Accepted Manuscript

Synthesis and pre-clinical studies of new amino-acid ester thiazolide prodrugs

Andrew V. Stachulski, Karl Swift, Mark Cooper, Stephen Reynolds, Daniel Norton,
Steven D. Slonecker, Jean-François Rossignol

PII: S0223-5234(16)30819-4
DOI: 10.1016/j.ejmech.2016.09.080
Reference: EJMECH 8946

To appear in: European Journal of Medicinal Chemistry

Received Date: 4 July 2016

Revised Date: 8 September 2016
Accepted Date: 25 September 2016

Please cite this article as: A.V. Stachulski, K. Swift, M. Cooper, S. Reynolds, D. Norton, S.D. Slonecker,
J.-F. Rossignol, Synthesis and pre-clinical studies of new amino-acid ester thiazolide prodrugs,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.080.

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 ACCEPTED MANUSCRIPT

Graphical Abstract

Synthesis and Pre-clinical Studies of New Amino-Acid Ester Thiazolide Prodrugs

Andrew V. Stachulski,* Karl Swift, Mark Cooper, Stephen Reynolds, Daniel Norton, Steven D.

Slonecker and Jean-François Rossignol

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Synthesis and Pre-clinical Studies of New Amino-Acid Ester Thiazolide

Prodrugs

Andrew V. Stachulski,*1 Karl Swift, 2

Mark Cooper, 2
Stephen Reynolds, 2
Daniel Norton, 3

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Steven D. Slonecker3 and Jean-François Rossignol4

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1
Robert Robinson Laboratories, Department of Chemistry, University of Liverpool, Liverpool

L69 7ZD, UK; 2Bio-Techne, The Watkins Building, Atlantic Road, Bristol BS11 9QD, UK;

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3
Calvert Laboratories, Inc., Scott Township, Pennsylvania 18447, 4Romark Laboratories, L.C.,

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Tampa, 33609 Florida AN
KEYWORDS: Antiviral agents, prodrugs, chemical synthesis, stability, biodisposition, oral

absorption, pharmacology, toxicology

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1 Phone, +44 0151 794 3482; Fax, +44 0151 794 3488; E-mail, stachuls@liv.ac.uk

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ABSTRACT: Thiazolides are polypharmacology agents with at least three mechanisms of action

against a broad spectrum of parasites, bacteria and viruses. In respiratory viruses they are protein

disulfide isomerase inhibitors, inhibiting the replication of orthomyxoviridae and

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paramyxoviridae at a post-translational level. Nitazoxanide 1a, the prototype thiazolide, was

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originally developed as an antiparasitic agent and later repurposed for the treatment of viral

respiratory infections. The second generation thiazolides following nitazoxanide, such as the 5-

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chloro analogue RM-5038 2a, are also broad-spectrum antiviral agents as we have reported. Both

1a and its effective circulating metabolite, tizoxanide 1b, are 5-nitrothiazole derivatives, while

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RM-5038 2a and its de-acetyl derivative RM-4848 2b are the corresponding 5-chloro derivatives.
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Recently 1a has completed phase II-III clinical trials in the United States, Canada, Australia and

New Zealand in a total of 2,865 adults and adolescents of at least 12 months of age with viral
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acute respiratory illness. Since its biodisposition is primarily seen in the gastro-intestinal tract, its
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efficacy in systemic viral diseases requires relatively high oral doses. The chemical synthesis of
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new derivatives with a better systemic absorption was therefore urgently needed. In order to

improve their systemic absorption, new amino-ester prodrug derivatives of 1b and RM4848 2b
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were prepared and tested for their animal pharmacology, pharmacokinetics and toxicology. RM-

5061 8a in rats showed 7-fold higher blood concentration compared to 1a: absolute
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bioavailability increased from 3 to 20%, with a good safety profile in animal safety
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pharmacology and toxicology.

