1H), 5.85−5.88 (m, 1H),

4.65−4.71 (m, 1H), 4.18−4.22 (m, 1H), 2.14 (s, 3H). ¹³C NMR

(CDCl₃, 100 MHz): δ 152.97, 150.91, 150.65, 149.63, 136.91, 135.65,

133.38, 126.45, 123.79, 122.96, 65.62, 58.02, 14.96. HRMS calcd for

C₁₃H₁₃N₅OS [M + H]⁺288.0919, found 288.0916.

β-Azido-4-methyl-5-thiazoleethanol (25). Epoxide 21 (400 mg,

2.8 mmol) was dissolved in acetonitrile and water (1:1, 10 mL). NaN₃

(553 mg, 8.4 mmol) was added. The reaction mixture was reﬂuxed for

1 h. Most of the solvent was removed under reduced pressure. The

residue was dissolved in DCM (20 mL) and washed with water. The

organic phase was separated and concentrated, and the crude product

was puriﬁed by column chromatography (SiO₂, hexane/ethyl acetate,

β-[4-(3-Pyridyl)phenyl]-4-methyl-5-thiazoleethanol (31). 22c

(350 mg, 1.4 mmol), 3-pyridineboronic acid (270 mg, 2.2 mmol),

Pd(OAc)₂(15 mg), (2-biphenyl)di-tert-butylphosphine (41 mg), and

KF (240 mg, 4.1 mmol) were placed in a ﬂask ﬁlled with argon. DMF

(10 mL) was added using a syringe. The mixture was heated at 120 °C

for 1 h. DMF was removed under vacuum, and the residue was diluted

with DCM (50 mL) and washed with water. The crude product was

puriﬁed with column chromatography (DCM/MeOH, 20:1, 58%). ¹H

NMR (CDCl₃, 400 MHz): δ 8.57−8.60 (m, 2H), 8.47 (d, 1H, J = 3.6

Hz), 7.80 (d, 1H, J = 7.6 Hz), 7.47 (d, 2H, J = 8.0 Hz), 7.31−7.37 (d,

3H), 4.50 (t, 1H, J = 6.0 Hz), 4.09−4.18 (m, 2H), 2.38 (s, 3H). ¹³C

NMR (CDCl₃, 100 MHz): δ 150.18, 149.57, 147.96, 147.61, 140.81,

136.27, 136.03, 134.38, 131.51, 128.74, 127.30, 123.57, 66.62, 45.98,

15.26. HRMS calcd for C₁₇H₁₆N₂OS [M + H]⁺297.1062, found

297.1071.

1

1:1, 410 mg, 61%). H NMR (CDCl₃, 400 MHz): δ 8.71 (s, 1H),

4.98−5.00 (m, 1H), 4.80 (bs, 1H), 3.76−3.87 (m, 2H), 2.49 (s, 3H).

¹³C NMR (CDCl₃, 100 MHz): δ 152.35, 150.67, 126.97, 65.28, 59.85,

15.01. HRMS calcd for C₆H₈N₄OS [M + H]⁺185.0497, found

185.0508.

[1-[β-(4-Methyl-5-thiazolyl)ethanol]-4-(3-pyridinyl)]-1H-

1,2,3-triazole (26). Compound 26 was prepared according to the

standard click chemistry procedure described above from 25 and 3-

ethynylpyridine. The crude product was puriﬁed by column

chromatography (SiO₂, DCM/MeOH, 20:1, 95%). ¹H NMR

(CDCl₃, 400 MHz): δ 9.05 (s, 1H), 9.01 (s, 1H), 8.80 (s, 1H), 8.52

(d, 1H, J = 3.6 Hz), 8.21 (d, 1H, J = 8.0 Hz), 7.45 (dd, 1H, J = 5.2 Hz,

7.6 Hz), 6.22−6.25 (m, 1H), 4.15−4.20 (m, 1H), 4.04−4.08 (m, 1H),

2.44 (m, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 153.49, 151.44,

148.90, 146.37, 143.36, 132.42, 126.56, 126.36, 123.96, 121.73, 63.87,

58.99, 15.07. HRMS calcd for C₁₃H₁₃N₅OS [M + H]⁺288.0919, found

288.0918.

β-Phenyl-(4-methyl-5-thiazolyl)ethane-1-nitrate (32a). 22a

(150 mg, 0.68 mmol) was dissolved in DCM (3 mL) and cooled in

an ice bath. Fuming nitric acid (120 μL) and acetic anhydride (280

μL) were added. The reaction mixture was stirred at 0−4 °C for 4 h.

