INFECTIONS

Field of the invention

The present invention relates to pneumoviruses such as respiratory syncytial virus (RSV) and in particular to compounds useful in the treatment of pneumovirus infection, and assays to identify inhibitors of pneumovirus replication.

Background to the Invention.

Pneumoviruses, such as human respiratory syncytial virus (hRSV), are viruses that cause respiratory tract infections. Infection with hRSV is widespread. Although infection in adults is usually mild (or even asymptomatic), with any symptoms similar to those of the common cold, the virus can cause severe lower respiratory tract infections in young children or the elderly.

Pneumovirus infections are also commonplace in animals, particularly cows, sheep and goats, with bovine RSV (bRSV), ovine RSV and caprine RSV respectively. An additional pneumovirus, pneumonia virus of mice (PVM), has been shown to infect a wide range of species including rodents, dogs and humans. The pneumoviruses display structural and functional homology, and similar respiratory tract infections in animals.

The current treatment options for hRSV are limited. There is therefore a need for new treatments for hRSV infection and other pneumovirus infections.

DEAD box RNA helicases are a family of ATP-dependent RNA helicase enzymes. These enzymes have a conserved sequence Asp-Glu-Ala-Asp (DEAD). DDX3X is a DEAD box RNA helicase, encoded by the DDX3X gene. DDX3X and homologs thereof can be found in humans and other mammals, such as cows and sheep. DEAD box proteins are associated with many processes ranging from RNA synthesis, RNA degradation and translation initiation. A DDX3X homologue expressed from a gene on the Y chromosome shares 91% sequence identity.

It is known that infection with HIV-1 or HCV (hepatitis C virus) can lead to DDX3X expression being induced, and that certain cancer types are associated with overexpression of DDX3X. Thus, WO 2011/039735 describes the use of certain DDX3X inhibitors to suppress in vitro activity of DDX3X, suppress HIV-1 replication, and suppress proliferation of tumour cell lines. DDX3X does not apparently play a role in regulating HIV-1 transcription, but apparently acts as a nucleo-cytoplasmic shuttling protein for the export of HIV-1 transcripts. Similarly, DDX3X is known to interact with HCV core proteins, and alter their intracellular location. However, no link has previously been suggested between DDX3X and pneumovirus infection.

Pneumovirus replication requires translation of two open reading frames (ORF) from the M2 mRNA. ORF-2 overlaps with ORF-1. Host ribosomes access and translate the pneumovirus M2 ORF-2 by using one of two or three AUG codons located upstream of the ORF-1 termination codon. Expression from these initiation codons requires the prior termination of M2 ORF-1 translation. The hRSV M2-2 protein produced by coupled translation has been proposed to be involved in the switch between virus transcription and replication.

Summary of the present invention

The present inventors have identified that DDX3X binds to the hRSV M2 mRNA. The region to which DDX3X binds has a large degree of complex RNA secondary structure which is essential for coupled translation. Blocking binding of DDX3X to this region prevents initiation of hRSV M2-2 translation, thus preventing the switch between virus transcription and replication. The interaction between hRSV M2 mRNA and DDX3X provides a novel target to identify agents which can inhibit hRSV replication, and suggests the use of DDX3X inhibitors in the treatment of hRSV infection and other pneumovirus infections.

According to one aspect of the invention, there is provided a DDX3X inhibitor for use in the treatment of pneumovirus infection, such as for example RSV infection, in a mammal. According to a further aspect of the invention, there is provided a method of treating a pneumovirus infection, such as for example hRSV infection, in a subject, wherein the method comprises the administration to the said subject of an effective amount of a DDX3X inhibitor.

In some embodiments, the DDX3X inhibitor is a human DDX3X inhibitor, a bovine DDX3X inhibitor, an ovine DDX3X inhibitor, and/or a caprine DDX3X inhibitor.

In some embodiments, the DDX3X inhibitor is a compound of Formula (I):

(I)

wherein y is 0, 1, or 2;

each Z is independently selected from substituted or unsubstituted C _1-3 alkyl, substituted or unsubstituted C _1-3 acyl, substituted or unsubstituted C _1-3 alkoxy, substituted or

unsubstituted C _1-3 alkylthio, substituted or unsubstituted C _1-3 alkylsulfoxide, substituted or unsubstituted C _1-3 alkylsulfonyl, halo, nitro, and cyano;

each X is independently O or S;

L is a bond or is selected from:

where n is 0, 1, 2, 3 or 4, and the wavy lines indicate the points of attachment to the rest of the molecule;

each R ¹ is independently selected from H and substituted or unsubstituted C _1-3 alkyl;

each R ² is a bond or is independently selected from substituted or unsubstituted C _1-3 alkyl; each R ³ is selected from substituted or unsubstituted Ci- ₆ alkyl; and

either:

R ^a is selected from H and substituted or unsubstituted C _1-3 alkyl, and R ^b is selected from: substituted or unsubstituted C _1-10 alkyl, substituted or unsubstituted C _2-10 alkenyl, substituted or unsubstituted C4-8 cycloalkyl, substituted or unsubstituted C4-8 cycloalkenyl, and

phenyl or 5- or 6-membered heteroaryl, each of which may be optionally substituted with up to 2 substituents Z,

or R ^a and R ^b , together with the nitrogen atom to which they are attached, form a 5- or 6- membered substituted or unsubstituted heterocyclyl ring;

or a tautomer or a pharmaceutically acceptable salt thereof.

In some further embodiments, each R ¹ is H.

In some further embodiments, y is 1.

In some further embodiments, each Z is independently selected from methyl, acetyl, methoxy, methylthio, methylsulfoxide, methylsulfonyl, bromo, nitro, cyano, chloro and substituted Ci alkyl. Preferably at least one Z is a meta or para substituent selected from methyl, methoxy, methylthio, cyano, nitro, bromo, chloro, and -CF ₃ . More preferably at least one Z is a meta or para substituent selected from methyl, methoxy, methylthio, cyano, chloro, and -CF ₃ .

In some further embodiments, each Z is independently selected from methyl, acetyl, methoxy, methylthio, methylsulfoxide, methylsulfonyl, bromo, nitro, and cyano.

Preferably at least one Z is a meta or para substituent selected from methyl, methoxy, methylthio, cyano, nitro and bromo.

In some further embodiments, L is a bond or is selected from:

In some further embodiments, n is 3.

In some further embodiments, R ^a is H. In some further embodiments, R ^b is selected from unsubstituted C _1-10 alkyl and unsubstituted C4-8 cycloalkyl. Preferably, R ^b is selected from 2-methylcyclohexyl and n- octanyl.

In some further embodiments, R ^b is phenyl or 5- or 6-membered heteroaryl, each of which may be optionally substituted with up to 2 substituents Z. Preferably, R ^b is selected from phenyl, pyridinyl, and pyrimidinyl, each of which is substituted with one substituent Z.

In some further embodiments, y is 1;

each Z is independently selected from methyl, acetyl, methoxy, methylthio,

methylsulfoxide, methylsulfonyl, bromo, nitro, cyano, chloro and substituted Ci alkyl.; each X is independently O or S;

L is a bond or is selected from:

where n is 3, and the wavy lines indicate the points of attachment to the rest of the molecule;

R ^a is H; and

R ^b is selected from:

2-methylcyclohexyl, ft-octanyl,

phenyl substituted one substituent Z,

pyridinyl, and

pyrimidinyl.

In some further embodiments, y is 1;

each Z is independently selected from methyl, acetyl, methoxy, methylthio,

methylsulfoxide, methylsulfonyl, bromo, nitro, and cyano;

each X is independently O or S;

L is a bond or is selected from:

X

where n is 3, and the wavy lines indicate the points of attachment to the rest of the molecule;

R ^a is H; and

R ^b is selected from:

2-methylcyclohexyl, ft-octanyl,

phenyl substituted one substituent Z,

pyridinyl, and

pyrimidinyl.

In some further embodiments, the DDX3X inhibitor is a compound of Formula (II):

(Π)

or a tautomer or a pharmaceutically acceptable salt thereof.

Preferably, R ^a is H and R ^b is selected from 2-methylcyclohexyl and «-octanyl.

Preferably, R ^a is H, R ^b is selected from:

phenyl substituted with 1 substituent Z,

pyridinyl and

pyrimidinyl;

and at least one Z is a meta or para substituent selected from methoxy, methylthio, cyano, nitro, bromo, chloro and -CF ₃ .

In some further embodiments, the DDX3X inhibitor is a compound of Formula (II):

(Π)

or a tautomer or a pharmaceutically acceptable salt thereof.

Preferably, R ^a is H and R ^b is selected from 2-methylcyclohexyl and «-octanyl.

Preferably, R ^a is H, R ^b is selected from:

phenyl substituted with 1 substituent Z,

pyridinyl and

pyrimidinyl;

and at least one Z is a meta or para substituent selected from methoxy, methylthio, cyano, nitro and bromo.

In some further embodiments, the DDX3X inhibitor is a compound of Formula

(III):

(III)

wherein each Z is a meta or para substituent independently selected from methoxy, methylthio, cyano and nitro;

or a tautomer or pharmaceutically acceptable salt thereof.

Preferably, each Z is the same substituent, and both Z are meta substituents or both Z are para substituents.

In some further embodiments, the DDX3X inhibitor is a compound of Formula (III) where one Z is an ortho methyl substituent, and the other Z is a meta or para substituent selected from methoxy, methylthio, cyano and nitro;

or a tautomer or pharmaceutically acceptable salt thereof.

In some further embodiments, the DDX3X inhibitor is a compound of Formula

(IV): wherein each Z is independently selected from methyl, methylthio and nitro;

or a tautomer or pharmaceutically acceptable salt thereof.

Preferably, each Z is the same substituent, and both Z are ortho substituents, both Z are meta substituents or both Z are para substituents.

Preferably, one Z is an ortho methyl substituent, and the other Z is a meta or para substituent selected from methylthio and nitro.

In some embodiments, the DDX3X inhibitor is selected from the group consisting of the compounds:

and tautomers and pharmaceutically acceptable salts thereof.

In other embodiments the DDX3X inhibitor is selected from the group consisting the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compounds:

and tautomers and pharmaceutically acceptable salts thereof. More preferably, the DDX3X inhibitor is selected from the group consisting of the com ounds:

and tautomers and pharmaceutically acceptable salts thereof.

Alternatively the DDX3X inhibitor is selected from the group consisting of the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compounds:

and tautomers and pharmaceutically acceptable salts thereof.

Most preferably, the DDX3X inhibitor is selected from the group consisting of the com ounds:

and tautomers and pharmaceutically acceptable salts thereof.

Alternatively the DDX3X inhibitor is selected from the group consisting of the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compound:

and tautomers and pharmaceutically acceptable salts thereof.

In some embodiments, the mammal is selected from humans, cattle, sheep and goats.

According to a further aspect of the invention, there is provided a compound of

Formula (I):

(I) wherein y, Z, X, L, n, R ¹ , R ² , R ³ , R ^a and R ^b are as defined above, or a tautomer or a pharmaceutically accept le salt thereof, provided that the compound is other than:

In some embodiments, the compound is selected from the group consisting of the com ounds:

and tautomers and pharmaceutically acceptable salts thereof.

In other embodiments, the compound is selected from the group consisting of the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compounds:

and tautomers and pharmaceutically acceptable salts thereof.

Preferably, the compound is selected from the group consisting of the compounds:

and tautomers and pharmaceutically acceptable salts thereof.

Alternatively, the compound is selected from the group consisting of the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compounds:

and tautomers and pharmaceutically acceptable salts thereof.

More preferably, the compound is selected from the group consisting of the com ounds:

and tautomers and pharmaceutically acceptable salts thereof.

Alternatively, the compound is selected from the group consisting of the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compound:

and tautomers and pharmaceutically acceptable salts thereof.

According to a further aspect of the invention, there is provided a compound of Formula (I):

(I)

wherein L is selected from

and y, Z, X, n, R ¹ , R ² , R ³ , R ^a and R ^b are as defined above, or a tautomer or a

pharmaceutically acceptable salt thereof.

According to a further aspect of the invention, there is provided a compound of Formula (I):

(I)

wherein X is S, and

y, Z, L, n, R ¹ , R ² , R ³ , R ^a and R ^b are as defined above, or a tautomer or a pharmaceutically acceptable salt thereof

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a compound as described above and a pharmaceutically- acceptable excipient.

According to a further aspect of the invention, there is provided a compound as described above for use in the treatment of the human or animal body.

Brief Description of the Figures

Fig. 1 shows two biological replicates carried out on different occasions for compound 1. Fig. 2 shows the effect of 3 on plaque size (relative to a DMSO control) after 5 days. Fig. 3 shows the results of further tests carried out to screen inhibition over a number of days for compound 4.

Fig. 4 shows results for compound 6 tested using assays stopped after 5, 6 and 7 days. Fig. 5 shows results for compound 35 tested in a 5 day assay.

Fig. 6 shows results for compound 39 tested in a 4 day assay using BSC1 cells.

Fig. 7 shows results for compound 40 tested in a 4 day assay using BSC1 cells.

Fig. 8 shows results for compounds 1, 4 and 6 tested against Bovine RSV with a 6-day assay.

Fig. 9 shows results of a full plaque assay carried out for compound 4, tested against PVM with assays of 5 days and 6 days after incubation.

Fig. 10 shows the yield of virus from cells treated with compounds 1, 4, 6 and 35 at the various concentrations as shown.

Fig. 11 shows results of an MTT assay for compound 1

Fig. 12 shows results of an MTT assay for compound 4

Fig. 13 shows results of an MTT assay for compound 6

Fig. 14 shows results of an MTT assay for compound 35

Fig. 15 shows results of an MTT assay for compound 37

Fig. 16 shows sequence (SEQ ID NO. 1) and coding capacity (SEQ ID NOs. 2 and 3) of the hRSV strain A2 M2 mRNA RSV strain A2 (subgroup A).