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Introduction

Nitazoxanide 1a, the first of thiazolides discovered in 1975 by Rossignol and Cavier is a broad-

spectrum antiparasitic agent effective against protozoa, nematodes, cestodes and trematodes. [1]

It was registered throughout Latin America, Egypt, India and Bangladesh for the treatment of

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intestinal protozoa and helminths while in the United States the drug was approved for the

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treatment of two intestinal protozoa, Cryptosporidium parvum and Giardia intestinalis. [2][3][4]

The antiprotozoal activity of 1a against anaerobic organisms such as some protozoa and bacteria

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is due to its interference with the pyruvate ferredoxin oxidoreductase (PFOR) enzyme-dependent

electron transfer reaction, which is essential to anaerobic metabolism. Against helminths and

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Mycobacterium tuberculosis it disrupts membrane potential and homeostasis of intramicro-
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organisms. [5][6][7][8] Thiazolides also inhibit protein disulfide isomerase (PDI) and possess a

broad spectrum of activity against parasites and viruses. In Neospora caninum, an apicomplexan
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emerging protozoa related to Cryptosporidium parvum, 1a binds to the NcPDI blocking the
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protozoan replication. [9] In respiratory viruses 1a binds to the protein-folding enzyme ERp57-
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PDI inhibiting the replication of respiratory viruses belonging to the classes of orthomyxoviridae

and paramyxoviridae at a post-translational level. [10][11] 1a and a number of structurally

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related thiazolides, e. g. RM5038 2a are also active against both hepatitis B and C viruses at low

micromolar concentrations. [12][13][14] For example, against a range of respiratory viruses

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including H1N1 influenza A strains, 1a exhibits IC50 values of 0.3-1.0 µg/ml; against a range of
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flaviviridae including hepatitis C, from 0.05-0.5 µg/ml; against hepatitis B (hepadnaviridae),

0.06 µg/ml.

Moving to in vivo antiviral activity, the antiviral activity of 1a and tizoxanide 1b, its

active circulating metabolite, was confirmed in well-controlled clinical trials carried out in more

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than 3,000 patients in the treatment of gastroenteritis caused by rotavirus and norovirus,

uncomplicated viral respiratory infections caused by influenza A and B and, alone or combined

with pegylated-interferon with and without ribavirin, in the treatment of chronic hepatitis C.

[15][16][17][18]

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1a is only partially absorbed from the gastro-intestinal tract: C-1a given to human

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volunteers showed that 33% of the oral dose was excreted via urine and 64% was excreted in

faeces. This is a perfect bio-disposition profile for a drug intended to treat intestinal pathogens,

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but much less desirable for a systemic antiviral agent. Upon oral absorption it is immediately

metabolized into deacetyl derivative 1b, which is subsequently metabolised in the liver as

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tizoxanide-glucuronide and rapidly eliminated via urine. [19][20] Ideally, the treatment of viral
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systemic infections as opposed to parasites or viruses infecting the intestinal tract calls for a

compound with a better oral biodisposition and metabolism than 1a but ideally liberating in the
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blood stream the same active circulating metabolite, 1b and its inactive glucuronide. A
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considerable amount of safety and efficacy data have been accumulated for these derivatives in
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the United States and abroad during the last 20 years.

In summary, the thiazolides are typified by nitazoxanide 1a, Scheme 1, and its active
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circulating metabolite tizoxanide 1b as shown. Among a large number of analogues synthesised,

the chloro analogue RM5038 2a and, similarly, its circulating metabolite RM4848 2b also have
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very good broad- spectrum antiviral activity. [12][13][14]

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Scheme 1. Thiazolide structures.

Although the O-acetates such as 1a are satisfactorily taken up by passive absorption,

behaving effectively as ester prodrugs, the oral bioavailability of 1a in the absence of food is

typically ≤ 30%. [20] We therefore set out to design a robust prodrug form of 1a/1b, so as to

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improve both the oral absorption and the solubility properties of the parent drug.

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Scheme 2. Valacyclovir, as its HCl salt, and acyclovir
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We were impressed by amino-acid based prodrug esters such as the antiviral agent

valacyclovir 3, Scheme 2 [21,22] which improves the oral bioavailability of acyclovir 4 from
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<20% to 54% and greatly improves its aqueous solubility; valacyclovir enters cells via the h-
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PEPT 1 transporter. [23] Initially, therefore, we prepared the direct analogue of tizoxanide,

namely valyl ester 5 (Scheme 3). This derivative was readily prepared as the HCl salt shown [cf.
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Scheme 4], but unfortunately its stability proved inadequate. After 3 weeks’ storage at room

temperature, hydrolysis of 5 was significant, with about 20% release of the parent drug 1b by
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NMR evidence.
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Scheme 3. The L-valyl ester prodrug of tizoxanide.