After dilution with ethyl acetate (20 mL), the reaction mixture was

neutralized by saturated aqueous NaHCO₃. The organic phase was

separated and concentrated, and the crude product was puriﬁed by

column chromatography (SiO₂, hexane/ethyl acetate, 3:1, 110 mg,

1

61%). H NMR (CDCl₃, 400 MHz): δ 8.64 (s, 1H), 7.25−7.37 (m,

1-Nitrooxy-β-azido-(4-methyl-5-thiazoyl)ethane (27). 25

(400 mg, 2.17 mmol) was dissolved in DCM (10 mL) and cooled

in an ice bath. Fuming nitric acid (130 μL) and acetic anhydride (245

5H), 4.89−4.94 (m, 1H), 4.80−4.85 (m, 1H), 4.68 (t, 1H, J = 7.2 Hz),

2.40 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 150.35, 150.21, 138.33,

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130.04, 129.06, 127.86, 127.49, 75.02, 41.34, 15.21. HRMS calcd for

C₁₂H₁₂N₂O₃S [M + H]⁺265.0647, found 265.0657.

NMR (CDCl₃, 400 MHz): δ 8.65 (s, 1H), 7.31−7.38 (m, 5H), 7.12 (s,

1H), 2.49 (s, 3H), 1.35 (s, 3H), 1.28−1.32 (m, 2H), 0.72−0.76 (m,

2H). ¹³C NMR (CDCl₃, 100 MHz): δ 174.50, 151.68, 150.40, 139.54,

131.62, 128.70, 128.29, 126.19, 70.16, 19.28, 18.70, 17.03, 17.00,

15.50. HRMS calcd for C₁₆H₁₇NO₂S [M + H]⁺288.1058, found

288.1070.

β-(4-Fluorophenyl)-(4-methyl-5-thiazolyl)ethane-1-nitrate

(32b). Compound 32b was prepared from 22b as described for 32a.

Product was puriﬁed by column chromatography (SiO₂, hexane/ethyl

1

acetate, 3:1, 69%). H NMR (CDCl₃, 400 MHz): δ 8.68 (s, 1H),

7.21−7.27 (m, 2H), 7.03−7.07 (m, 2H), 4.86−4.91 (m, 1H), 4.79−

4.84 (m, 1H), 4.68 (t, 1H, J = 7.2 Hz), 2.40 (s, 3H). ¹³C NMR

(CDCl₃, 100 MHz): δ 163.39, 160.93, 150.56, 150.27, 134.18, 134.14,

129.83, 129.25, 129.17, 116.17, 115.95, 74.85, 40.69, 15.22. HRMS

calcd for C₁₂H₁₁FN₂O₃S [M + H]⁺283.0553, found 283.0565.

α-(Phenylmethyl)-(4-methyl-5-thiazolyl)methanenitrate

(33). Compound 33 was prepared from 23a as described for 32a

5-(Methoxyphenylmethyl)-4-methylthiazole (38). To a sol-

ution of 2 (200 mg, 0.87 mmol) in DMF (4 mL) was added NaH (52

mg, 60%, 0.87 mmol). The reaction mixture was stirred at room

temperature for 15 min. CH₃I (65 μL, 1 mmol) was added, and the

reaction mixture was stirred for another 3 h. The reaction was

quenched with MeOH (1 mL), and then most of the solvent was

removed under vacuum. The residue was dissolved in ethyl acetate (50

mL) and washed with water. The organic phase was separated and

concentrated. The crude product was puriﬁed by column chromatog-

raphy (SiO₂, hexane/ethyl acetate, 2:1, 150 mg, 70%). ¹H NMR

(acetone-d₆, 400 MHz): δ 8.80 (s, 1H), 7.42−7.44 (m, 2H), 7.35−7.39

(m, 2H), 7.27−7.31 (m, 1H), 5.68 (s, 1H), 3.34 (s, 3H), 2.43 (s, 3H).

HRMS calcd for C₁₂H₁₃NOS [M + H]⁺220.0796, found 220.0792.