Fig. 17 shows sequence alignment of RSV M2 mRNAs (SEQ ID NOs. 4 to 7).

Fig. 18 shows amino acid sequence of human ATP-dependent RNA helicase DDX3X isoform 1. GenBank accession number NP 001347.3. (SEQ ID NO. 8).

Fig. 19 shows alignment of the amino acid sequences of mammalian ATP-dependent RNA helicase DDX3X (SEQ ID NOs. 9 to 13).

Description of the Sequence Listing

SEQ ID NO. 1 shows sequence of the hRSV strain A2 M2 mRNA RSV strain A 2

(subgroup A).

SEQ ID NOs. 2 and 3 show coding capacity of the hRSV strain A2 M2 mRNA RSV strain A2 (subgroup A).

SEQ ID NO. 4 shows sequence of human respiratory syncytial virus strain A2.

SEQ ID NO. 5 shows sequence of human respiratory syncytial virus strain Bl . SEQ ID NO. 6 shows sequence of bovine respiratory syncytial virus strain A51908.

SEQ ID NO. 7 shows sequence of ovine respiratory syncytial virus.

SEQ ID NO. 8 shows amino acid sequence of human ATP-dependent RNA helicase DDX3X isoform 1.

SEQ ID NO. 9 shows sequence of ATP-dependent RNA helicase DDX3X isoform 1

[Homo sapiens].

SEQ ID NO. 10 shows sequence of Ddx3x protein [Mus musculus].

SEQ ID NO. 11 shows sequence of ATP-dependent RNA helicase DDX3X isoform 1

[Ovis aries].

SEQ ID NO. 12 shows sequence of ATP-dependent RNA helicase DDX3X isoform X2 [Bos taurus].

SEQ ID NO. 13 shows sequence of ATP-dependent RNA helicase DDX3X-like isoform X2 [Capra hircus]. Detailed description

The present invention enables the treatment of pneumovirus (such as hRSV) infection, involving the use of a DDX3X inhibitor. The invention further provides a number of compounds which are of interest for use as DDX3X inhibitors.

As used herein, the term "alkyl" refers to hydrocarbons, particularly saturated hydrocarbons, having the specified number of carbon atoms, and includes straight-chain and branched-chain groups and combinations thereof.

As used herein, the term "acyl" refers to hydrocarbons having the specified number of carbon atoms and including at least one carbonyl moiety, and includes straight-chain and branched-chain groups and combinations thereof. The carbonyl may be located at any position in the group, but is typically located adjacent to a point of attachment to the remainder of the molecule.

As used herein, the term "alkoxy" refers to hydrocarbons having the specified number of carbon atoms and including one or more oxygen atoms linked by single bonds to carbon, and includes straight-chain and branched-chain groups and combinations thereof. The oxygen atoms may be located at any position in the group, but one oxygen atom is typically located adjacent to a point of attachment to the remainder of the molecule.

As used herein, the term "alkylthio" refers to hydrocarbons having the specified number of carbon atoms and including one or more sulfur atoms linked by single bonds to carbon, and includes straight-chain and branched-chain groups and combinations thereof. The sulfur atoms may be located at any position in the group, but one sulfur atom is typically located adjacent to a point of attachment to the remainder of the molecule.

As used herein, the term "alkylsulfoxide" refers to hydrocarbons having the specified number of carbon atoms and including at least one sulfinyl moiety, and includes straight-chain and branched-chain groups and combinations thereof. The sulfinyl moiety may be located at any position in the group, but is typically located adjacent to a point of attachment to the remainder of the molecule.

As used herein, the term "alkylsulfonyl" refers to hydrocarbons having the specified number of carbon atoms and including at least one sulfonyl moiety, and includes straight-chain and branched-chain groups and combinations thereof. The sulfonyl moiety may be located at any position in the group, but is typically located adjacent to a point of attachment to the remainder of the molecule.

As used herein, the term "halo" refers to an element found in Group 13 of the periodic table. Typical examples of halo substituents include fluoro, chloro and bromo. Another typical example is iodo.

As used herein, the term "cycloalkyl" refers to hydrocarbons having the specified number of carbon atoms and including at least one cyclic structure, and includes straight- chain and branched-chain groups, and combinations thereof.

As used herein, the term "alkenyl" refers to hydrocarbons having the specified number of carbon atoms, and including at least one carbon-carbon double bond, and includes straight-chain and branched-chain groups, and combinations thereof.

As used herein, the term "cycloalkenyl" refers to hydrocarbons having the specified number of carbon atoms, and including at least one carbon-carbon double bond and at least one cyclic structure. The double bond may be incorporated wholly or partially within the cyclic structure or may be distinct from the cyclic structure. The term includes straight- chain and branched-chain groups, and combinations thereof.

As used herein, the term "substituted" refers to a moiety in which one or more hydrogen atoms, typically 1, 2 or 3 hydrogen atoms, has each been replaced by a substituent independently selected from hydroxyl, methoxy, thio, methylthio, amino, methylamino, dimethylamino, fluoro, chloro, bromo, iodo, cyano, nitro, sulfonate (or sulfonic acid), sulfonamide, carboxylate (or carboxylic acid) and carbonamide.

The present invention also relates to a method of identifying an agent which modulates hRSV replication comprising contacting an agent with (a) an RNA construct comprising the sequence of nucleotides 392 to 511 of the hRSV M2 transcript and (b) a protein comprising DDX3X, and determining whether the agent modulates the interaction between the construct and DDX3X.

The M2 mRNAs of pneumoviruses contain two open reading frames, referred to herein as M2-1 and M2-2. The region of nucleotides 392-511 of the hRSV M2 mRNA has strong secondary structure. The present inventors show that DDX3X interacts with this structure, to allow initiation of translation from the second ORF. The M2-2 protein translated from the M2 mRNA ORF-2 is required for the switch from transcription of the viral genome to replication, leading to virus particle assembly and release. Thus agents which interfere with this interaction are identified as useful in the treatment of infection by hRSV and other pneumoviruses.

The RNA constructs of the present invention comprise nucleotides 392 to 511 of the human M2 transcript, or an equivalent region in other pneumoviruses, including but not limited to bovine RSV, ovine RSV, caprine RSV. The sequence of the human ORF-1 region of the hRSV (strain A2) M2 transcript is shown in Figure 16 (SEQ ID NOs. 1-3). M2 transcripts from other pneumoviruses show a high degree of homology in this region seen in Figure 17 (SEQ ID NOs. 4-7). The construct may include additional sequences, for example flanking sequences from the hRSV M2 transcript. Assay methods which monitor for translation of the M2-2 ORF will include this ORF. Alternatively the construct may be provided with a reporter gene provided in frame with one of the AUG codons of the second ORF.

DDX3X is a DEAD box RNA helicase. The sequence of human DDX3X is provided in Figure 18 (SEQ ID NO. 8). DDX3X is highly conserved across mammalian species as shown in Figure 19 (SEQ ID NOs. 9-13). In view of the high degree of homology between pneumoviral M2 transcripts and DDX3X across species, the methods of the present invention can use pneumovirus constructs and DDX3X derived from other species, and can also be used to identify modulators which can be used to interfere with pneumovirus infection in a variety of mammalian species.

The compounds tested may be enhancers or inhibitors of the interaction between the construct and DDX3X, and thus act to enhance or inhibit pneumovirus replication, although preferably the method is used to identify an inhibitor of RSV replication. An inhibitor of pneumovirus replication interferes with the interaction between the construct and DDX3X. Any compound(s) can be used in the method of the invention. The compound(s) may be any chemical compound(s) used in drug screening programmes. They may be natural or synthetic. Extracts of plants which contain several characterised or

uncharacterised components may also be used. Typically, organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 Daltons. Compounds can be biomolecules including peptide and peptide mimetics, oligonucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate compounds may be obtained from a wide variety of sources including libraries of synthetic or natural substances. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. The compound(s) may be the product(s) of a combinatorial library such as are now well known in the art (see e.g. Newton (1997) Expert Opinion Therapeutic Patents, 7(10): 1183-1194). Natural product libraries, such as display (e.g. phage display libraries), may also be used.

The methods of the invention allow the screening of one or more compounds for their ability to act as modulator of pneumovirus replication. The methods are preferably carried out in vitro or ex vivo.

Techniques for determining the effect of compound(s) on the interaction between the RNA construct and DDX3X protein are within the skill of those in the art. Any of those techniques may be used in accordance with the invention. The method may be carried out in vitro, such as in a cell-free system or alternatively in a cell-based system.

In accordance with the methods of the present invention, the activity of an agent may be determined by investigating directly the effect on the binding interaction between DDX3X and the RNA construct. Alternatively, the methods may be conducted to determine translation from the second open reading frame (ORF-2) of M2. Reporter systems may also be used, for example to provide a construct which comprises a reporter gene cloned in frame from the start codon of ORF-2. Expression of the reporter gene will only occur when DDX3X is able to interact with the construct. The agents which interfere with the interaction will prevent translation of the reporter gene.

Preferred in vitro translation systems utilise cell extracts which provide components necessary for the process of translation. These typically include macromolecular components such as ribosomes, tRNAs, aminoacyl tRNA synthetases, initiation, elongation and termination factors. The cell extracts may be of any origin provided they allow for translation of the reporter coding sequence. Suitable cell extracts may be obtained from reticulocytes, such as rabbit reticulocytes, wheat germ and bacterial extracts, such as E. coli extracts. The cell extracts are suitably supplemented with additional components required for translation, such as amino acids, nucleotide triphosphate energy sources and other co-factors. The skilled person is familiar with the use of such systems.

In preferred embodiments, the RNA is added to the in vitro translation system directly in RNA form. Alternatively, a coupled or linked transcription/translation system may be utilised in which a DNA construct encoding the RNA is first transcribed prior to translation of the resulting RNA.

Techniques for measuring translation of a reporter coding sequence are well known in the art. Any suitable technique may be used. Preferred methods of measuring reporter coding sequence translation involve luminescence, fluorescence, or an immunoassay. For example, a reporter coding sequence may encode a luminescent or fluorescent protein such that the level of translation may be monitored through measurement of a luminescent or fluorescent signal. A suitable example of a luminescent reporter coding sequence is luciferase. A suitable example of a fluorescent reporter coding sequence is green fluorescent protein.

Measuring levels of translated protein using an immunoassay is also well known in the art. Any suitable immunoassay which allows for detection of a reporter coding sequence by an antibody may be used. Any suitable commercially available antibody for a given target may be used. An example of a suitable immunoassay is Enzyme-Linked Immunosorbent Assay (ELISA). In some embodiments, the ELISA assay may be performed in flat plates where wells are coated with binding proteins or antibodies which can bind and allow for detection of the translated reporter polypeptide. Other types of immunoassay include immunoprecipitation and Western blotting.

Modulators of pneumovirus replication can be formulated into pharmaceutical compositions. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes. Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

For delayed release, the modulators of pneumovirus replication may be included in a pharmaceutical composition which is formulated for slow release, such as in

microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

The dose of a modulator of pneumovirus replication may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated and the frequency and route of administration.

Preferably, daily dosage levels are from 5 mg to 2 g. That dose may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered daily.

Embodiments of the invention are illustrated by the following examples.

Library 1 X = O

Library 2 X = S

Scheme 1 Synthesis of ligands in Ta

Library 3 X = O

Library 4 X = S

Scheme 2 Synthesis of ligands in Tables 3 and 4.

Table 1 : Library 1 diarylureas prepared and summary of activities in cellular RSV TCID ₅₀ assay. Solubility refers to a solution of ligand in DMSO (20mM) diluted in cell media to a working concentration of 100 μΜ and ranges from 'very poor' (almost insoluble) to 'very good' remaining freely soluble over extended periods at 4 °C.

Table 2 Library 2, diarylthioureas prepared and summary of activities in cellular RSV TCID ₅₀ assay. Solubility refers to a solution of ligand in DMSO (20mM) diluted in cell media to a working concentration of 100 μΜ and ranges from 'very poor' (almost insoluble) to 'very good' remaining freely soluble over extended periods at 4 °C.

Table 3 Library 3, alkylaryl ureas prepared and summary of activities in cellular RSV TCID ₅₀ assay. Solubility refers to a solution of ligand in DMSO (20mM) diluted in cell media to a working concentration of 100 μΜ and ranges from 'very low' (almost insoluble) to 'very good' remaining freely soluble over extended periods at 4 °C.

Table 4 Library 4 alkylaryl thioureas prepared and summary of activities in cellular RSV TCID ₅₀ . Solubility refers to a solution of ligand in DMSO (20mM) diluted in cell media to a working concentration of 100 μΜ and ranges from 'very low' (almost insoluble) to 'very good' remaining freely soluble over extended periods at 4 °C.

Library 5 X = O, X = S

Scheme S3 Synthesis of ligands in Table 5.

Table 5 Library 5, oligomeric ureas and thioureas prepared and summary of activities in cellular RSV TCID ₅₀ assay. Solubility refers to a solution of ligand in DMSO (20mM) diluted in cell media to a working concentration of 100 μΜ and ranges from 'very low' (almost insoluble) to 'very good' remaining freely soluble over extended periods at 4

°C.