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Chemistry AN
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Scheme 4. Synthesis of L-tert-leucyl thiazolide prodrugs. Reagents: i) Boc-Tle-OH 6, EDC,

DMAP, THF or DMF, 65%; ii) HCl-dioxane, 0-20 °C, 95%. Abbreviation: Tle= L-tert-leucine,

(2S)- 2-amino-3,3-dimethylbutanoic acid. [24]

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We considered that a more stable ester should result on increasing the steric bulk of the

amino-acid side chain, and therefore turned next to the corresponding derivative of L-tert-

leucine, [24] Scheme 4. Reaction of 1b with Boc-Tle-OH [25] 6 using EDC catalysed by DMAP

in THF afforded protected ester 7a in satisfactory yield after chromatography. We later found that

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DMF was a superior solvent, especially for 1b, and performed well on scale-up, delivering a 90%

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yield of 7a. Deprotection was very conveniently achieved by treatment of 7a with HCl-dioxane,

as the HCl salt RM5061 8a of the product could be crystallised directly from the reaction

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medium and was obtained in excellent yield. Importantly, RM5061 8a showed no appreciable

hydrolysis after standing at 20°C for three months (<1% of 1b seen by NMR analysis and by

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HPLC); its aqueous solubility was approximately 5 mg/ mL. [16] It was of paramount importance
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to assay the chiral purity of RM5061 8a; for comparison, valacyclovir is typically obtained from

acyclovir in about 92% e.e. [26] From Boc-Tle-OH (S-enantiomer), using DMF as solvent,
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RM5061 8a was obtained in 99% purity by HPLC and the e.e. was 99.8%. Starting from the
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corresponding derivative of D-tert-leucine, by chiral HPLC analysis the e. e. of ent-8a was

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determined to be 99.5%.

As noted above, the 5´-chlorothiazolide RM4848 2b is another important compound: its

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corresponding pro-drug was similarly made, Scheme 1. The intermediate 7b was isolated as a

crystalline solid and deprotection again proceeded smoothly to afford HCl salt RM5064 8b as a
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white solid in excellent yield. This product showed very similar stability behaviour to RM5061
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8a (<1% hydrolysis after 3 months at 20°C) and its aqueous solubility was greater, approximately

20 mg/ mL.

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9 10

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Scheme 5. Isoleucyl and allo-isoleucyl prodrugs.

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The stability of valacyclovir has been extensively studied. [27] In order to probe further

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the issues of stability and chiral purity of products, we also prepared the isoleucine and allo-
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isoleucine derivatives 9 and 10, Scheme 5 (see supporting information). No scrambling of NMR

peaks could be seen in either product at the NMR detection level; here, epimerisation would have
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generated diastereoisomeric mixtures, possibly to different extents for the two products. As might

have been intuitively expected, the stabilities of these compounds by NMR, ca.5% hydrolysis
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after 3 months at 20°C, were greater than 5 but less than RM5061 8a/ RM5064 8b.
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Comparative pharmacokinetics of nitazoxanide 1a, RM-5061 8a, RM-5038 2a and RM-5064 8b

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in rats
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Thiazolides are poorly soluble compounds. In rodents they are inefficiently absorbed from
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the gastrointestinal tract: most of the compounds given orally remain in the gut. In order to assess

the absolute bioavailability of our new amino-acid ester derivatives, RM-5061 8a and RM-5064

8b, in comparison with 1a and RM5038 2a we carried out four parallel studies, each one

including six Sprague-Dawley rats weighing about 300 g divided into two groups of three

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animals. In each of the four studies a group of three rats was treated with a single oral dose of

each of the four compounds while the second group of three rats received a single intravenous

injection of each of the same four test compounds.

For each of the four compounds the oral dose was calculated to be 30 mg/kg while the

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intravenous injection was 6 mg/kg, viz. 5 times less. Serial blood samples were obtained from

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each animal at 5, 10, 15, 30 min and 1, 2, 4, 8, 23, and 24 h post-dose. As noted above, [19]

tizoxanide glucuronide 11a, Scheme 6, is the major in vivo metabolite of 1b and significant

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concentrations of 11a were noted after 5 min upon oral administration of either 1a or RM-5061

8a; this was also the time of maximum plasma concentration.

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11a R = NO2, 11b R = Cl

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Scheme 6. Thiazolide glucuronides.