5-[[2-(2-Bromoethoxy)ethoxy]phenylmethyl]-4-methylthia-

zole (39). To a solution of 2 (500 mg, 2.4 mmol) in DMF (5 mL) was

added NaH (117 mg, 2.92 mmol). The reaction mixture was stirred at

room temperature for 15 min. Bis(2-bromomethyl) ether (2.2 g, 9.6

mmol) was added, and the reaction mixture was stirred for another 3

h. The reaction was quenched with MeOH (1 mL), and then most of

the solvent was removed. The residue was dissolved in ethyl acetate

(50 mL) and washed with water. The organic phase was separated and

concentrated. The crude product was puriﬁed by column chromatog-

raphy (SiO₂, hexane/ethyl acetate, 2:1, 740 mg, 86%). ¹H NMR

(CDCl₃, 300 MHz): δ 8.65 (s, 1H), 7.26−7.39 (m, 5H), 5.75 (s, 1H),

3.81(t, 2H, J = 6.1 Hz), 3.62−3.73 (m, 4H), 3.46 (t, 2H, J = 6.1 Hz),

2.45 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 151.50, 149.60, 140.83,

133.74, 128.60, 128.09, 126.58, 77.31, 71.25, 70.65, 68.36, 30.45,

15.57.

1-Nitrooxy-2-[2-[(4-methyl-5-thiazolyl)phenylmethoxy]-

ethoxy]ethane (40). Silver nitrate (2.0 g, 11.8 mmol) was added to a

solution of 39 (1.1 g, 3.1 mmol) in CH₃CN (15 mL). The reaction

was reﬂuxed for 2 h and then diluted with ethyl acetate (50 mL). After

ﬁltration and concentration, the crude product was puriﬁed by column

chromatography (SiO₂, hexane/ethyl acetate, 1.5:1, 720 mg, 69%). ¹H

NMR (CDCl₃, 400 MHz): δ 8.62 (s, 1H), 7.26−7.38 (m, 5H), 4.59 (t,

2H, J = 4.4 Hz), 3.77 (t, 2H, J = 4.4 Hz), 3.67−3.69 (m, 2H), 3.62−

3.64 (m, 2H), 2.45 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ 151.40,

149.46, 140.63, 133.55, 128.49, 127.99, 126.39, 77.22, 72.03, 70.69,

68.26, 67.15, 15.36. HRMS calcd for C₁₅H₁₈N₂O₅S [M + H]⁺

339.1014, found 339.1014.

1

(86%). H NMR (CDCl₃, 400 MHz): δ 8.55 (s, 1H), 7.32−7.34 (m,

3H), 7.26−7.28 (m, 2H), 5.88 (t, 1H, J = 7.2 Hz), 3.38−3.44 (m, 1H),

3.24−3.29 (m, 1H), 2.22 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz): δ

150.90, 150.41, 136.15, 129.12, 128.66, 126.22, 124.04, 84.51, 31.27,

14.51. HRMS calcd for C₁₂H₁₂N₂O₃S [M + H]⁺265.0647, found

265.0657.

1-[(Nitrooxy)methyl]cyclopropanemethanol (34). 1,1-Bis-

(hydroxymethyl)cyclopropane (5.0 g, 48.9 mmol) was dissolved in

DCM (100 mL) and cooled in an ice bath. Fuming nitric acid (2.5

mL) followed by acetic anhydride (5 mL) was added slowly. The

mixture was stirred at 4 °C for 4 h and quenched by aqueous

NaHCO₃. The organic phase was separated and washed with water.

The crude product was puriﬁed by column chromatography (hexanes/

1

ethyl acetate, 4:1, 1.2 g, 17%). H NMR (acetone-d₆, 400 MHz): δ

4.53 (s, 2H), 3.44 (s, 2H), 0.59−0.66 (m, 4H). ¹³C NMR (acetone-d₆,

100 MHz): 78.35, 65.36, 21.35, 8.92.

1-[(Nitrooxy)methyl]cyclopropanecarboxylic Acid (35).

Compound 34 (1.9 g, 12.9 mmol) was dissolved in DCM (100

mL). TEMPO (0.4 g, 2.6 mmol) and PhI(OAc)₂(4.5 g, 14.2 mmol)