2 H H H H good toxic at

100 μΜ 3 H H H H poor ++ toxic at

100 μΜ 4 poor ++ toxic at

100 μΜ 5 H H H H good toxic at

-^^ ^N y ^N ° o ° ^N γ- ^N Y^r' ^N ° ²

100 μΜ 6 good 7 good +++ toxic at

H ₃ CS ^A ^ ^J ° ° ^SCH ₃ 50 μΜ 8 I 1— 1 H H H good toxic at

50 μΜ

General Experimental. Reagents and chemicals were purchased from Sigma- Aldrich and Acros and were used as supplied; solvents were purchased from Fisher and were used as supplied. All reactions were carried out under an atmosphere of nitrogen. Silica flash chromatography was carried out under low pressure using Merck silica gel 60 H (230-400 mesh). MR spectra were acquired on Bruker DPX 300 or DPX 400 spectrometers with tetramethylsilane, δ = 0 ppm as an internal standard. Assignments were made using 2D methods including COSY, HMBC and HMQC NMR and coupling constants quoted to the nearest 1 Hz. Low-resolution mass spectra were measured on an Agilent 6130B ESI-quad instrument with an electrospray ionization (ESI) mass selective detector in positive ion or negative ion mode as indicated. MALDI mass spectra were acquired on a Bruker Ultraflex II instrument. High-resolution mass spectra (HRMS) were acquired on a Bruker MicroTOF instrument with an ESI mass selective detector in either positive or negative ion mode. Infrared spectra were acquired with a Bruker Alpha FT-IR spectrophotometer. Melting points (mps) were determined on a Stuart SMP10 melting point apparatus; mp > 250 °C were measured on an Olympus BH2 microscope equipped with a Linkam HFS 91 heating stage, or differential scanning calorimeter (DSC, Mettler Toledo DSCl-400). Warwick Analytical Service Limited, performed CHN microanalysis using a CE440 Elemental Analyser.

Preparation of compounds

SAFETY Aromatic amines are highly toxic and considered as known or suspected human carcinogens. Isocyanates are sensitizers and toxic or highly toxic and their endpoint of hydrolysis is the parent amines. All procedures were carried out in an efficient fumehood, wearing standard laboratory protection. In addition, all reagents and reaction vessels were handled wearing a pair of thin nitrile gloves overgloved with medium weight nitrile gloves. All waste, including aqueous was disposed in specifically labeled containers.

General Procedure I -Addition of aromatic amines to isocyanates

Aromatic ureas or thioureas were prepared using a previously reported general method. ¹ To a solution of the amine (1 equiv., 5 mmol) in dry dichloromethane (5 mL) under dry N ₂ was added the isocyanate (1 equiv., 5 mmol) in dry dichloromethane (-10 mL) with stirring, and left overnight at room temperature. The precipitate was collected by filtration and washed with a little ethanol, or if no precipitate, volatiles were removed under reduced pressure on a rotary evaporator. The resulting mixture was then purified through either recrystallization or a flash silica column (details specified) to provide the desired product.

Preparation of 3-(2-methylphenyl)-l-(3-nitrophenyl)urea (1),

Compound 1 was prepared from o-toluidine and 3-nitrophenylisocyanate using General Procedure I on 20 mmol scale to give, after recrystallization from ethanol, the title product 1 as a cotton wool-like solid (3.782 g, 69%); mp 216 - 218 °C; 1H MR (400 MHz, DMSO-i ) δ 9.53 (br. s., 1H), 8.59 (t, J= 2 Hz, 1H), 8.08 (br. s., 1H), 7.83 (ddd, J= 8, 2, 1 Hz, 1H), 7.80 (dd, J= 8, 1 Hz, 1H), 7.71 (ddd, J= 8, 2, 1 Hz, 1H), 7.58 (t, J= 8 Hz, 1H), 7.15 - 7.23 (m, 2H), 7.01 (dt, J= 7, 1 Hz, 1H), 2.27 (s, 3H) ppm; mlz C ₁₄ H ₁₃ N ₃ O ₃ (ESI pos. mode) 294.0 (MNa ⁺ ); mlz (ESI neg. mode) 306.0 (M ³⁵ C1 ^" ), 270.1 (M-H ⁺ ).

In agreement with literature data: Radi, M.; Falchi, F.; Garbelli, A.; Samuele, A.; Bernardo, V.; Paolucci, S.; Baldanti, F.; Schenone, S.; Manetti, F.; Maga, G.; Botta, M. Bioorg. Med. Chem. Lett. 2012, 22, 2094-2098. Preparation of l-(3-acetylphenyl)-3-(2-methylphenyl)urea (2)

o-Toluidine and 3-acetylphenylisocyanate were combined using General Procedure I on a 3.10 mmol scale to give, after recrystallization from ethanol, the title product 2 as off- white crystals (0.454 g, 55%); mp 198 - 199 °C; 1H NMR (300 MHz, DMSO- ) δ 9.26 (s, 1H), 8.07 (t, J = 1 Hz, 1H), 7.96 (s, 1H), 7.83 (d, J= 8 Hz, 1H), 7.70 (dd, J = 8, 1 Hz, 1H), 7.58 (d, J= 8 Hz, 1H), 7.44 (t, J = 8 Hz, 1H), 7.1 1 - 7.22 (m, 2H), 6.96 (dt, J = 7, 1 Hz, 1H), 2.57 (s, 3H), 2.25 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) δ 197.7, 152.6, 140.3, 137.4, 137.2, 130.2, 129.2, 127.7, 126.1, 122.9, 122.5, 121.7, 121.2, 1 17.1, 26.7, 17.8 ppm; Vmax (solid) 3282, 3080, 3026, 2974, 1670, 1641, 1586, 1560, 1433, 1360, 1274, 1239, 915, 795, 765, 662 cm ^"1 , mlz (ESI neg. mode) 267.1 (M-H) ^~ Elemental analysis calcd for Ci ₆ Hi ₆ N ₂ 0 ₂ : C, 71.62; H, 6.01 ; N, 10.44. Found: C, 71.41 ; H, 5.93, N, 10.28.

Preparation of 3-(3-cyanophenyl)-l-(2-methylphenyl)urea (3)

o-Toluidine and 3-cyanophenylisocyanate were combined using General Procedure I on a 3.47 mmol scale to give, after recrystallization from ethanol, the title product 3 as a cotton wool-like solid; mp 200 - 20FC; 1H NMR (300 MHz, DMS0 ) δ 9.35 (br. s., 1H), 8.08 (br. s., 1H), 8.01 (s, 1H), 7.78 (d, J = 8 Hz, 1H), 7.65 (d, J = 9 Hz, 1H), 7.50 (t, J= 8 Hz, 1H), 7.42 (d, J = 7 Hz, 1H), 7.12 - 7.22 (m, 2H), 6.98 (t, J = 7 Hz, 1H), 2.25 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) δ 152.5, 140.7, 136.9, 130.2 (2C), 128.2, 126.1, 125.1, 123.2, 122.6, 121.6, 120.5, 1 18.9, 1 1 1.6, 18.2 ppm; methyl peak missing but seen on

HMQC at 18.2 ppm. Peak at 130.2 ppm determined to be 2C (non identical) by HMQC; Vmax (solid) 3323, 3271, 2232, 1643, 1585, 1556, 1458, 1433, 1289, 1248, 795, 774, 753, 658 cm ^"1 ; mlz (ESI pos. mode) 274.1 (MNa ⁺ ); mlz 288.0 (ESI neg. mode) (M ³⁷ C1 ^" ), 286.0 (M ³⁵ C1 ^" ), 250.1 (M-H ⁺ ). Elemental analysis calcd for C ₁₅ H ₁₃ N ₃ O: C, 71.70; H, 5.21 ; N, 16.72. Found: C, 71.65; H, 5.16; N, 16.64.

Preparation of l-(2-methylphenyl)-3-[3-(methylsulfanyl)phenyl]urea (4)

o-Toluidine and 3-methylthiophenylisocyanate were combined using General Procedure I on a 3.03 mmol scale to give, after recrystallization from ethanol, the title product 4 as an amorphous solid (0.362 g, 44%); mp 175 - 176 °C; 1H NMR (400 MHz, DMSO- ) δ 9.06 (s, 1H), 7.93 (s, 1H), 7.82 (dd, J = 8, 1 Hz, 1H), 7.49 (t, J = 2 Hz, 1H), 7.23 (t, J = 8 Hz, 1H), 7.12 - 7.21 (m, 3H), 6.96 (dt, J= 7, 1 Hz, 1H), 6.87 (td, J = 8, 1 Hz, 1H), 2.47 (s, 3H), 2.25 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO-i ) δ 152.6, 140.4, 138.6, 137.2, 130.2, 129.3, 127.6, 126.1, 122.8, 121.1, 1 19.1, 1 14.9, 1 14.5, 17.8, 14.6 ppm; (neat) 3285, 1632, 1606, 1555, 1478, 1459, 1288, 1260, 1232, 926, 848, 772, 743, 665 cm ^"1 ; miz (ESI pos. mode) 295.0 (MNa ⁺ ); miz (ESI neg. mode) 307.0 (M ³⁵ C1 ^" ), 271.1 (M-H ⁺ ). HRMS (ESI pos. mode) miz Ci ₅ Hi ₆ N ₂ NaOS required 295.0876. Found miz 295.0877. Preparation of 3-(3-methanesulfinylphenyl)-l-(2-methylphenyl)urea (5)

To a stirred solution of l-(2-methylphenyl)-3-[3-(methylsulfanyl)phenyl]urea 4 (0.25 g, 0.92 mmol) in acetone - water (2: 1 v/v, 15 mL) at 0 °C was added dropwise a solution of sodium periodate (0.96 g, 0.92 mmol) in water (8 mL). A thick white precipitate of sodium iodate formed, which was removed by filtration after 4 hours and washed with a small portion of acetone. The filtrate was extracted with ethyl acetate (3 x 20 mL), with the addition of brine (15 mL) for the final extraction. The combined organics were washed with water, dried (MgS0 ₄ ) and evaporated under reduced pressure to give the crude product. Purification by flash chromatography (Si0 ₂ , 1% MeOH - EtOAc) gave a solid which was recrystallized from hot ethanol - water (1 :2 v/v) to give the title product 5 as a white powder (0.098 g, 35%); mp 139 - 144 °C; 1H NMR (300 MHz, DMSO- ) δ 9.35 (s, 1H), 7.97 (s, 1H), 7.87 (t, J= 2 Hz, 1H), 7.83 (d, J= 8 Hz, 1H), 7.51 - 7.57 (m, J= 8, 2 Hz, 1H), 7.47 (t, J= 8 Hz, 1H), 7.23 (td, J= 7, 1 Hz, 1H), 7.11 - 7.21 (m, 2H), 6.97 (dt, J= 7, 1 Hz, 1H), 2.73 (s, 3H), 2.25 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO- ) δ 152.5, 147.1, 140.8, 137.1, 130.2, 129.7, 127.7, 126.1, 122.9, 121.2, 119.7, 116.5, 112.3, 43.3, 17.8 ppm; Vmax (solid) 3438, 3292, 3056, 2917, 1640, 1585, 1457, 1425, 1242, 1019, 789, 761 cm ^"1 ; miz (ESI pos. mode) 311.0 (MNa ⁺ ); (ESI neg. mode) 323.0 (M ³⁵ C1 ^" ), 287.1 (M-H ⁺ ) ^" .

HRMS (ESI pos. mode) miz Ci ₅ Hi ₇ N ₂ 0 ₂ S (MH ⁺ ) required 289.1005. Found miz 289.1001 Preparation of l-(4-cyanophenyl)-3-(2-methylphenyl)urea (6)

o-Toluidine and 4-cyanophenylisocyanate were combined using General Procedure I on a 3.47 mmol scale to give, after recrystallization from ethanol (< 20 mL), the title product 6 as white needles (0.548 g, 63%); mp 207 - 210 °C; 1H NMR (400 MHz, DMSO-i ) δ 9.50 (br. s., 1H), 8.11 (br. s., 1H), 7.78 (dd, J= 8, 1 Hz, 1H), 7.74 (td, J= 9, 2 Hz, 2H), 7.65 (td, J= 9, 3 Hz, 2H), 7.15 - 7.23 (m, 2H), 7.00 (dt, J= 8, 1 Hz, 1H), 2.26 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO-i ) δ 152.2, 144.3, 136.8, 133.3, 130.2, 128.3, 126.2, 123.3, 121.6, 119.3, 117.8, 103.1, 17.8 ppm; v _max (solid) 3285, 3067, 3024, 2227, 1637, 1583, 1540, 1456, 1407, 1294, 1241, 1175, 847, 836, 755, 648, 552 cm ^"1 ; miz (ESI pos. mode) 274.0 (MNa ⁺ ), 252.1 (MH ⁺ ); miz (ESI neg. mode) 286.0 (M ³⁵ C1 ^" ), 250.1 (M-H ⁺ ) ^~ Elemental analysis calcd for C15H13N3O: C, 71.70; H, 5.21; N, 16.72. Found: C, 71.51; H, 5.16; N, 16.64.

Preparation of 3-(3-cyanophenyl)-l-(pyridin-3-yl)urea (7)

3-Aminopyridine and 3-cyanophenylisocyanate were combined using General Procedure I on a 3.47 mmol scale to give, after slow recrystallization from ethanol (- 30 mL), the product as red-tinged crystals (0.269 g, 33%). A second recrystallization from a minimum of hot ethanol gave the title product 7 as a white cotton wool-like mass of crystals (0.255 g, 31%); mp 197-198 °C (dec); 1H MR (400 MHz, DMSO-^ ₆ ) δ 9.17 (s, 1H), 9.03 (s, 1H), 8.63 (d, J= 3 Hz, 1H), 8.23 (dd, J= 5, 2 Hz, 1H), 7.99 (t, J= 2 Hz, 1H), 7.95 (ddd, J= 8, 3, 2 Hz, 1H), 7.71 (ddd, J= 8, 2, 1 Hz, 1H), 7.52 (t, J= 8 Hz, 1H), 7.45 (td, J= 8, 1 Hz, 1H), 7.34 (dd, J= 8, 5 Hz, 1H) ppm; ¹³ C NMR (101 MHz, DMS0 ) δ 152.5, 143.2, 140.4, 140.3, 136.0, 130.2, 125.6, 125.5, 123.6, 123.1, 121.0, 118.8, 111.6 ppm; Vmax (solid) 3285, 3249, 3068, 2230, 1712, 1582, 1537, 1475, 1424, 1326, 1278, 1247, 1199, 785, 739, 705, 679 cm ^"1 ; mlz (ESI pos. mode) 239.0 (MH ⁺ ); (ESI neg. mode) 283.0 (M ³⁵ C1 ^" ), 237.1 (M-H ⁺ ). Elemental analysis calcd for C ₁₃ H ₁₀ N ₄ O: C, 65.54; H, 4.23; N, 23.52. Found: C, 65.47; H, 4.18; N, 23.42.