The comparison of the AUC of 1b calculated from the pharmacokinetics parameters obtained
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after oral and intravenous administrations of 1a and RM-5061 8a showed a 2.8% absolute

bioavailability for 1a, 2.04 versus 70.5 after correction for the dose given to 30 mg/kg, and 20%
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for RM-5061 8a, 3.12 versus 26.0 after correction for the dose to 30 mg/kg. Interestingly, the
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chloro-derivative are much less bioavailable: the comparison of the AUCs of RM4848 2b shows

essentially no oral absorption for RM5038 2a while the amino-ester derivative, RM5064 8b,

shows a 22% absolute bioavailability (0.23 versus 17.4 and 0.71 versus 3.15 for corrected values

of the intravenous dose). This again demonstrates a better pharmacokinetic profile for the amino-

acid ester derivatives than the corresponding O-acetyl compounds. Here again, the glucuronide

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metabolite 11b [12] was quickly observed on administration of RM5038 2a or RM 5064 8b and

is the main in vivo metabolite of RM4848 2b. The bioavailabilities of the four compounds are

summarised in Table 1. For detailed post oral and intravenous blood levels, see supporting

information.

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Compound Bioavailability F%

Nitazoxanide 1a 2.8

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RM5038 2a ~0

RM5061 8a 20

RM5064 8b
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Table 1. Absolute bioavailabilities F for compounds 1a, 8a, 2a and 8b.

We therefore decided to proceed with the complete pre-clinical development of RM 5061

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8a in order to perform phase 1 human trials. Full pharmacokinetic data for all four compounds
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1a, RM 5038 2a, RM 5061 8a and RM 5064 8b are given in supporting information.

Additionally, the new derivatives are active in vitro antivirals in their own right: a direct
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comparison between 1a and RM5061 8a for two strains of influenza virus is given in Table 2.
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Viral strain Compound EC50a SI50b

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H1N1A/ Puerto Rico/8/34c Nitazoxanide 1ad 0.3 >50

H1N1A/ Puerto Rico/8/34 RM5061 8ad 1.0 >50

H5N9A/ Chicken/Italy/9097/97c Nitazoxanide 1a 0.5 >100

H5N9A/ Chicken/Italy/9097/97 RM5061 8a 0.32 >150

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Table 2. Antiviral activity of RM5061 8a compared to nitazoxanide 1a. a EC50 values are in mg/ml; for
details of antiviral assays, see ref. [10] b Cell safety index figure. c See ref. [10] for details of strains used. d
1a was administered as a DMSO solution whereas 8a was administered as an aqueous solution.

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Safety pharmacology

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Two safety pharmacology studies evaluated the effect of RM-5061 8a on the central nervous

system and on the respiratory function respectively in the rats.

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Single oral doses of 100, 300 and 1,000 mg/kg of RM5061 8a were administered to three

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groups of 10 rats, 5 males and 5 females, by oral gavage: one group of untreated animals was
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kept as a control. There were no abnormal signs recorded at 4 and 24 hours after treatment at the

100 and 300 mg/kg doses but at the 1,000 mg/kg dose level there was decreased activity,
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decreased abdominal tone, labored breathing, tremors and no pain responses observed in all

animals in the group suggesting that this dose level was producing CNS toxicity but without
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mortality of the animals treated at this high dose.

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In a second study, single oral doses of 100, 300 and 1,000 mg/kg of RM5061 8a were

given by oral gavage to three groups of six conscious rats, 3 males and 3 females to study the
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effects of the test drugs on respiratory function. One group of untreated animals was kept as
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controls. Some minor changes on the respiratory rate expressed as breaths per minute and the
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tidal volume were observed for the low 100 mg/kg and the 300 mg/kg oral doses without

affecting the Minute Volume dose, but more pronounced effects on the three parameters recorded

and described above were observed at the 1,000 mg/kg dose suggesting that this level of RM5061

8a has some effects on the respiratory function of the rats. However, no mortality was recorded

at the three dose levels tested. The dosing schedules are summarised in Table 3.

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Indication Grouping Dose employed mg/kg

CNS 3 groups, 10 rats each (5M/5F) 100 300 1000

Respiratory 3 groups, 6 rats each (3M/3F) 100 300 1000

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Table 3. Dosing schedules for safety pharmacology study of RM5061 8a in rats. One untreated group was
kept as a control; for full details, see supporting information.