were added successively. The reaction mixture was stirred until the

alcohol was no longer detectable by TLC. The reaction mixture was

washed with aqueous Na₂S₂O₃, NaHCO₃, and H₂O. The organic

phase was separated and concentrated. The residue was puriﬁed by

column chromatography (hexanes/ethyl acetate, 2:1). The starting

material was oxidized to an aldehyde as a white solid (1.3 g, 70%). ¹H

NMR (acetone-d₆, 400 MHz): δ 8.84 (s, 1H), 4.78 (s, 2H), 1.36−1.47

(m, 4H). ¹³C NMR (acetone-d₆, 100 MHz): 199.35, 74.15, 30.35,

12.56. The aldehyde (1.1 g, 7.6 mmol) and 2-methyl-2-butene (8.8

mL) were dissolved in t-BuOH (50 mL). NaH₂PO₄·H₂O (10.0 g) and

NaClO₂(9.0 g) were mixed with H₂O (25 mL), and then the mixture

was added to the t-BuOH solution. The reaction mixture was stirred

overnight. Solvent was removed under reduced pressure, and the

residue was diluted with ethyl acetate (200 mL) and washed with

aqueous Na₂S₂O₃(25 mL), NaHCO₃(25 mL), and H₂O (2 × 40

mL). The organic phase was separated, dried over anhydrous MgSO₄,

and concentrated. Product was obtained as a white solid (1.1 g, 90%).

¹H NMR (acetone-d₆, 400 MHz): δ 10.3 (bs, 1H), 4.73 (s, 2H), 1.36−

[2-[(4-Methyl-5-thiazolyl)phenylmethoxy]ethoxy]ethanol

(41). To a solution of 40 (70 mg, 0.21 mmol) in THF (5 mL) was

added LiAlH₄(35 mg, 0.85 mmol). The mixture was reﬂuxed for 1 h

and then quenched with methanol. Solvent was removed under

reduced pressure, and the residue was diluted with ethyl acetate and

washed with water. The crude product was puriﬁed by column

1.40 (m, 2H), 1.16−1.20 (m, 2H). ¹³C NMR (acetone-d₆, 100 MHz):

174.21, 76.96, 21.58, 14.48.

1

chromatography (SiO₂, hexane/acetone, 2:1, 25 mg, 41%). H NMR

[1-[(Nitrooxy)methyl]cyclopropanecarboxyl]-[(4-methyl-5-

thiazolyl)phenylmethyl] Ester (36). Compounds 2 (236 mg, 1.15

mmol) and 35 (222 mg, 1.38 mmol) were dissolved in a mixture of

DCM (5 mL) and DIPEA (570 μL). EDCI (330 mg, 1.7 mmol),

HOBt (233 mg, 1.7 mmol), and DMAP (15 mg, 0.13 mmol) were

added. The reaction mixture was stirred overnight and then diluted

with ethyl acetate (50 mL) and washed with water. The organic phase

was separated and concentrated, and the crude product was puriﬁed by

column chromatography (SiO₂, hexane/ethyl acetate, 2:1, 160 mg,

(CDCl₃, 400 MHz): δ 8.64 (s, 1H), 7.29−7.38 (m, 5H), 5.70 (s, 1H),

3.71−3.74 (m, 4H), 3.60−3.66 (m, 4H), 2.45 (s, 3H). ¹³C NMR

(CDCl₃, 100 MHz): δ 151.60, 149.56, 140.71, 133.66, 128.65, 128.16,

126.52, 72.38, 70.37, 68.49, 61.80, 15.52. HRMS calcd for

C₁₅H₁₉NO₃S [M + H]⁺294.1164, found 294.1169.

N-[(4-Methyl-5-thiazolyl)phenylmethyl]-[1-[(nitrooxy)-

methyl]cyclopropyl]methyl Carbamate (42). Compound 4 (160

mg, 0.78 mmol) and triphosgene (297 mg, 1.0 mmol) were dissolved

in ethyl acetate (10 mL) and reﬂuxed for 2 h. Solvent was removed.

The residue was redissolved in a mixture of THF (5 mL) and NEt₃

(0.25 mL, 1.8 mmol), and 34 (147 mg, 1 mmol) was added. The

reaction mixture was stirred at 40 °C for 1 h. Solvent was removed,

and the product was puriﬁed by column chromatography (SiO₂,

hexane/ethyl acetate, 2:1, 150 mg, 51%). ¹H NMR (CDCl₃, 400

MHz): δ 8.55 (s, 1H), 7.27−7.36 (m, 5H), 6.19 (s, 1H), 5.98 (s, 1H),

4.35 (s, 2H), 3.98 (s, 2H), 2.39 (s, 3H), 0.67−0.71 (m, 4H). ¹³C NMR

(CDCl₃, 100 MHz): δ 155.21, 150.51, 149.68, 140.32, 133.53, 128.69,

1

40%). H NMR (CDCl₃, 400 MHz): δ 8.66 (s, 1H), 7.29−7.39 (m,

5H), 7.15 (s, 1H), 4.62−4.68 (m, 2H), 2.48 (s, 3H), 1.45−1.52 (m,

2H), 1.06−1.10 (m, 2H). ¹³C NMR (CDCl₃, 100 MHz): δ 170.67,

151.94, 150.66, 138.77, 130.72, 128.76, 128.50, 126.06, 75.06, 71.18,

21.54, 15.39, 14.85, 14.78. HRMS calcd for C₁₆H₁₆N₂O₅S [M + H]⁺

349.0858, found 349.0854.