Preparation of 3-(4-cyanophenyl)-l-(pyridin-2-yl)urea (8)

2-Aminopyridine and 4-cyanophenylisocyanate were combined using General Procedure I on a 3.46 mmol scale to give the title product 8 as a white solid (0.20 g, 24%).

Recrystallization of 160 mg from a minimum of hot ethanol gave a colourless solid (98 mg, 12%); mp 212 - 213 °C (dec); 1H NMR (400 MHz, DMS0 ) δ 10.90 (br. s., 1H), 9.62 (s, 1H), 8.31 (ddd, J= 5, 2, 1 Hz, 1H), 7.76 - 7.81 (m, 3H), 7.73 (td, J= 9, 2 Hz, 2H), 7.54 (d, J= 9 Hz, 1H), 7.06 (ddd, J= 7, 5, 1 Hz, 1H); ¹³ C NMR (101 MHz,

DMSO-i ) 5 152.4, 151.9, 147.0, 143.5, 138.7, 133.3 (2C), 119.2, 118.7 (2C), 118.0, 112.1, 104.0; v _max (solid) 3203, 3060, 2973, 2221, 1702, 1581, 1476, 1414, 1305, 1243, 1155, 840, 770 cm ^"1 ; mlz (ESI pos. mode) 261.0 (MNa ⁺ ), 239.0 (MH ⁺ ); (ESI neg. mode) 273.0 (M ³⁵ C1 ^" ), 237.1 (M-H ⁺ ) ^~ Elemental analysis calcd for C ₁₃ H ₁₀ N ₄ O: C, 65.54; H, 4.23; N, 23.52. Found: C, 65.29; H, 4.13; N, 23.30.

Preparation of l-(2-methylphenyl)-3-(pyridin-2-yl)urea (9)

2-Aminopyridine and o-tolyl isocyanate were combined using General Procedure I on a

3.76 mmol scale to give, after rcrystallization from hot ethanol, the title product 9 as a white solid (0.301 g, 35%); mp 212 - 213 °C; 1H NMR (400 MHz, OMSO-d ₆ ) δ 10.94 (br. s., 1H), 9.78 (s, 1H), 8.29 (ddd, J= 5, 2, 1 Hz, 1H), 8.05 (dd, J= 8, 1 Hz, 1H), 7.77 (ddd, J = 8, 7, 2 Hz, 1H), 7.30 (d, J = 8 Hz, 1H), 7.22 (d, J = 8 Hz, 1H), 7.18 (dt, J= 8, 2 Hz, 1H), 7.02 (ddd, J= 7, 5, 1, Hz, 1H), 6.98 (dt, J= 7, 1 Hz, 1H) , 2.34 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO- ) δ 153.1, 152.3, 146.3, 138.8, 137.3, 130.2, 126.9, 126.2, 122.7, 120.3, 117.2, 111.9, 18.1 ppm; Vmax (solid) 3199, 3057, 2982, 2915, 1686, 1605, 1478, 1458, 1415, 1289, 1240 cm ^"1 ; mlz (ESI pos. mode) 250.0 (MNa ⁺ ), 228.0 (MH ⁺ ); (ESI neg. mode): mlz 226.1 (M-H ⁺ ) ^" . Elemental analysis calcd for C ₁₃ H ₁₃ N ₃ O: C, 68.70; H, 5.77; N, 18.49. Found: C, 68.61; H, 5.78; N, 18.43.

Preparation of l-(2-methylphenyl)-3-(pyridin-3-yl)urea (10)

o-Tolyl isocyanate and 3-aminopyridine were combined using General Procedure I on a 3.76 mmol scale to give, after recrystallization from a minimum of hot ethanol, the title product 10 as white needles (0.278 g, 32%); mp 164 - 165°C; 1H NMR (400 MHz, DMSO-i ) δ 9.18 (s, 1H), 8.62 (d, J= 3 Hz, 1H), 8.20 (dd, J= 5, 2 Hz, 1H), 8.06 (s, 1H), 7.98 (ddd, J= , 8, 3, 2 Hz, 1H), 7.82 (dd, J= 8, 1 Hz, 1H), 7.32 (dd, J= 8, 5 Hz, 1H), 7.14 - 7.22 (m, 2H), 6.98 (dt, J= 7, 1 Hz, 1H), 2.26 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO- de) 6 152.7, 142.7, 139.8, 137.1, 136.6, 130.2, 127.9, 126.2, 124.9, 123.6, 123.0, 121.4, 17.8 ppm; v _max (solid) 3406, 2890, 1700, 1610, 1584, 1556, 1509, 1449, 1424, 1286, 1250, 802, 753, 703 cm ^"1 ; mlz (ESI pos. mode): 250.0 (MNa ⁺ ), 228.1 (MH ⁺ ); (ESI neg. mode): mlz 226.1 (M-H ⁺ ). Elemental analysis calcd for C ₁₃ H ₁₃ N ₃ O: C, 68.70; H, 5.77; N, 18.49. Found: 68.58; H, 5.78; N, 18.37.

Preparation of 3-(3-cyanophenyl)-l-(pyridin-2-yl)urea (11)

2-Aminopyridine and 3-cyanophenyl isocyanate were combined using General Procedure I on a 3.46 mmol scale to give a white solid (220 mg). Recrystallization from hot ethanol, gave the title product 11 as white cotton wool-like crystals (0.140 g, 17%); mp 164 - 165°C; 1H NMR (400 MHz, DMSO-d ₆ ) δ 10.93 (br. s., 1H), 9.63 (s, 1H), 8.31 (ddd, J= 5, 2, 1 Hz, 1H), 8.08 (t, J= 2 Hz, 1H), 7.75 - 7.82 (m, 2H), 7.54 (t, J= 8 Hz, 1H), 7.46 - 7.50 (m, 2H), 7.05 (ddd, J= 7, 5, 1 Hz, 1H) ppm; ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 152.5,

152.2, 146.9, 139.9, 138.7, 130.2, 126.0, 123.5, 121.4, 118.7, 117.8, 112.0, 111.7 ppm;

Vmax (solid) 3207, 3059, 2982, 2228, 1707, 1609, 1582, 1562, 1479, 1417, 1317, 1150, 771,

725 cm ^"1 ; mlz (ESI pos. mode) 487.2 (M ₂ Na ⁺ ). Elemental analysis calcd for C ₁₃ H ₁₀ N ₄ O: C,

65.54; H, 4.23; N, 23.52. Found: C, 65.48; H, 4.21; N, 23.61. Preparation of 3-(3-cyanophenyl)-l-(pyrimidin-2-yl)urea (12)

2-Aminopyrimidine and 3-cyanophenyl isocyanate were combined using General

Procedure I on a 3.47 mmol scale to give a white solid (-100 mg). Recrystallization from hot ethanol (poor solubility, 150 mL), gave the title product 12 as a white powder (0.077 g, 9%); mp 260 - 266 °C; 1H MR (400 MHz, DMSO-d ₆ ) δ 11.80 (br. s., 1H), 10.37 (br. s., 1H), 8.70 (d, J= 5 Hz, 2H), 8.14 (t, J= 2 Hz, 1H), 7.95 (td, J= 8, 2 Hz, 1H), 7.57 (t, J= 8 Hz, 1H), 7.53 (td, J= 8, 2, Hz, 1H), 7.18 (t, J= 5 Hz, 1H) ppm. Insufficient solubility for carbon MR. v _max (solid) 3140, 3065, 2980, 2916, 2223, 1693, 1615, 1582, 1561, 1515, 1407, 1300, 1240, 1225, 785, 725 cm ^"1 ; mlz (ESI neg. mode) 274.0 (M ³⁵ C1 ^" ), 238.1 (M- H ⁺ ) ^" ; HRMS (ESI pos. mode) mlz Ci ₂ H ₉ N ₅ ONa required 262.0699 (MNa ⁺ ). Found mlz 262.0701. Barely soluble in CH ₂ C1 ₂ , insoluble in 80:20 MeOH:H ₂ 0. Elemental analysis calcd for Ci ₂ H ₉ N ₅ 0: C, 60.25; H, 3.79; N, 29.27. Found: C, 60.10; H, 3.670; N, 29.23. Preparation of 3-(3-methoxyphenyl)-l-(2-methylphenyl)urea (13)

o-Toluidine and 3-methoxyphenylisocyanate using General Procedure I on a 3.35 mmol scale to give, after recrystallization from hot ethanol, the title product 13 as white needles (0.327 g, 39%); mp 162 - 162 °C; 1H NMR (300 MHz, DMS0 ) δ 9.03 (s, 1H), 7.91 (s, 1H), 7.84 (d, J= 8 Hz, 1H), 7.10 - 7.22 (m, 4H), 6.91 - 6.98 (m, 2H), 6.55 (dd, J= 8, 2 Hz, 1H), 3.73 (s, 3H), 2.24 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) δ 159.7, 152.5, 141.1, 137.3, 130.1, 129.5, 127.4, 126.1, 122.6, 121.0, 110.2, 107.1, 103.6, 54.8, 17.8 ppm; v _max (solid) 3274, 2923, 2826, 1633, 1590, 1545, 1455, 1293, 1225, 1160, 1055, 759 cm ^"1 ; mlz (ESI pos. mode) 279.0 (MNa ⁺ ), 257.0 (MH ⁺ ); (ESI neg. mode) 291.0 (M ³⁵ C1 ^" ), 255.1 (M- H ⁺ ) ^~ Elemental analysis calcd for Ci ₅ Hi ₆ N ₂ 0 ₂ : C, 70.29; H, 6.29; N, 10.93. Found: 70.12; H, 6.23; N, 10.88.

Preparation of 3-(2-methylphenyl)-l-[4-(methylsulfanyl)phenyl]urea (14)

o-Toluidine and 4-methylthiophenylisocyanate were combined using General Procedure I on a 3.46 mmol scale to give, after recrystallization from hot ethanol, the title product 14 as a white powder (0.315 g, 38%); mp 205 - 206 °C; 1H NMR (250 MHz, DMS0 ) δ

8.98 (s, 1H), 7.86 (s, 1H), 7.80 (d, J = 8 Hz, 1H), 7.38 - 7.45 (m, J = 9 Hz, 2H), 7.18 - 7.25

(m, J= 8 Hz, 2H), 7.08 - 7.18 (m, 2H), 6.93 (dt, J= 7, 1 Hz, 1H), 2.42 (s, 3H), 2.23 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO- ,) δ 152.6, 137.6, 137.3, 130.2, 129.7, 127.8, 127.5,

126.1, 122.6, 121.0, 118.7, 17.9, 16.0 ppm; v _max (solid) 3283, 2977, 2919, 1637, 1581, 1543, 1489, 1282, 1236, 826, 796, 760, 740 cm ^"1 ; mlz (ESI pos. mode) 295.0 (MNa ⁺ ); (ESI neg. mode) 307.0 (M ³⁵ C1) ^" , 271.0 (M-H) ^~ Elemental analysis calcd for Ci ₅ Hi ₆ N ₂ OS: C, 66.15; H, 5.92; N, 10.29. Found: C, 65.96; H, 5.84; N, 10.17. Preparation of l-(4-methoxyphenyl)-3-(2-methylphenyl)urea (15)

o-Toluidine and 4-methoxyphenylisocyanate were combined using General Procedure I on a 3.35 mmol scale to give, after recrystallization from hot ethanol, the title product 15 as a white powder (0.300 g, 35%); mp 200 - 201°C; 1H NMR (400 MHz, DMSO- ) δ 8.83 (s, 1H), 7.86 (d, J= 8 Hz, 1H), 7.82 (s, 1H), 7.35 - 7.40 (m, 2H), 7.11 - 7.19 (m, 2H), 6.94 (dt, J= 8, 1 Hz, 1H), 6.86 - 6.91 (m, J= 9 Hz, 2H), 3.73 (s, 3H), 2.25 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO-i ) δ 154.3, 152.8, 137.6, 132.9, 130.1, 127.2, 126.1, 122.4, 120.8, 119.7, 114.0, 55.1, 17.9 ppm; v _max (solid) 3292, 3042, 2837, 1638, 1598, 1555, 1509, 1455, 1239, 1032, 828, 799 cm ^"1 ; mlz (ESI pos. mode) 279.0 (MNa ⁺ ), 257.0 (MH ⁺ ); (ESI neg. mode) 291.0 (M ³⁵ C1) ^" , 255.1 (M-H ⁺ ) ^~ Elemental analysis calcd for Ci ₅ Hi ₆ N ₂ 0 ₂ : C, 70.29; H, 6.29; N, 10.93. Found: C, 70.02; H, 6.13; N, 10.80.

Preparation of l-(3-bromophenyl)-3-(2-methylphenyl)urea (16).

o-Tolyl isocyanate (666 mg, 5 mmol) and 3-bromoaniline (860 mg, 5 mmol) were combined using General Procedure I to produce the title compound 16 as a white solid (1.011 g, 66%); mp 215-218 °C; 1H NMR (300 MHz, DMSO-i ) δ 9.24 (s, 1H), 8.02 (s, 1H), 7.90 (t, J= 2 Hz, 1H), 7.78 (d, J= 8 Hz, 1H), 7.10 (m, 5H), 6.97 (t, J= 8 Hz, 1H), 2.24 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO) δ 176.63, 147.86, 111.54, 77.57, 70.33, 40.29, 40.01, 39.74, 39.61, 39.46, 39.18, 38.90, 38.62 ppm; v _max (solid) 3280, 1635, 1542 cm ^"1 ; m/z (ESI pos. mode) 383.981 (MHBr ⁺ , 100%), 405.956 (MHNaBr ⁺ , 93%); HRMS (ESI pos. mode) m/z Ci ₄ Hi ₃ BrN ₂ ONa required: 327.0103. Found: 327.0167. Elemental analysis calcd for Ci ₄ Hi ₃ BrN ₂ 0: C 55.10%, H 4.29%, N 9.18%. Found: C 54.88%, H 4.23%, N 9.01%.