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Animal toxicology of RM5061 8a

Two sub-acute toxicity studies of RM 5061 8a were carried out in Sprague-Dawley rats and

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Beagle dogs. The first study involved dosage of rats at three separate dose levels of 10, 30 and 75
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mg/kg of RM 5061 8a once a day for 28 consecutive days, to evaluate systemic exposure to the
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drug. No terminal adverse effects were noted with the rats. Bright yellow urine was commonly

observed, linked to the formulations of RM 5061 8a, which were yellow suspensions. Weight
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losses were observed, especially in the male group, but these were within acceptable limits. It
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was concluded that the no observed adverse effect level (NOAEL) was achieved with a dose of

10mg/kg. Full toxicological details are given in supporting information; the dosing and group
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numbers are summarised in Table 4.

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Indication Grouping Dose employed mg/kg

Systemic 4 groups, ≤ 30 rats each (15M/15F) 10 30 75

exposure to drug

Table 4. Dosing schedules for sub-acute toxicology study of RM5061 8a in rats. The doses shown were
administered for 28 consecutive days; for full details, see supporting information.

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The second study evaluated the systemic exposure of RM-5061 8a when administered orally via

gelatin capsule at three separate dose levels of 5, 15 and 25 mg/kg once a day for 28 consecutive

days to Beagle dogs. Again, there were no early deaths with the dogs during the study period.

Bright yellow urine was commonly observed and there were cases of emesis, with yellow

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particulate material in a few cases. However, emesis was not observed in any control animal. In

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both animal studies, exposure to 1b and 11a was apparent. Based on the overall study data, the

high dose level of 25 mg/kg/day of RM 5061 8a administered in a single gelatin capsule for 28

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consecutive days to Beagle dogs was considered a no observed adverse effect level (NOAEL).

Full details are given in supporting information: the dosing and group numbers are summarised in

Table 5.
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Indication Grouping Dose employed mg/kg
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Systemic 4 groups, ≤10 dogs each (5M/5F) 0 10 30 75

exposure to drug
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Table 5. Dosing schedules for sub-acute toxicology study of RM5061 8a in beagle dogs. The doses shown
were administered for 28 consecutive days; for full details, see supporting information.
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Conclusions

Nitazoxanide 1a was originally designed as a broad-spectrum antiparasitic drug for the treatment
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of intestinal protozoan and helminthic infections, for which the drug has been marketed around
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the world for more than 15 years. It was recently re-purposed as a broad-spectrum antiviral agent

in the treatment of viral acute respiratory infections. Phase III clinical trials carried out in 2,865

adults and adolescents with uncomplicated influenza A and B showed that the drug reduced the

duration of the influenza illness when compared to placebo with a p value < 0.05. Additionally

the studies showed that nitazoxanide compared to placebo was effective in the treatment of the

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common cold caused by rhinovirus and coronavirus. Further studies in patients with viral

respiratory infections at risk of developing complications, children, and adults and children with

severe acute respiratory infections (SARI) are currently underway. It was important to identify

new derivatives with better systemic absorption, and we have now shown that RM-5061 8a is a

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second prodrug for tizoxanide 1b. RM5061 8a is more soluble and better absorbed in laboratory

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animals than 1a and is now undergoing Phase I clinical trials. It may provide an oral dose

effective at a lower dosage, and more importantly an injectable form of tizoxanide that

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nitazoxanide was unable to do.

Acknowledgements
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We are grateful to Romark Laboratories LC for funding this work at the University of Liverpool

(AVS) and at Calvert Laboratories (DN, SDS).

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Experimental
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Chemistry

Organic extracts were washed finally with satd. aq. NaCl and dried over anhydrous Na2SO4 prior
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to rotary evaporation at <30 °C. Analytical thin-layer chromatography was performed using

Merck Kieselgel 60 F 254 silica plates. Preparative column chromatography was performed on
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Merck 938S silica gel. Unless otherwise stated, 1H and 13

C NMR spectra were recorded on
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CDCl3 solutions using either Bruker 250 or 400 MHz (100 MHz for C) instruments with

tetramethylsilane as internal standard. Both low- and high-resolution mass spectra were obtained

by direct injection of sample solutions into a Micromass LCT mass spectrometer operated in the

electrospray mode, +ve or -ve ion as indicated. CI mass spectra (NH3) were obtained on a Fisons

Instruments Trio 1000. Analytical HPLC was performed using an Ascentia Express C-18 column,

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eluting with a gradient of 10-100% MeCN aq.+ 0.1% v/v CF3CO2H and monitored at 345 nm.