[1-Methylcyclopropanecarboxyl]-[(4-methyl-5-thiazolyl)-

phenylmethyl] Ester (37). Compound 37 was prepared from 2 and

1-methylcyclopropane-1-carboxylic acid as described for 36 (85%). ¹H

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127.93, 126.39, 77.06, 68.31, 51.97, 30.68, 18.33, 15.08, 9.41. HRMS

calcd for C₁₇H₁₉N₃O₅S [M + H]⁺378.1124, found 378.1114.

N-[(4-Methyl-5-thiazolyl)phenylmethyl]-[4-(nitrooxy)butyl]

Carbamate (43). Compound 43 was prepared from 4 and 4-

nitrooxybutan-1-ol⁹⁰using the same procedure described for 42.

Product was puriﬁed by column chromatography (SiO₂, hexane/ethyl

throughout the housing in the facility. Animals were acclimated for at

least 1 week in BRL before running any experiment. Also, 1−2 h prior

to each testing, mice were habituated to the animal study room in the

facility. All protocols using live animals were reviewed and approved by

Institutional Animal Care and Use Committee (IACUC) at UIC

(Animal Care Committee). Injection volumes were adjusted based on

the animal weight (0.1 mL/25 g mouse).

1

acetate, 2:1, 65%). H NMR (CDCl₃, 400 MHz): δ 8.60 (s, 1H),

7.27−7.38 (m, 5H), 6.19 (bs, 1H), 5.45 (bs, 1H), 4.45 (bs, 2H), 4.13

(t, 2H, J = 5.6 Hz), 2.41 (s, 3H), 1.60−1.80 (m, 4H). ¹³C NMR

(CDCl₃, 100 MHz): δ 155.35, 150.46, 149.51, 140.31, 133.60, 128.60,

127.83, 126.35, 72.40, 64.10, 51.81, 25.01, 23.18, 14.98. HRMS calcd

for C₁₆H₁₉N₃O₅S [M + H]⁺366.1124, found 366.1117.

Double transgenic APP/PS1 mice expressing both the human APP

(K670M:N671L) and PS1 (M146L) (line 6.2) mutations were used

for the LTP study. They were obtained by crossing APP with PS1

animals.³⁸The animals were maintained on a 12 h light/dark cycle

(with light onset at 6:00 a.m.) in temperature- and humidity-controlled

rooms of the Columbia University Animal Facility. Food and water

were available ad libitum.

Primary Neuronal Cultures. Primary cultures of rat cortical

neurons were prepared as described.⁹¹Brieﬂy, brains were removed

from embryonic day 17 Sprague−Dawley rat embryos, cortices were

dissected, and meninges were removed. Neurons were mechanically

dissociated in basal medium Eagle 1× containing glucose (33 mM),

glutamine (2 mM), 10% horse serum, and 10% FBS and then plated at

3 × 10⁵cells/cm²in poly-L-lysine-precoated plates. The medium was

replaced 24 h later with serum-free neurobasal medium (NBM)

supplemented with 0.5 mmol/^L-glutamine and 2% complete B27

supplement. Four days later, 50% of the medium was replaced with

fresh NBM. Neurons were used for OGD or NMDA cytotoxicity

experiments after being grown for 8−10 days in NBM.

Primary Neurons Subject to Transient OGD Followed by

Reoxygenation. Primary cortical cultured neurons were subjected to

a transient OGD as described.⁹²On days 8−10 of culture, cultured

neurons were placed in a humidiﬁed 37 °C incubator (95% N₂, 5%

CO₂) and the culture medium was replaced with deoxygenated,

glucose-free balanced salt solution, pH 7.4, containing the following

(in mM): NaCl 116, CaCl₂1.8, MgSO₄0.8, KCl 5.4, NaH₂PO₄1,

NaHCO₃14.7, and HEPES 10. Following 2 h of OGD incubation,

OGD was ended by replacing OGD medium with testing compound

conditioned fresh NBM (supplemented with 0.5 mmol/^L-glutamine

and B27 without antioxidants). Cultures were returned to the

normoxic 37 °C incubator (5% CO₂) for 24 h prior to evaluation of

cell viability and neuronal death by MTT and LDH assays. The test

compounds (50 μM) were present during the 2 h OGD and 24 h

reoxygenation.