Preparation of 3-(2-methylphenyl)-l-(3-nitrophenyl)thiourea (17)

o-Toluidine (0.54 g, 5 mmol) and 3-nitrophenylisothiocyanate (0.90 g, 5 mmol) were combined with General Procedure I to give, after recrystallization from hot methanol, the title product 17 as white crystals (0.41 g, 45 %); mp 171 - 173 °C; 1H NMR (300 MHz, chloroform-ύ δ 8.17 (t, J= 2 Hz, 1H), 8.01 - 7.91 (m, 1H), 7.92 - 7.79 (m, 1H), 7.71 (s, 1H), 7.42 (t, J = 8 Hz, 1H), 7.35 - 7.20 (m, 4H), 7.16 (s, 1H), 2.29 (s, 3H) ppm; ¹³ C (75 MHz, chloroform-ύ 180.5, 147.4, 141.0, 137.3 132.2, 134.9, 130.5, 129.6, 127.9, 126.8, 126.3, 118.5, 117.6, 17.8 ppm; Vmax (solid) 3340, 3149, 2956, 1534, 1497, 1459, 1339, 1283, 1258, 1237, 1208, 1155, 1108, 1089, 987, 945, 853, 805, 751, 741, 709, 687 cm ^"1 ; mlz = 286.0, (M ⁺ ), 310.0 (MNa ⁺ ); HRMS (ESI pos. mode) m/z Ci ₄ Hi ₃ N ₃ Na0 ₂ S (MNa ⁺ ) required 310.0621. Found: 310.0615.

Preparation of l-(3-cyanophenyl)-3-(2-methylphenyl)thiourea (18)

o-Toluidine (0.54 g, 5 mmol) and 3-cyanophenylisothiocyanate (0.90 g, 5 mmol) were combined with General Procedure I to give, after recrystallization from hot ethanol, the title product 18 as pale yellow crystals (0.42 g, 31%); mp 166 - 168 °C; 1H MR (300 MHz, chloroform-ύ δ 7.45 - 7.30 (m, 3H), 7.35 - 7.20 (m, 6H), 7.17 (s, 1H), 2.29 (s, 3H) ppm; ¹³ C (75.5 MHz, DMSO- ) δ 180.5, 140.6, 137.4, 134.9, 130.4, 129.7, 128.4, 127.9, 127.7, 126.8, 126.6, 126.2, 118.6, 111.0 17.8 ppm; v _max (solid) 3217, 3145, 2979, 2240, 1586, 1543, 1519, 1481, 1455, 1429, 1358, 1320, 1297, 1283, 1270 cm ^"1 ; mlz (ESI pos. mode) 268.0, (MH ⁺ ), 290.0 (MHNa ⁺ ); HRMS (ESI pos. mode) m/z Ci ₅ Hi ₃ N ₃ NaS (MNa ⁺ ) required 290.0722. Found: 290.0729.

Preparation of l-(2-methoxyphenyl)-3-(2-methylphenyl)thiourea (19)

o-Toluidine (0.54 g, 5 mmol) and 4-methylthiophenylisothiocyanate (0.91 g, 5 mmol) were combined with General Procedure I to give, after recrystallization from hot ethanol the title product 19 as white crystals (0.52 g, 38%); mp 140 - 142 °C; 1H NMR (300 MHz, chloroform-ύ δ 8.18 (d, J= 8 Hz, 1H), 7.75 (s, 1H), 7.53 (s, 1H), 7.35 - 7.18 (m, 4H), 7.09 (t, J= 8, 2 Hz, 1H), 6.93 (t, J= 8, 1 Hz, 1H), 6.81 (d, 1H), 3.68 (s, 3H), 2.28 (s, 3H); ¹³ C (75.5 MHz - DMSO-i ) δ 180.0, 151.6, 137.5, 134.9, 130.3, 128.2, 127.7, 126.6, 126.1, 125.8, 125.3, 119.8, 111.4, 55.6, 17.7 ppm; (solid) 3336, 3125, 2936, 1598, 1533, 1505, 1484, 1459, 1434, 1276, 1248, 1202, 1179, 1159, 1044, 1026, 745, 725, 699 cm ^"1 ; mlz 273.0 (MH ⁺ ), 295.0 (MHNa ⁺ ); HRMS (ESI pos. mode) m/z Ci ₅ Hi ₆ N ₂ NaOS (MHNa ⁺ ) required 295.0876. Found: 295.0874. Preparation of 3-(2-methylphenyl)-l-[4-(methylsulfanyl)phenyl]thiourea, (20) o-Toluidine (0.54 g, 5 mmol) and 4-methylthiophenylisothiocyanate (0.91 g, 5 mmol) were combined with General Procedure I to give, after recrystallization from hot ethanol, the title product 20 as white crystals (0.93 g, 62%); mp 149 - 151°C; 1H NMR (300 MHz,

DMSO-i ) δ 9.64 (s, 1H), 9.33 (s, 1H), 7.48 - 7.37 (m, 2H), 7.30 - 7.10 (m, 6H), 2.46 (s, 3H), 2.24 (s, 3H); ^li C (75.5 MHz, DMSO- ,) δ 180.3, 164.0, 137.7, 136.7, 134.8, 133.5, 130.3, 127.9, 126.4, 126.4, 126.1, 124.6, 124.4, 17.9, 15.2 ppm; Vmax (solid) 3223, 1580 (w), 1498 (s), 1347, 1255, 1201, 1089, 1016, 853, 790, 751, 718, 627 cm ^"1 ; mlz 31 1.0 (MHNa ⁺ ); HRMS (ESI pos. mode) m/z Ci ₅ Hi ₆ N ₂ NaS ₂ (MHNa ⁺ ) required 31 1.0647.

Found: 31 1.0637.

Preparation of 3-(2-methylcyclohexyl)-l-(3-nitrophenyl)urea 21

2-Methylcyclohexylamine and 3-nitrophenylisocyanate were combined using General Procedure I on a 3.05 mmol scale, to give after recrystallization from hot ethanol, the title product 21 as yellow needles (0.202 g, 24%); mp 173 - 175 °C; 1H NMR (400 MHz,

DMSO-i ) δ 8.85 (s, 1H), 8.53 (t, J = 2 Hz, 1H), 7.74 (dd, J= 2, 8 Hz, 1H), 7.61 (d, J = 9 Hz, 1H), 7.50 (t, 7= 8 Hz, 1H), 6.18 (d, 7 = 9 Hz, 1H), 3.16 (ddt, 7 = 4, 9, 1 1 Hz, 1H), 1.86 (dd, 7= 3, 13 Hz, 1H), 1.72 (dt, 7 = 2, 13 Hz, 2H), 1.62 (d, 7= 12 Hz, 1H), 0.98 - 1.37 (m, 5H), 0.91 (d, 7 = 7 Hz, 3H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) δ 154.4, 148.1, 141.9, 129.8, 123.5, 1 15.3, 1 1 1.3, 53.9, 37.6, 34.0, 33.5, 25.3, 25.2, 19.2 ppm; v _max (solid) 2195, 3072, 2927, 2856, 1655, 1638, 1519, 1346 cm ^"1 ; mlz (ESI pos. mode) 300.0 (MNa ⁺ ); (ESI neg. mode) 312.1 (M ³⁵ C1 ^" ), 276.1 (M-H ⁺ ). Elemental analysis calcd for C14H19N3O3 : C, 60.63; H, 6.91 ; N, 15.15. Found: C, 60.53; H, 6.85; N, 15.04. Preparation of l-(4-cyanophenyl)-3-(2-methylcyclohexyl)urea (22)

4-Cyanophenyl isocyanate (721 mg, 5 mmol) and l-amino-2-methyl cyclohexane (566 mg, 5 mmol) were combined with General Procedure I to produce a white solid. This product was recrystallized from hot ethanol and a small volume of water to give the title compound 11 as a white powder (529 mg, 49%), mp 190-192 °C; 1H NMR (300 MHz, chloroform-i ) δ 7.63 (s, 1H), 7.56 - 7.42 (dd, 7 = 9 Hz, 5 Hz, 4H), 5.24 (d, 7 = 9 Hz, 1H), 3.26 (dq,

7 = 1 1, 4 Hz, 1H), 1.98 (dd J = 14, 3 Hz) 1.76 - 1.61 (m, 4H), 1.16 - 0.96 (m, 4H) 0.94 (d, 7 = 7 Hz, 3H) ppm; ¹³ C NMR (75.5 MHz, chloroform-i ) δ 154.8, 144.0, 133.3, 1 19.4, 1 18.3, 104.5, 58.6, 55.1, 38.7, 34.3, 25.6, 25.4, 19.2 ppm; (solid) 3302, 2924, 2226, 1634, 1558, 1516, 1310, 1228, 1 175, 835 cm ^"1 ; mlz (ESI pos. mode) 258.1 (MH ⁺ , 36%), 280.1 (MNa ⁺ , 100%); HRMS mlz (ESI pos. mode) C15H20N3O required 258.1601. Found 258.1600. Elemental analysis calcd for C15H19N3O: C 70.01%, H 7.44%, N 16.33%. Found C 69.98%, H 7.44%, N 16.20%.

Preparation of l-(3-cyanophenyl)-3-(2-methylcyclohexyl)urea (23) To a stirred solution of 2-methylcyclohexylamine (0.57 g, 5 mmol) in dichloromethane (5 mL) at 0 °C under nitrogen was added a solution of 3-cyanophenyl isocyanate (0.72 g, 5 mmol) in dichloromethane (5 mL) from a pressure equalized dropping funnel. The reaction was stirred overnight and the crude product was isolated by evaporation under reduced pressure. Trituration with ethanol - water and brief contact with dry ice / acetone gave the title product 23 as a white solid (0.38 g, 30%); mp 168 - 174°C; 1H (300 MHz, DMSO-i ) δ 8.65 (s, lH), 7.94 (s, 1H), 7.55 (d, J= 9 Hz, 1H), 7.41 (t, J= 8 Hz, 1H), 7.30 (J= 10 Hz, 1H), 6.18 (d, J= 9 Hz, 1H) 3.14 (dq, J= 10, 4 Hz, 1H), 1.85 (m, 1H), 1.78 - 1.51 (m, 4H), 1.35 - 1.00 (m, 6H), 0.90 (d, J= 6 Hz, 3H) ppm; ¹³ C (75.5 MHz, DMS0 ) δ 154.5, 141.5, 130.0, 124.2, 122.0, 119.9, 119.0, 111.4, 53.8, 37.7, 34.0, 33.5, 25.3, 25.2, 19.2 ppm; v _max (solid) 3395, 3300, 2928, 2850, 1653, 1547, 1481, 1411, 1310, 1232, 1029, 880, 809, 686, 591, 475 cm ^"1 ; mlz (ESI pos. mode) 280.1 (MNa ⁺ ), 258.1 (MH ⁺ ). HRMS (ESI pos. mode) mlz C ₁ 5H ₂ 0N3 ₀ (MH ⁺ ) required 258.1601. Found 258.1602. Preparation of l-(4-cyanophenyl)-3-octylurea (24).

4-Cyanophenyl isocyanate (544 mg, 5 mmol) and octylamine (642 mg, 5 mmol) were combined using General Procedure I to produce a white solid. This product was purified using flash column chromatography (Si0 ₂ , 30:70 EtOAc : petroleum ether 40-60°) to give the title compound 24 as a white powder (887 mg, 76%); mp 115-120 °C; 1H MR (300 MHz, chloroform-ύ δ 7.64 (s, 1H), 7.56 - 7.41 (m, 4H), 5.47 (t, J= 6 Hz, 1H), 3.29 - 3.16 (q, J= 7 Hz, 2H), 1.50 (m, 2H), 1.37 - 1.17 (m, 10H), 0.86 (t, J= 7 Hz, 3H) ppm; ¹³ C NMR (75 MHz, chloroform-i ) δ 164.4, 132.7, 117.9, 42.7, 31.2, 29.4, 28.6, 26.3, 22.2 ppm; Vmax (solid) 3374, 2923, 2219, 1685, 1657, 1592, 1535, 1507, 1320, 1231, 1172, 835 cm ^"1 ; m/z (ESI pos. mode) 272.1 (M-H ⁺ , 100%). Elemental analysis calcd for Ci ₆ H ₂ 3N ₃ 0: C 70.30%, H 8.48%, N 15.37%. Found: C 70.39%, H 8.46%, N 15.63%.

Preparation of l-(2-methylphenyl)-3-octylurea, (25)

To a stirred solution of octylamine (0.65 g, 5 mmol) in dichloromethane (5 mL) at 0 °C under dry nitrogen was added a solution of 2-methylphenyl isocyanate (0.67 g, 5 mmol) in dichloromethane (5 mL) from a pressure equalized dropping funnel. The reaction was stirred overnight and the white solid was collected by filtration under reduced pressure on a sintered glass funnel. Recrystallization from hot ethanol, with addition of a small quantity of water, gave the title product 25 as a white solid (0.46 g, 36%); mp 113 °C; 1H (300 MHz, chloroform-ύ δ 7.35 (1H, d, J= 8 Hz), 7.20-7.05 (4H, m), 5.85 (1H, br. s.), 4.50 (1H, br with app. t structure), 3.20 (2H, dd, J= 10, 7 Hz), 2.25 (3H, s), 1.60 (2H, s, bound H ₂ 0), 1.48 (2H, m), 1.30 - 1.20 (10H, m), 0.85 (3H, t, J= 7 Hz) ppm; ¹³ C (75.5 MHz, chloroform-ύ δ 164.6, 155.9, 135.5, 130.4, 126.5, 125.4, 125.0, 39.8, 31.2, 29.6, 28.7, 28.6, 26.2, 22.0, 17.3, 13.5 ppm; v _max (solid) 3336, 3300, 2960, 2926, 2853, 1585, 1552, 1481, 1458, 1303, 1290, 1260, 1236, 1213, 1190, 1106, 1082, 1043, 758, 734 cm ^"1 ; mlz (ESI neg. mode) 261 (M-H ⁺ ). HRMS (ESI pos. mode) mlz Ci ₆ H ₂ 6N ₂ NaO required

285.1937, found 285.1932.