Chiral HPLC was performed using a Chiralpak AD-H column, eluting with n-C7H16: PriOH, 4:1.

Microanalytical data were obtained using an Elementar Vario micro cube instrument.

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(2S)-[2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]-2-(t-butoxycarbonyl)amino-3,3-

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dimethylbutanoate (7a).

A mixture of Boc-Tle-OH 2 (0.21 g, 0.97 mmol) and tizoxanide 1b (0.25 g, 0.94 mmol) was

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stirred at 20°C in anhydrous THF (7.5 mL). N-ethyl-N´-3-(dimethylamino)propyl

carbodiimide.HCl (EDC; 0.19 g, 1 mmol) was added in one portion, followed immediately by 4-

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dimethylaminopyridine (DMAP; 0.12 g, 1 mmol). After 20 h, the mixture was filtered through
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Celite and the precipitate washed with further THF, then diluted with ethyl acetate (25 mL). The

combined filtrate and washings were washed with 7% aq. citric acid, saturated aq. NaHCO3,
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water and brine, then dried over anhydrous Na2SO4. Evaporation afforded a yellow foam which
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was chromatographed on silica gel, being applied in CH2Cl2 and eluted with 1:1 ethyl acetate:
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hexane. Appropriate fractions were combined and evaporated to afford the title compound 7a as

an off-white solid (280 mg, 64%); 1H NMR [400 MHz, (CD3)2SO] δH1.02 (9 H, s, Me3C), 1.40 (9
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H, s, Me3CO), 4.05 (1 H, d, J = 7.6 Hz, CHNH), 7.25 (1H, d, J = 8.0 Hz, ArH), 7.31 (1 H, d, J =

7.6 Hz, CHNH), 7.47 (1 H, t, J = 8.0 Hz, ArH), 7.70 (1 H, t, J = 8.0 Hz, ArH), 7.78 (1 H, d, J =
C

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8.0 Hz, ArH), 8.70 (1 H, s, thiazole 4-H) and 13.67 (1 H, br s, NH); C NMR [100 MHz,
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(CD3)2SO] δC 26.9, 28.6, 34.0, 63.5, 79.0, 123.4, 126.6, 127.2, 129.9, 133.6, 142.6, 143.0, 148.4,

156.3, 162.4, 165.8 and 170.5; m/z (ES +ve mode) 501 (MNa+, base peak). Found: m/z,

501.1417. C21H26N4O7SNa requires m/z, 501.1420.

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We later found that, by using DMF as solvent and 1.5 eq. of both EDC and DMAP, all reagents

could be fully dissolved and a conversion of 88% of 7a was obtained after 6h at 0°C; the final

isolated yield was very similar.

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(2S)-[2-[(5-chloro-1,3-thiazol-2-yl)carbamoyl]phenyl]-2-(t-butoxycarbonyl)amino-3,3-

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dimethylbutanoate (7b). This compound was prepared similarly to 7a; from 2b (0.51 g, 2 mmol)

was obtained 7b (0.62 g, 67%) as a solid which could be crystallised from EtOAc-hexane. Found:

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C, 53.8; H, 5.5; N, 9.1: S, 6.5. C21H26ClN3O3S requires C, 53.9; H, 5.6; N, 9.0: S, 6.85%; 1H

NMR (400 MHz, CDCl3) δH1.10 (9 H, s, Me3CC), 1.43 (9 H, s, Me3CO), 4.30 (1 H, d, J = 7.6 Hz,

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CHNH), 5.28 (1 H, br d, J = 7.6 Hz, CHNH), 6.82 (1 H, s, thiazole 4-H), 7.35-7.45 (2 H, m,
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2xArH), 7.62 (1 H, t, J = 8.0 Hz, ArH), 7.89 (1 H, d, J = 8.0 Hz, ArH) and 11.66 (1 H, br s, NH);