NMDA and Glutamate Induced Cytotoxicity in Primary

Neurons. Primary cortical cultured neurons were subjected to NMDA

induced cytotoxicity as described.^93,94On day 11 of culture, primary

neurons were preincubated with testing compounds (50 μM) for 30

min in neurobasal culture mudium and were exposed to NMDA (100

μM) in HEPES-buﬀered salt solution containing the following (in

mM): NaCl 20, KCl 5.4, MgCl₂0.8, CaCl₂1.8, glucose 15, and

HEPES 20 (pH 7.4). Twenty-four hours later, cell viability and

neuronal death were evaluated by MTT and LDH assays. The testing

compounds (50 μM) were presented during the preincubation and 24

h chronic NMDA cytotoxicity. MTT assay for cell viability and LDH

assay of cell death were performed as described previously.⁹⁵

To probe the mechanism of neuroprotection elicited by MZ

derivatives, after 10−11 DIV (days in vitro), picrotoxin (100 μM) was

added to primary neuronal cultures 1 h before glutamate (100 μM)

insult. One hour after glutamate addition, MZ derivatives or muscimol

were supplied at a ﬁnal concentration of 50 μM. After incubation for

24 h, ﬁnal cell survival was assayed using MTT. For comparison,

picrotoxin treatment was omitted in another experiment.

N-[(4-Methyl-5-thiazolyl)phenylmethyl]-1-[(nitrooxy)-

methyl]cyclopropanecarboxamide (44). Compound 44 was

prepared from 4 and 35 as described for 36. The crude product was

puriﬁed by column chromatography (SiO₂, hexane/ethyl acetate, 1:1,

1

49%). H NMR (CDCl₃, 300 MHz): δ 8.59 (s, 1H), 7.23−7.38 (m,

5H), 6.61 (d, 1H, J = 7.2 Hz), 6.45 (d, 1H, J = 7.3 Hz), 4.58 (s, 2H),

2.39 (s, 3H), 1.35−1.39 (m, 2H), 0.94−0.97 (m, 2H). ¹³C NMR

(CDCl₃, 100 MHz): δ 169.97, 150.58, 150.04, 139.98, 133.04, 128.97,

128.18, 126.50, 76.03, 50.59, 22.68, 15.26, 13.74, 13.66. HRMS calcd

for C₁₆H₁₇N₃O₄S [M + H]⁺348.1018, found 348.1008.

Stability of NO-Chimera in Neutral Phosphate Buﬀer. Stock

solutions of 36, 37, and 42 were made in DMSO. 36 or 37 (10 μM)

and 5i (internal standard, 10 μM) were dissolved in phosphate buﬀer

(100 mM, pH 7.4), and the ﬁnal concentration of DMSO was 2% (v/

v). The solution was kept in the autosampler of the HPLC instrument

at room temperature, and an aliquot (50 μL) was injected into HPLC

column. Injection occurred about every 7 min for 36 and 67 min for

37 because of the diﬀerent stability of the two compounds. HPLC

analysis was performed using a Shimadzu UFLC instrument with UV

absorbance detection at 254 nm. Separation of parent compound,

hydrolysis product (compound 2), and internal standard (IS) was

achieved using a Hypersil BDS C18 column (2.1 mm × 30 mm, 3

μm). The composition of the HPLC mobile phase was a mixture of

water with 10% methanol, 0.1% formic acid (solution A), and

acetonitrile with 0.1% formic acid (solution B). The mobile phase was

initially composed of solvent A/solvent B (90:10), held for 1 min, and

then a linear gradient of solution B from 10% to 95% over the next 3

min. The solvent was held at 95% of solution B over 1 min, and the

column was ﬁnally balanced using 10% solvent B for 1.5 min. Flow rate

was 0.5 mL/min. Peak area ratio of parent compound vs internal

standard was calculated at each time point and divided by the time

zero value to give the percentage of compound remaining.