Preparation of 3-[2-(dimethylamino)ethyl]-l-(2-methylphenyl)urea (26)

To a stirred solution of N,N-dimethylethylamine (0.44 g, 5 mmol) in dichloromethane (5 mL) at 0 °C under dry nitrogen was added a solution of 2-methylphenyl isocyanate (0.67 g, 5 mmol) in dichloromethane (5 mL) dropwise from a pressure equalized dropping funnel. The reaction was stirred overnight then evaporated under reduced pressure. The solid product was recrystallized from warm diethyl ether with a few drops of dichloromethane, which gave after 4 days, the title product 26 as a white solid (0.72 g, 65%); mp 120 -

123°C; 1H (300 MHz, chloroform-i ) δ 7.50 (dd, J= 8, 1 Hz, 1H), 7.20 - 7.10 (m, 3H) 6.95 (dd, J= 7, 6 Hz, 1H), 5.65 - 5.58 (1H, m), 3.30 (2H, q, J= 5.5 Hz), 2.40 (t, J= 5.8 Hz, 2H), 2.24 (s, 3H), 2.17 (s, 6H) ppm; ¹³ C (75.5 MHz, chloroform-i ) δ 156.4, 136.2, 130.6, 130.1, 126.2, 124.3, 123.9, 58.4, 44.6, 37.4, 17.3 ppm; v _max (solid) 3300, 2900, 2760, 1630, 1562, 1458, 1292, 1243, 1096, 1057, 850, 754, 665 cm ^"1 ; mlz (ESI pos. mode) 222 (MH ⁺ ). HRMS (ESI pos. mode) mlz Ci ₂ H ₂₀ N ₃ O (MH ⁺ ) required 222.1601. Found 222.1605.

Preparation of N-(2-methylphenyl)morpholine-4-carboxamide (27)

To a stirred solution of morpholine (0.45 g, 5 mmol) in dichloromethane (5 mL) at 0 °C under nitrogen, was added o-tolyl isocyanate (0.67 g, 5 mmol) in a solution of

dichloromethane (5 mL) dropwise from a pressure equalized dropping funnel. The reaction was stirred overnight and the white precipitate collected by filtration under reduced pressure (0.38 g, 38%), estimated >95% pure by MR and thin layer

chromatography. This solid was recrystallized with some difficulty from ethanol - water to give the title product 27 as colourless crystals (0.06 g, 5%); mp 160 - 163°C; 1H (300 MHz, chloroform-ύ δ 7.60 (d, J 8.1 = Hz, 1H), 7.20 - 7.10 (m, 2H), 7.00 (1H, dd, J= 8 Hz), 6.10 (br. S, 1H), 3.72 (dd, J= 5, 4 Hz, 4H), 3.48 (4H, dd, J= 5, 4 Hz), 2.22 (s, 3H) ppm; ¹³ C (75.5 MHz, chloroform-i ) δ 155.5, 136.7, 130.4, 129.2, 126.8, 124.4, 123.1, 66.5, 44.4, 17.8 ppm; v _max (solid) 3286, 3000, 2850, 1630, 1510, 1456, 1379, 1300, 1255, 1112, 996, 888, 744, 714, 565 cm ^"1 ; mlz (ESI pos. mode) 243. 1, (MNa ⁺ ), 463.2 (M ₂ Na ⁺ ); HRMS (ESI pos. mode) mlz Ci ₂ Hi ₆ N ₂ Na0 ₂ (MNa ⁺ ) required 243.1104. Found 243.1108.

Preparation of l-(2-methylphenyl)-3-octylthiourea (28)

General Procedure I was used with o-tolyl isothiocyanate (0.75 g, 5 mmol) and octylamine (0.65 g, 5 mmol) to give, after purification by flash column chromatography (Si0 ₂ , 15 :85 EtOAc : 40-60° petroleum ether) the title product 28 as a colourless oil (0.12 g, 9 %); mp 95 - 97 °C; ¹ H MR (300 MHz, chloroform-i ) δ 7.36 - 7.12 (m, 5H), 5.59 (s, 1H), 3.57 (dd, J= 8, 6 Hz, 2H), 2.26 (s, 3H), 1.32 - 1.18 (m, 10H), 0.85 (t, J = 7 Hz, 3H) ppm; ¹³ C (75.5 MHz, chloroform-ύ δ 180.3, 135.3, 131.2, 128.0, 127.0, 126.9, 45.0, 28.5, 31.1,

28.6, 28.5 26.2, 22.0, 17.2, 13.5 ppm; v _max (oil) 3157, 2953, 2925, 2849, 1536, 1504, 1456, 1282, 1249, 1214, 1175, 1152, 1115, 1086, 1036, 740, 707 cm ^"1 , mlz (ESI pos. mode) 279.1 (MH ⁺ ), 301.1 (MHNa ⁺ ); HRMS (ESI pos. mode) mlz Ci ₆ H ₂₆ N ₂ NaS (MNaH ⁺ ) required 301.1709. Found 301.1695.

Preparation of 3-[2-(dimethylamino)ethyl]-l-(2-methylphenyl)thiourea (29)

General Procedure I was used with o-tolyl isothiocyanate (0.75 g, 5 mmol) and NJT- dimethylenediamine (0.44 g, 5 mmol) to give, after purification by flash column chromatography (Si0 ₂ , 15:85 EtOAc : 40-60° petroleum ether) the title product 29 as a colourless oil (0.80 g, 67 %); 1H MR (300 MHz, chloroform-i ) δ 7.75 (s, 1H), 7.29 - 7.12 (m, 4H), 6.57 (s, 1H), 3.64 - 3.55 (m, 2H), 2.38 (t, J= 6 Hz, 2H), 2.25 (s, 3H), 2.04 (s, 6H) ppm; ¹³ C NMR (75.5 MHz, chloroform-i ) δ 181.3, 134.5, 130.6, 129.7, 127.2, 122.9, 120.1, 43.6, 40.3, 26.5, 17.4 ppm; mlz (ESI pos mode) 238.1 (M ⁺ H). HRMS (ESI pos. mode) mlz Ci ₂ H ₂₀ N ₃ S (MH ⁺ ) required 238.1372, found 238.1362.

Preparation of N-(2-methylphenyl)morpholine-4-carbothioamide, (30)

General Procedure I was used with o-tolyl isothiocyanate (0.75 g, 5 mmol) and morpholine

(0.44 g, 5 mmol) to give, after recrystallization from hot ethanol the title product 30 as white crystals (1.08 g, 92 %); mp 145 - 147 °C; 1H NMR (300 MHz, chloroform-i ) δ 7.26 - 7.03 (m, 4H), 6.95 (s, 1H), 3.81 - 3.64 (m, 8H), 2.24 (s, 3H) ppm; ¹³ C (75.5 MHz, chloroform-i δ 183.7, 138.0 131.6, 130.5, 126.3, 125.8, 124.2, 65.5, 49.0, 17.4 ppm;

Vmax (oil) 3159, 2854, 1518, 1492, 1461, 1401, 1318, 1295, 1281, 1264, 1218, 1204, 1114,

1064, 1027, 942, 910, 864, 751, 719, 693 cm ^"1 ; mlz 237.0 (MH ⁺ ), 259.0 (MNaH ⁺ ). HRMS

(ESI pos. mode) mlz Ci ₂ Hi ₆ N ₂ NaOS (MNaH ⁺ ) required 259.0876. Found 259.0863. General Procedure II - addition of isocyanate to bifunctional amines.

Aromatic bis-ureas and -thioureas were prepared using a previously reported method (Radi, M.; Falchi, F.; Garbelli, A.; Samuele, A.; Bernardo, V.; Paolucci, S.; Baldanti, F.; Schenone, S.; Manetti, F.; Maga, G.; Botta, M., Discovery of the first small molecule inhibitor of human DDX3X specifically designed to target the RNA binding site: Towards the next generation HIV-1 inhibitors. Bioorg. Med. Chem. Lett. 2012, 22 (5), 2094-2098). To a solution of the amine (1 equiv., 5 mmol) in dry dichloromethane (5 mL) under dry N ₂ was added the isocyanate (2 equiv., 10 mmol) in dry dichloromethane (-10 mL) with stirring, and left overnight at room temperature. The precipitate was collected by filtration and washed with hot ethanol on a fluted filter paper. The resulting solid was insoluble in most solvents except dimethylformamide and dimethylsulfoxide.

Preparation of l,l'-(l ₉ 3-phenylene)bis(3-(3-nitrophenyl)urea) (31).

Compound 31 was prepared using General Procedure II with w-phenylenediamine

(540 mg, 5 mmol) and nitrophenyl isocyanate (1.64 g, 10 mmol) to produce the title compound 31 as a white-yellow solid (1.948 g, 85%); mp 289 ± 1 °C; 1H NMR (300 MHz, DMSO-i ) δ 9.15 (s, 2H), 8.93 (s, 2H), 8.60 (t, J= 2 Hz, 2H), 7.88 - 7.75 (m, 3H), 7.57 (t, J = 8 Hz, 2H) 7.20 (dd, J = 9, 7 Hz 1H), 7.10 (dd, J= 7, 2 Hz, 2H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) 5 152.3, 148.1, 141.0, 139.7, 130.0, 129.1, 124.3, 116.2, 112.4, 112.1, 108.5 ppm; v _max (solid) 3291, 1651, 1595, 1551 cm ^"1 ; m/z (ESI pos. mode) 459.124 (MNa ⁺ , 41%), 461.140 (MNaH ₂ ⁺ , 100%), 463.159 (MNaH ₄ ⁺ , 25%), 465.227 (MNaH ₆ ⁺ , 13%). HRMS (ESI pos. mode) mlz C ₂₀ Hi ₆ N ₆ NaO ₆ required 459.1024. Found 459.0812.

Elemental analysis calcd for C ₂₀ Hi ₆ N ₆ O6: C 55.05%, H 3.70%, N 19.26%. Found: C 54.31%, H 3.67%, N 19.13%.

Preparation of l,l'-(l ₉ 3-phenylene)bis(3-(3-nitrophenyl)thiourea), (32)

General Procedure II was used with w-phenylenediamine (0.38 g, 3.5 mmol) and 3- nitrophenylisothiocyanate (1.27 g, 5 mmol) to give after recrystallisation from hot ethanol the title product as yellow crystals (0.97 g, 60%); mp 190 - 193 °C; 1H NMR (300 MHz,

DMSO-i ) δ 10.29 - 10.22 (m, 2H), 10.08 (s, 2H), 8.45 (t, J= 2 Hz, 2H), 7.97 - 7.80 (m,

4H), 7.68 (t, J= 2 Hz, 1H), 7.54 (t, J= 8 Hz, 2H), 7.37 (dd, J= 9, 7 Hz, 1H), 7.31 - 7.21

(m, 2H) ppm; ¹³ C (75.5 MHz, DMSO- ,) δ 179.5, 164.0, 147.3, 140.8, 139.1, 129.8, 129.4,

129.0, 120.0, 118.6, 117.8, ppm; v _max (oil) 3016, 2364, 1523, 1455, 1438, 1347, 1319, 1281, 1234, 1091, 1000, 970, 935, 878, 862, 831, 787, 742, 730, 682, 665 cm ^"1 . HRMS (ESI pos. mode) mlz C ₂ oHi ₆ N6Na0 ₄ S2 required 491.0567. Found 491.0569.

Preparation of l,l'-(l ₉ 3-phenylene)bis(3-(3-(methylthio)phenyl)urea) (33).

Compound 33 was prepared using general procedure II with w-phenylenediamine (600 mg, 5 mmol) and 3-(methylthio)phenyl isocyanate (1.76 g, 10 mmol) to give the title compound 33 as a white powder (1.54 g, 65%); mp 305 ± 1 °C; 1H NMR (300 MHz, DMSO-^ ₆ ) δ 8.76 (br. s, 1H), 8.66 (br. s, 1H), 7.67 (d, J= 2 Hz, 1H), 7.49 (t, J= 2 Hz, 1H), 7.20 - 7.01 (m, 5H), 6.86 (d, J = 8 Hz, 1H), 5.76 (s, 4H), 2.46 (s, 6H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) δ 164.0, 152.3, 140.2, 138.6, 129.2, 129.1, 119.1, 115.0, 114.6, 111.8, 107.9, 54.9 ppm; v _max (solid) 3276, 1633, 1553, 1479, 1395, 1281, 1222, 681, 647 cm ^"1 ; m/z (ESI pos. mode) 439.180 (MH ⁺ , 12%), 461.159 (MNa ⁺ , 100%); HRMS (ESI pos. mode) mlz C ₂₂ H ₂₂ N ₄ Na0 ₂ S2 required 461.1076. Found 461.1232. Elemental analysis calcd for C22H22N4O2S2: C 60.25%, H 5.06%, N 12.78%. Found: C 60.10%, H 5.02%, N 12.77%.

Preparation of l,l'-(l ₉ 3-phenylene)bis(3-(3-methoxyphenyl)urea) (34).