C NMR (100 MHz, CDCl3) δC 26.6, 28.2, 34.4, 62.7, 80.3, 120.8, 123.2, 125.9, 126.4, 130.1,
13
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133.1, 134.5, 148.3, 155.7, 156.6, 163.3 and 170.0; m/z (ES +ve mode) 490, 492 (MNa+, base
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peaks for 35Cl/37Cl). Found: m/z, 490.1166. C21H2635ClN3O3SNa requires m/z, 490.1179.
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(S)-[2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]-2-amino-3,3-dimethylbutanoate,
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hydrochloride RM-5061 (8a). The preceding Boc derivative 7a (0.254 g, 0.53 mmol) was

suspended in CH2Cl2 (5 mL) and 4M HCl in dioxane (2 mL) was added with stirring at 20°C. A
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solution resulted after a few minutes, but solid soon began to precipitate. After 16 h, the reaction
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was diluted with ether, briefly stirred, then cooled to 0°C to complete precipitation; filtration

afforded the title compound RM5061 8a (0.205 g, 93%); 1H NMR [400 MHz, (CD3)SO] δH 1.10

(9 H, s, Me3C), 4.00 (1 H, br s, CHNH3+), 7.54 (1 H, d, J = 8.0 Hz, ArH), 7.62 (1 H, t, J = 8.0 Hz,

ArH), 7.75 (1 H, t, J = 8.0 Hz, ArH), 7.85 (1 H, d, J = 8.0 Hz, ArH), 8.73 (1 H, s, thiazole 4-H),

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8.86 (3 H, br s, NH3+) and 13.85 (1 H, br s, NH); 13C NMR [100 MHz, (CD3)SO] δC 26.6, 33.9,

61.5, 124.0, 126.6, 127.1, 130.0, 133.7, 142.6, 143.0, 147.8, 162.2, 165.8 and 167.5; m/z (ES +ve

mode) 379 (base peak, ammonium ion). Found: C, 46.1; H, 4.6; N, 13.6. C16H19N4O5SCl requires

C, 46.3; H, 4.6; N, 13.5%; Found: m/z, 379.1060. C16H19N4O5S requires m/z, 379.1076.

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The highest ee’s were observed when DMF was used in the coupling step. Following

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deprotection, RM5061 8a was obtained with an HPLC area purity of 99.0% and a chiral purity of

99.8%. The corresponding (R) enantiomer, viz. derived from D-tert-leucine, was similarly made;

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this material had an HPLC purity of 99.5% and a chiral purity of 99.5%.

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(S)-[2-[(5-chloro-1,3-thiazol-2-yl)carbamoyl]phenyl]-2-amino-3,3-dimethylbutanoate,
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hydrochloride RM-5064 (8b). This compound was prepared similarly to 8a. From 7b (600 mg,

1.28 mmol) there was obtained HCl salt RM 5064 8b (490 mg, 94%); 1H NMR [400 MHz,
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(CD3)SO] δH 1.07 (9 H, s, Me3C), 3.96 (1 H, d, J = 7.6 Hz, CHNH), 7.47 (1 H, t, J = 8.0 Hz,
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ArH), 7.56 (1 H, d, J = 8.0 Hz, ArH), 7.60 (1 H, s, 4´-H), 7.68 (1 H, t, J = 8.0 Hz, ArH), 7.75 (1
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H, d, J = 8.0 Hz, ArH) and 8.82 (3 H, br s, NH3+); 13C NMR [100 MHz, (CD3)SO] δC 26.6, 33.8,

61.6, 119.1, 123.8, 127.0, 127.5, 129.7, 133.0, 136.2, 147.7, 156.3, 164.8 and 167.4; m/z (ES +ve
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mode) 368 (base peak, ammonium ion). Found: m/z, 368.0833. C16H19N3O3S35Cl requires m/z,

370.0806.
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For the synthesis and characterization of compounds (5a), (9) and (10) and their Boc
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precursors, together with details of the pharmacokinetic and toxicological methods employed, see

Supporting Information.

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HIGHLIGHTS

• An effective phenolic prodrug for the antiviral agent tizoxanide and a 5-Cl analogue is described

• These derivatives employ the amino-acid L-tertiary-leucine

• The stability of this prodrug significantly exceeds that of the Val or Ile analogues

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• The pharmacokinetics, bioavailability and pre-clinical toxicology of the prodrug are reported

• Good blood levels are obtainable by oral or IV administration

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• The compounds show a good safety profile

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