To detect the decomposition products of 42, the compound (100

μM) was dissolved in phosphate buﬀer (100 mM, pH 7.4). The ﬁnal

concentration of DMSO was 2% (v/v). The sample was placed in the

dark at room temperature for 48 h. An aliquot (10 μL) of the sample

was analyzed using an Agilent 6310 ion trap mass spectrometer

(Agilent Technologies, Santa Clara, CA) equipped with Agilent 1100

HPLC system and electrospray ionization (ESI) source operated in

positive mode. HPLC was performed using an Agilent Zorbax Bonus

reverse phase C18 column (3.0 mm × 150 mm, 3.5 μm) with UV

absorbance detection at 254 nm. The composition of the HPLC

mobile phase was a mixture of water with 10% methanol, 0.1% formic

acid (solution A), and acetonitrile with 0.1% formic acid (solution B).

The mobile phase was initially composed of solvent A/solvent B

(90:10), held for 1 min, and then a linear gradient of solution B from

10% to 80% over 17 min, 0.5 min gradient of solution B from 80% to

95% and held for 5 min. Flow rate was set at 0.5 mL/min.

Animals. Male C57BL/6 mice aged 8−10 weeks (20−25 g, Charles

River Laboratories, IL) were used for step-through passive avoidance

(STPA) task and brain bioavailability assay. Sprague−Dawley rat

(gestational days 17−18, Charles River Laboratories, IL) were used for

the primary neuronal cultures. Animals were housed at the BRL

(biological resources laboratories) animal facility at the University of

Illinois at Chicago (UIC). The animals were kept under standard

laboratory conditions with free access to dry food and water but no

nesting materials (Nestlets) supplied (not enriched environment).

Regular light−dark cycle with at least 12 h of light was maintained

LTP Measurement. Hippocampal slice preparation was performed

as described.³⁸Brieﬂy, APP/PS1 trangenic mice were decapitated and

their hippocampi were quickly removed. Transverse hippocampal

slices (400 μm) were cut and transferred to an interface chamber,

where they were maintained at 29 °C. They were perfused (1−3 mL/

min) with saline solution (124.0 mM NaCl/4.4 mM KCl/1.0 mM

Na₂HPO₄/25.0 mM NaHCO₃/2.0 mM CaCl₂/2.0 mM MgSO₄/10

mM glucose) continuously bubbled with 95% O₂and 5% CO₂. Slices

were maintained at 29 °C for 90 min before recording. The CA1

region of the hippocampus ﬁeld excitatory postsynaptic potentials

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(fEPSPs) were recorded by placing both the stimulating and the

recording electrodes in CA1 stratum radiatum. A 15 min baseline was

recorded every minute at an intensity that evokes a response of

approximately 35% of the maximum evoked response. LTP was

induced using θ-burst stimulation at 20 min. Responses were recorded

for 1 h after tetanization.

Statistical Analyses. For all experiments, data were expressed as

the mean SEM. LTP results were analyzed with two-way ANOVA.

STPA results were analyzed with one-way ANOVA followed by

Bonferroni multiple comparison test. The level of signiﬁcance was set

for p < 0.05.

Step-Through Passive Avoidance (STPA) Task. The STPA

apparatus consists of a two-compartment acrylic box with an

illuminated side (15 cm × 12 cm × 12 cm) connected to a dark

side (15 cm × 12 cm × 12 cm) by a vertical door. The dark side is

equipped with an electric grid ﬂoor. Animals was habituated to

translocating to the dark from the light side until latency of entering

the dark side for each animal is <30 s. During training, animals

received an aversive electrical shock (0.5 mA, 2 s), triggered by entry

to the dark compartment, until the latency of entering the dark side

reaches 300 s. To test procognitive activity of NO-chimeras,

scopolamine (1.0 mg/kg, ip) was given 30 min before training and

drug (4.5 μmol/kg, ip) was administered 20 min before training. At 24

h after training, the animals were again individually placed in the light

compartment and the latency to enter the dark comparment, without

shock stimulus, was recorded. Statistical analysis of the latency data

was performed with one-way ANOVA followed by Bonferroni multiple

comparison test.

ASSOCIATED CONTENT

* Supporting Information

■

S

HPLC chromatogram of the incubation of 42 in phosphate

buﬀer (100 mM, pH 7.4) at room temperature for 48 h and the

proposed mechanism of decomposition (Figure S1); inhibition

of excitotoxic cell death from treatment with NMDA (100 μM)

by CMZ and by a number of MZ derivatives (Table S1). This

material is available free of charge via the Internet at http://

pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: 312-355-5282. Fax: 312-996-7107. E-mail: thatcher@

uic.edu.

■

Notes

Brain Bioavailability of NO-Chimeras. Mice were injected (ip)

with drug at a dosage that was equimolar with 1 (4.5 μmol/kg, ip).