Compound 34 was prepared using general procedure II using w-phenylenediamine

(541 mg, 5 mmol) and 3-methoxyphenylisocyanate (1.49 g, 10 mmol) to produce the title compound M as a white solid (440 mg, 22%); mp 294-297 °C; 1H NMR (300 MHz, DMSO-i ) δ 8.61 (s, 2H), 8.41 (s, 2H), 7.62 (t, J= 2 Hz, 1H), 7.35 (d, J= 7 Hz, 4H), 7.12 (m, 1H), 7.00 (d, J= 7 Hz, 1H), 6.86 (d, J = 7 Hz, 4H), 3.71 (s, 6H) ppm; ¹³ C NMR (75 MHz, DMSO-i¾) 5 163.8 154.4, 129.0, 119.9, 113.9, 111.4, 55.1 ppm; (solid) 3305, 1639, 1599, 1562, 1512, 1493, 1300, 1242, 1035, 879, 648 cm ^"1 ; m/z (ESI pos. mode) 429.214 (MNa ⁺ , 100%); HRMS (ESI pos. mode) mlz C ₂₂ H ₂₂ N ₄ Na0 ₄ required 429.1533. Found 429.1669. Elemental analysis calcd for C ₂₂ H ₂₂ N ₄ 0 ₄ : C 65.01%, H 5.46%, N

14.78%. Found: C 64.71%, H 5.56%, N 14.78%.

Preparation of l-(2-nitrophenyl)-3-(3-{2-[2-(3-{[(2-nitrophenyl)carbamoyl]a mino}- propoxy)ethoxy] ethoxy} propyl)ur ea (35)

To a stirred solution of 4,7, 10-trioxa-l, 13-tridecanediamine (0.82 g, 10 mmol) in dichloromethane (10 mL) at 0 °C under nitrogen was added a solution of 3-nitrophenyl isocyanate (3.28 g, 20 mmol) in dichloromethane (10 mL) from a pressure equalized dropping funnel. The reaction was stirred overnight and the crude product was isolated a by removal of volatiles on a rotary evaporator in an efficient fumehood. Purification by flash column chromatography (Si0 ₂ , 1% MeOH - EtOAC gave the title product 35 as a white solid (3.703 g, 68%) mp 99 - 101 °C; 1H (300 MHz, DMSO-i ) δ 9.00 (s, 1H), 8.50 (t, J= 2 Hz, 1H), 7.72 (dd, J= 7, 1 Hz, 1H), 7.52 (dd, J 8, 1 = Hz, 1H), 7.49 (t, J= 8 Hz, 1H), 6.32 (t, J = 6 Hz, 1H), 3.60 - 3.50 (m, 4H), 3.48 (m, 4H), 3.15 (q, J = 7 Hz, 2H), 1.70 (q, J= 7 Hz, 2H) ppm; ¹³ C (75.5 MHz, DMSO-i ) δ 154.9, 148.1, 141.9, 129.8, 123.6, 123.6, 115.3, 111.4, 69.7, 69.5, 68.1, 36.6, 29.8 ppm; Vmax (solid) 3326, 3300, 2950, 2900, 1634, 1553, 1481, 1347, 1259, 1102, 968, 874, 832, 801, 728, 690, 640 cm ^"1 ; mlz (ESI pos. mode) 571.1 (MNa ⁺ ); ESI (neg. mode) 547.1 (M-H) ^" , 583.1 (M ³⁵ C1) ^" , 585.1 (M ³⁷ C1) ^~ . HRMS (ESI pos. mode) mlz C24H33N6O9 (MNa ⁺ ) required 549.2304, found 549.2309.

Preparation of l-[3-(methylsulfanyl)phenyl]-3-[3-(2-{2-[3-({[3-(methylsulfa nyl)- phenyl] carbamoyl}amino)propoxy] ethoxy}ethoxy)propyl] urea, (36)

To a solution of 4,7, 10-trioxa-l, 13-tridecanediamine (1.13 g, 5.1 mmol) in

dichloromethane (10 mL) at 0 °C was added dropwise 3-(methylthio)phenyl isocyanate (1.67 g, 10.1 mmol) in dichloromethane (10 mL). The solution was allowed to warm to room temperature and stirred for 3 hrs, then evaporated under reduced pressure. The reaction mixture was purified by flash column chromatography (Si0 ₂ , 5:95 MeOH:EtOAc) to give the title product 36 as a white solid (1.91 g, 3.47 mmol, 68.7%); mp 106-108 °C; 1H MR (300 MHz, CDC1 ₃ ) δ 7.40 (t, J= 2 Hz, 2H), 7.33 (s, 2H), 7.15 (t, J= 8 Hz, 2H), 7.05 (dd, J= 7, 2 Hz, 2H), 6.84 (dd, J= 8, 1 Hz, 2H), 5.62 (t, J= 6 Hz, 2H), 3.70-3.65 (m, 4H), 3.63-3.58 (m, 4H), 3.57 (t, J= 6 Hz, 4H), 3.30 (quartet, J= 6 Hz, 4H), 2.42 (s, 6H), 1.73 (quintet, J= 6 Hz, 4H); ¹³ C NMR (101 MHz, CDC1 ₃ ) δ 156.1, 140.2, 139.3, 129.2, 120.4, 116.7, 115.7, 70.3, 69.9, 69.3, 38.4, 28.9, 15.6 ppm; v _max (solid) 3303, 2865, 1628, 1562, 1481, 1117, 613 cm ^"1 ; mlz (ESI pos. mode) 573.1 (MNa ⁺ ); Elemental analysis calcd for C ₂₆ H ₃₈ N ₄ 0 ₅ S ₂ : C 56.70% H 6.69% N 10.17%. Found: C 56.70% H 6.70% N 10.20%.

Preparation of l-[4-(methylsulfanyl)phenyl]-3-[3-(2-{2-[3-({[4-(methylsulfa nyl)- phenyl] carbamoyl} amino)propoxy] ethoxy}ethoxy)propyl] urea, (37)

To a solution of 4,7, 10-trioxa-l, 13-tridecanediamine (1.23 g, 5.58 mmol) in

dichloromethane (10 mL) at 0 °C was added dropwise a solution of 4-(methylthio)phenyl isocyanate (1.68 g, 10.2 mmol) in dichloromethane (10 mL). The solution was allowed to warm to room temperature and stirred for 24 hrs, then evaporated under reduced pressure.

The reaction mixture was purified by flash column chromatography (Si0 ₂ , 10:90

MeOH:EtOAc), followed by recrystallization from hot ethanol to give the title product 37 as a white solid (0.464 g, 0.84 mmol, 16.5%); mp 137-139 °C; 1H MR (400 MHz, CDC1 ₃ ) δ 7.32 (d, J= 9 Hz, 4H), 7.22 - 7.18 (br. s, 2H), 7.20 (d, J= 9 Hz, 4H), 5.55 (s, 2H), 3.73 - 3.68 (m, 4H), 3.65 - 3.62 (m, 4H), 3.60 (t, J= 6 Hz, 4H), 3.35 (quartet, J= 6 Hz, 4H), 2.44 (s, 6H), 1.76 (quintet, J= 6 Hz, 4H) ppm; ¹³ C MR (101 MHz, CDCI ₃ ) δ 156.1, 137.4, 131.2, 128.7, 120.0, 70.3, 69.9, 69.2, 38.5, 28.9, 17.2 ppm; v _max (solid) 3299, 2868, 1634, 1594, 1558, 1136, 817, 506 cm ^"1 ; mlz (ESI pos. mode) 573.1 (MNa ⁺ ). Elemental analysis calcd for C ₂₆ H ₃₈ N ₄ O ₅ S ₂ C 56.70% H 6.96% N 10.17%. Found C 56.30% H 6.92% N 10.13%. Preparation of l-(2-methylphenyl)-3-(3-{2-[2-(3-{[(2-methylphenyl)carbamoyl ]- amino}propoxy)ethoxy]ethoxy}propyl)urea, (38)

To a solution of 4,7, 10-trioxa-l, 13-tridecanediamine (1.12 g, 5.08 mmol) in

dichloromethane (10 mL) at 0 °C, was added dropwise a solution of o-tolyl isocyanate (1.45 g, 10.9 mmol) in dichloromethane (10 mL). The solution was allowed to warm to room temperature and stirred for 24 hrs, then evaporated under reduced pressure. The reaction mixture was then purified by flash column chromatography (S1O ₂ , 10:90

MeOH:EtOAc) to give the title product 38 as a white solid (1.83 g, 3.76 mmol, 74.0%); mp 78-82 °C; 1H NMR (400 MHz, CDC1 ₃ ) δ 7.53 (d, J= 8 Hz, 2H), 7.16 (t, J= 7 Hz, 4H), 7.02 (t, J= 6 Hz, 2H), 6.40 (s, NH, 2H), 5.41 (t, J= 6 Hz, NH, 2H), 3.60-3.51 (m, 12H), 3.32 (quartet, J = 6 Hz, 4H), 2.24 (s, 6H), 1.73 (quintet, J = 6 Hz, 4H) ppm; ¹³ C NMR

(101 MHz, CDCI ₃ ) δ 156.7, 136.9, 130.6, 126.7, 124.5, 124.0, 70.4, 69.7, 69.5, 38.1, 29.5, 17.9 ppm; v _max (solid) 3306.5, 1629.4, 1561.9, 1243.0, 1104.4, 753.3, 652.2 cm ^"1 , mlz (ESI pos mode) 509.2 (MNa ⁺ ). Elemental analysis calcd for C ₂₆ H ₃₈ N ₄ O ₅ C 64.16% H 7.88% N 11.52%. Found C 64.03% H 7.88% N 11.48%.

Preparation of l-(3-chlorophenyl)-3-(o-tolyl)urea, (39)

o-Toluidine and 3-chlorophenylisocyanate were combined using General Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 39 (1.02 g, 78%); mp 201-202 °C; 1H NMR (300 MHz, DMS0 ) δ 9.21(s, 1H), 7.99 (s, 1H), 7.80 (d, J=8 Hz, 1H), 7.76-7.72 (m, 1H), 7.34-7.09 (m, 4H), 7.04-6.92 (m, 2H), 2.24 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.0, 152.5, 141.4, 137.0, 133.2, 130.4, 130.2, 127.9, 126.1, 123.0, 121.3, 117.3, 116.4, 17.8 ppm; v _max (solid) 3287, 1633, 1585, 1546, 1480, 1457, 1269, 1259, 1235, 760 cm ^"1 ; mlz Ci ₄ Hi ₃ ClN ₂ 0 (ESI pos. mode) Calcd for [MNa] ⁺ = 283.06 Found: 283.0. Preparation of l-(o-tolyl)-3-(3-(trifluoromethyl)phenyl)urea, (40)

o-Toluidine and 3-trifluoromethylphenylisocyanate were combined using General

Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 40 (1.27 g, 86%); mp 185 °C; 1H MR (300 MHz, DMSO): δ 9.37(s, 1H), 8.03 (s, 2H), 7.78 (d, J=8 Hz, 1H), 7.60-7.48 (m, 2H), 7.31 (d, J=7 Hz, 1H), 7.22-7.08 (m, 2H), 6.97 (t, J=8 Hz, 1H), 2.25 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.0, 152.6, 140.7, 136.9, 130.2, 129.9, 129.7, 129.3, 128.1, 126.1, 123.1, 121.5, 117.9, 113.8, 17.8 ppm; Vmax (solid) 3298, 1637, 1560, 1331, 1155, 1124, 1067 cm ^"1 ; mlz C ₁₅ H ₁₃ F ₃ N ₂ O (ESI pos. mode) Calcd for [MNa] ⁺ = 317.09 Found: 317.1.

Preparation of l-(4-bromophenyl)-3-(i>-tolyl)urea (41)

o-Toluidine and 3-bromophenylisocyanate were combined using General Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 41 (1.19 g, 78%); mp 240 °C; 1H NMR (300 MHz, DMSO): δ 9.14(s, 1H), 7.95 (s, 1H), 7.81 (d, J=8 Hz, 1H), 7.44 (s, 4H), 7.21-7.09 (m, 2H), 6.95 (t, J=8 Hz, 1H), 2.23 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.1, 152.5, 139.3, 137.1, 131.5, 130.2, 127.7, 126.1, 122.9, 121.2, 119.9, 113.0, 17.8 ppm; v _max (solid) 3288, 1637, 1584, 1546, 1478, 1457, 1238, 1071, 1009, 835, 796 cm ^"1 ; mlz Ci ₄ Hi ₃ BrN ₂ 0 (ESI pos. mode) Calcd for [MNa] ⁺ = 329.01 Found: 329.0.

Preparation of l-(o-tolyl)-3-(4-((trifluoromethyl)thio)phenyl)urea (42)

o-Toluidine and 4-thiotrifluoromethylphenylisocyanate were combined using General Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 42 (1.24 g, 76%) mp 222-224 °C; 1H NMR (300 MHz, DMSO): δ 9.39(s, 1H), 8.05 (s,

1H), 7.81 (d, J=8 Hz, 1H), 7.62 (s, 4H), 7.21-7.11 (m, 2H), 6.97 (m, 1H), 2.24 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.0, 152.3, 143.1, 137.5, 136.9, 131.7, 130.2, 128.0, 126.2, 123.1, 121.4, 119.1, 118.7, 113.7, 17.8 ppm; v _max (solid) 3349, 3251, 3180, 3115, 3069, 1655, 1587, 1540, 1489, 1452, 1148, 1107, 1086 cm ^"1 ; mlz Ci ₅ Hi ₃ F ₃ N ₂ OS (ESI pos. mode) Calcd for [MNa] ⁺ = 349.06 Found: 349.1.