Four mice were used for the injection of each drug. Brain tissues were

collected 20 min after ip injection. Analytes were extracted as follows:

to a mixture of blank brain tissue (100 mg) and cold acetonitrile/

MeOH (1:1, 600 μL), internal standard (400 ng/mL, 10 μL) in

acetonitrile/MeOH (1:1) was added. Brain tissue was homogenized

manually using a disposable pestle, vortexed, and then centrifuged at 4

°C, 13 000 rpm for 15 min. The supernatant was transferred to

another vial. The remaining pellet was washed and extracted with cold

acetonitrile/MeOH (300 μL,1:1 v/v) and centrifuged as described

above. Supernatants were combined and stored at −20 °C for 1 h,

allowing further protein precipitation. The clear supernatant from

centrifugation was collected and concentrated using a ﬂow of N₂. The

residue was reconstituted with solvent (200 μL, H₂O/MeOH/

acetonitrile, 50/25/25, v/v), vortexed, centrifuged, and analyzed

using LC−MS/MS. To construct standard curves, working solutions in

MeOH/acetonitrile/H₂O (0.5:0.5:1) containing NO-chimera and

corresponding hydroxyl compound at diﬀerent concentrations were

prepared. Concentrations of working solutions were 2000, 1000, 500,

200, 80, 40, 20 ng/mL. To a mixture of blank brain tissue (100 mg)

and cold acetonitrile/MeOH (1:1, 600 μL) were added each analyte

working solution (10 μL) and internal standard (400 ng/mL, 10 μL).

Calibration samples were extracted as described above, and the ﬁnal

concentrations of analytes after reconstitution were 100, 50, 25, 10, 4,

2, 1 ng/mL. The ﬁnal concentration of IS was 20 ng/mL.

All LC−MS/MS measurements were carried out using the positive

mode electrospray ionization method on an API 3000 (Applied

Biosystem, Foster City, CA) triple quadrupole mass spectrometer

attached to an Agilent 1200 HPLC (Agilent Technologies) instru-

ment. MRM transitions for the detection of analytes and internal

standards are described in Table 2. HPLC was performed with a

Waters XBridge C18 column (3.5 μm, 3 mm × 100 mm). Flow rate

was set at 0.35 mL/min. The composition of the HPLC mobile phase

was a mixture of water with 10% methanol, 0.1% formic acid (solution

A), and acetonitrile with 0.1% formic acid (solution B). To analyze

32a and 22a, the mobile phase was initially composed of solvent A/

solvent B (90:10), held for 1 min, and a linear gradient of solution B

from 10% to 60% over the next 1 min. The composition of B was then

increased to 99% in 13 min. The solvent was held at 99% of solution B

over 5 min, and the column was ﬁnally balanced using 10% solvent B

for 5 min. To analyze 26 and 28, the mobile phase was initially

composed of solvent A/solvent B (95:5), held for 1 min, and a linear

gradient of solution B from 10% to 60% over next 1 min. Composition

of B was then increased to 99% in 11 min. The solvent was held at

99% of solution B over 5 min, and the column was ﬁnally balanced

using 5% solvent B for 5 min.

The authors declare the following competing ﬁnancial

interest(s): G.R.J.T. has a consulting relationship with sGC

Pharma, an entity that currently licenses intellectual property

associated with nomethiazoles from the University of Illinois.

ACKNOWLEDGMENTS

The work was supported by NIH Grant U01 AG031294. Hong

Zhang and Ian J. Orozco are thanked for technical assistance.

■

ABBREVIATIONS USED

■

NO-GC, NO-sensitive or soluble guanylyl cyclase; cGK,

cGMP-dependent protein kinase; CREB, cyclic adenosine 5′-

monophosphate response element binding protein; LTP, long-

term potentiation; Aβ, amyloid β; AD, Alzheimer’s disease;

BDNF, brain derived neurotropic growth factor; MAPK,

mitogen activated protein kinase; ESI, electrospray ionization;

MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide; MZ, 4-methylthiazole; NMDA, N-methyl-^D-aspartate;

OGD, oxygen glucose deprivation; DIV, day in vitro; NO_x, a

−

nitrogen oxide (including NO, NO₂, and NO₃); STPA, step-

through passive avoidance; MRM, multiple reaction monitoring

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dx.doi.org/10.1021/jm300353r | J. Med. Chem. 2012, 55, 6784−6801

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Design and synthesis of neuroprotective methylthiazoles and modification as NO-chimeras for neurodegenerative therapy

DOI: 10.1021/jm300353r

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