Preparation of l-(o-tolyl)-3-(4-(trifluoromethoxy)phenyl)urea (43)

o-Toluidine and 4-trifluoromethoxyphenylisocyanate were combined using General

Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 43 (1.10 g, 71%); mp 190-191 °C; 1H NMR (300 MHz, DMSO): δ 9.21(s, 1H), 7.97 (s, 1H), 7.81 (d, J=8 Hz, 1H), 7.59-7.50 (m, 2H), 7.30 (d, J=9 Hz, 2H), 7.20-7.10 (m, 2H), 7.00-6.91 (m, lH), 2.24 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.1, 164.1, 154.41,

152.6, 139.1, 137.1, 130.2, 128.7, 127.8, 126.2, 122.9, 121.8, 121.2, 119.1, 17.8 ppm; Vmax (solid): 3279, 1640, 1551, 1263, 1202, 1152 cm ^"1 ; mlz C ₁₅ H ₁₃ F ₃ N ₂ O ₂ (ESI pos. mode)

Calcd for [MNa] ⁺ = 333.08 Found: 333.1.

Preparation of l-(4-iodophenyl)-3-(o-tolyl)urea (44)

o-Toluidine and 4-iodophenylisocyanate were combined using General Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 44 (1.43 g,

81%); mp 240-242 °C; 1H NMR (300 MHz, DMSO): δ 9.12(s, 1H), 7.94 (s, 1H), 7.81 (d, J=8 Hz, 1H), 7.63-7.56 (m, 2H), 7.35-7.28 (m, 2H), 7.21-7.09 (m, 2H), 6.99-6.91 (m, 1H), 2.23 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.3, 152.4, 139.7, 137.3, 137.1, 130.2,

127.7, 126.1, 122.8, 121.1, 120.2, 17.8 ppm; (solid) 3275, 1637, 1583, 1548, 1280, 1239, 1006, 833 cm ^"1 ; mlz Ci ₄ Hi ₃ IN ₂ 0 (ESI pos. mode) Calcd for [MNa] ⁺ = 375.0 Found: 375.0

Preparation of l-(3,4-dimethoxyphenyl)-3-(o-tolyl)urea (45).

o-Toluidine and 4-iodophenylisocyanate were combined using General Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 45 (1.24 g, 87%); mp 192-194 °C; 1H NMR (300 MHz, DMSO): δ 8.87 (s, 1H), 7.87-7.79 (m, 2H), 7.21-7.08 (m, 3H), 6.97-6.83 (m, 3H), 3.76 (s, 3H), 3.72 (s, 3H), 2.23 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.3, 152.7, 148.8, 143.8, 137.5, 133.5, 130.1, 127.2, 126.1, 122.4, 120.8, 112.5, 109.7, 103.5, 55.8, 17.8 ppm; 3296, 1631, 1549, 1512, 1269, 1230, 1137, 1025 cm ^"1 ; MS: mlz Ci ₆ Hi ₈ IN ₂ 0 ₃ (ESI pos. mode) Calcd for [MNa] ⁺ = 309.12 Found: 309.1.

Example: Plaque inhibition assay

Viruses

An identical assay procedure was carried out for hRSV (strain A2) bovine RS

(strain Snook) and PVM (strain 15). Virus stocks were diluted 10 fold in GMEM

(Invitrogen) so that the wells containing cells received sufficient virus to generate approximately 2000, 200 and 20 plaques per well. Growth medium

GMEM plus 10% FCS (Biosera), penicillin (4 units/ml) and streptomycin (4 μ§/ιη1) (Invitrogen), 3 mM glutamine (Invitrogen).

Overlay medium

GMEM containing 2% (v/v) FCS, 1% (w/v) carboxymethylellulose (CMC) (Sigma), penicillin (4 units/ml), streptomycin (4 μg/ml), 3 mM glutamine.

The overlay medium also contained the compound to be tested at appropriate concentrations. Typically the 6 concentrations of the test compounds used were: 100 μΜ, 31.6 μΜ, 10 μΜ, 3.16 μΜ, 1 μΜ and 0.316 μΜ.

Test compounds

Stocks of the test compounds were prepared in DMSO (Sigma) (carrier). Each stock compound was diluted 1 in 200 in overlay medium to give dilutions stated above.

Plaque Assay

Cells of a standard cell line known to support pneumovirus replication (typically HEp2 or BSC1 cells) were grown in standard 12 well plates at 37 °C in an atmosphere of 5% (v/v) C0 ₂ until confluent. Medium was aspirated from the cells and replaced with 200 μΐ aliquots of virus. Aliquots were prepared by diluting virus stocks 10-fold in GMEM (Invitrogen) as described above with no FCS. A mock (no virus) was also set up to test cell viability. The virus was incubated with the cells for 1 hour at 33 °C with gentle rocking every 5-10 minutes. The inocula were removed and 2 ml of overlay medium was added. Overlay medium was either test (plus compound) or control (medium alone or medium containing DMSO). The plates were incubated at 33 °C for 4-8 days to allow plaques to develop as indicated. A volume of 1 ml of additional overlay medium

(containing the test compound as appropriate) was added every 3 days. All virus dilutions were carried out with each concentration of test compound, or treated as controls, and each was carried out in triplicate.

Following incubation, cells were fixed by addition of an equal volume of 4% (v/v) glutaraldehyde (Sigma) in PBS with further incubation for a minimum of 2 hours. The fixing solution was washed off under a slow flowing stream of water and 1 ml of crystal violet (0.075% w/v: Sigma) was added and left for 20 minutes at room temperature. The crystal violet was removed and the fixed cell monolayers washed. Plates were air dried. Plaques counts were recorded from wells with >20 and <200 in number. These numbers provide the most robust statistical reliability for viable plaque counts.

This experimental approach screens the ability of compounds to block the formation of plaques. A lower number of plaques than the DMSO carrier control shows that the compound is inhibiting virus growth.

96 well plate TCID ₅₀

This method provides a rapid approach to initially screen compounds and was developed as an increased number of compounds were synthesised. Cells of a standard cell line known to support pneumovirus replication (typically HEp2 or BSC1 cells) were grown to 95% confluence in a 96 well plate. Virus was then added over 5 different half log dilutions starting at a multiplicity of infection of 0.02 plus mock control. Three dilutions of compound plus a DMSO control were screened per plate. Cells were fixed once the highest dilution of virus showed signification destruction or cell death in the DMSO control. Identical plate repeats for each compound dilution were fixed in subsequent days as indicated.

Stained plates were read at 595 nM, higher optical density (OD) readings indicate more viable cells remain. Therefore this application not only shows if virus growth is blocked but if the compound is detrimental to cells (reading the mock control).

Compound 1

Figure 1 shows two biological replicates carried out on different occasions for compound 1 using HEp2 cells.

We observed that plaques that were present in the treated wells at concentrations of compound 1 above 1 μΜ were smaller than in the DMSO control. The level of inhibition seen between the repeats clearly differs between the repeats. This may be because the plaques that counted are only just developing, making biological variables more pronounced. Both assays were stopped after 5 days but a small difference in host cell viability can delay viral growth and give the observed results. Compound 1 is relatively insoluble and a concentration between 5-10 μΜ represents an upper limit, consistent with no increase in activity seen at > 10 μΜ in the TCID50 assay. Note the required quantity of 1 gave the appearance of insolublity in cell medium at 100 μΜ and a virus inhibition experiment was not performed. Following these initial results, further plaque assays were carried out with compounds 3, 4, 6, 35, 37, 39 and 40.

Compound 3

Figure 2 shows the effect of 3 on plaque size (relative to a DMSO control) after 5 days using F£Ep2 cells. Where plaques were present, their size was much smaller than those seen with the DMSO control. In particular, plaques observed at 31.6 μΜ and to some extent 10 μΜ were very small.

As before, the total number of plaques counted from 6 different wells in the presence of DMSO alone was set at 100% (standard deviation of the mean of DMSO control 5.4%). Plaques counted in the presence of 3 at the concentrations indicated are given as a percentage of the DMSO control. The IC ₅₀ was estimated to be 50 μΜ, although further experiments at concentrations of 30-80 μΜ are required to confirm this.

Compound 4

It was hypothesised that these compounds are delaying plaque formation, so that, at lower dilutions, virus plaques will appear. Further tests using HEp2 cells were carried out to screen inhibition over a number of days for compound 4. The results are shown in Figure 3. This confirmed the delayed plaque formation hypothesis.

IC ₅₀ values were calculated as 4.1 μΜ (Day 5), 8.5 μΜ (Day 6), and 15 μΜ

(Day 7). Thus, the data show an increasing IC ₅₀ value with time of incubation of the assay, as expected. This emphasises that the compound does not inhibit virus replication completely, but significantly limits it. This is consistent with our understanding of the role of the M2-2 protein in the virus replication cycle.

Compound 4 showed toxicity at 100 μΜ and to some degree at 31.6 μΜ. No obvious toxicity was seen at 10 μΜ, although this concentration was also less effective.

Compound 6

This compound was tested using assays stopped after 5, 6 and 7 days using HEp2 cells. The results are shown in Figure 4. The IC ₅₀ on day 5 was 10 μΜ. A 50% reduction was not reached on days 6 and 7

Compound 35 This compound was tested in a 5 day assay using HEp2 cells. The results are shown in Figure 5. The IC ₅₀ was 12.5 μΜ. At 100 μΜ, compound 35 showed toxicity and showed some signs of precipitating out of solution. Subsequent dilutions also showed a reduction in plaque numbers (as shown) and size.

Compound 37

This compound was tested in a 5 day assay using F£Ep2 cells. The IC ₅₀ was 17.5 μΜ. At 100 μΜ, compound 37 showed toxicity and showed some signs of precipitating out of solution.

Compound 39

This compound was tested in a 4 day assay using BSC1 cells. The results are shown in Figure 6. The IC ₅₀ was 16.5 μΜ. At 100 μΜ, compound 39 showed some toxicity and was moderately soluble in cell medium. Subsequent dilutions also showed a reduction in plaque numbers (as shown) and size.

Compound 40

This compound was tested in a 4 day assay using BSC1 cells. The results are shown in Figure 7. This compound showed moderate inhibition of plaque formation. 100 μΜ, compound 40 showed low toxicity.

Example: activity against other pneumoviruses

Compounds 1, 4 and 6 were also tested against bovine RSV and pneumonia virus of mice (PVM).

Bovine RSV

The compounds were tested with a 6-day assay using F£Ep2 cells. The results are shown in Figure 8. IC ₅₀ values of 80 μΜ (compound 4) and 15 μΜ (compound 6) were calculated. Compound 1 again precipitated out before 50% reduction was seen.

Pneumonia virus of mice (PVM)

A full plaque assay was carried out for compound 4 using F£Ep2 cells, with assays of 5 days and 6 days after incubation. The results are shown in Figure 9. ICsovalues of 32 μΜ (day 5) and 100 μΜ (day 6) were obtained. Compounds 1 and 6 were not tested fully, but appear to have similar IC ₅₀ values to compound 4 for the dilutions tested.

The compounds (1, 4, 6 and 35) also significantly reduce the size of plaques of hRSV, bRSV and PVM. These smaller size plaques are still counted in the assay, but indicate a significant reduction in virus replication and spread.

Example: Effect on virus yield

The initial studies on compounds assessed their ability to reduce the number of plaques detected in a conventional plaque assay. When the plaques were observed it was noted that the plaque size was significantly reduced in the presence of some compounds. These data suggested that the compounds reduced the amount of virus produced from infected cells and hence slowed the rate of plaque development. A reduction in virus yield is a desirable attribute for antiviral compounds and this was therefore tested directly.

A cell monolayer was infected at a low multiplicity of infection (a small amount of virus relative to the total number of cells) such that only a few cells were infected. CMC was not added to the overlay medium, so that virus particles released from cells could spread throughout the dish and infect new cells. If left, this process will continue until eventually the whole cell monolayer is destroyed. The supernatant which contains the released virus from cells can be collected and the virus titre can be measured using a standard plaque assay. This gives an indication of released (circulating) virus.

The compounds were therefore tested to determine whether they affected the amount of infection virus particles released into the supernatant. Method

A monolayer of cells of a standard cell line known to support pneumovirus replication (typically HEp2 or BSC 1 cells) were infected at a moi of 0.02 for an hour at

33 °C in 200 μΐ of medium lacking serum. The inoculum was removed and 2 ml of overlay medium (GMEM 2% FCS) was added with either carrier or compounds as indicated. After a designated period, 1.5 ml of supernatant was carefully collected without disturbing the cells and stored at -80 °C. Individual plates were used for each time point.

The virus titre in the medium was calculated by carrying out a standard plaque assay with the exception of the following change. During the initial 1 hour step where the virus is first added to the cells the volume of inoculum added was 2 ml instead of 200 μΐ. Thus the supernatant was diluted at least 1 in 20 taking the final concentration of any compound present to a level below its active range. Following virus adsorption period (1 hour) the inoculum was removed and replaced with 2% CMC overlay medium (without compounds).

Four compounds were tested. Compound 1 was tested at concentrations of 32 μΜ and 10 μΜ. Compounds 4, 6 and 35 were each tested at 100 μΜ, 32 μΜ and 10 μΜ. The carrier DMSO alone was used as a control. Supernatants were collected on day 4, day 5 and day 6 post infection. Data were processed by establishing the yield of virus in cells treated with DMSO carrier alone. This was set at 100% and the yield of virus from cells treated with the four compounds at the various concentrations were calculated relative to this value. The data are shown in Figure 10.

Conclusion

The data show a clear dose-dependent effect of all four compounds with yield of virus reduced as the concentration increased. All compounds show a statistically significant reduction in released virus at 32μΜ (p<0.05, compared to the carrier treated control, assessed using a Mann- Witney test and indicated by an asterisk in Figure 10). It is anticipated that in infected animals this reduction in virus production would give the immune system an advantage in combating infection and reducing clinical disease while allowing the establishment of long-term protective immune memory. Compound 35 at ΙΟΟμΜ appears to be the most active compound with no released virus detected when infected cells were treated with 100 μΜ of compound. This represents a reduction in virus yield of at least 2.3 x 10 ³ -fold. However a high level of cellular toxicity of this compound was noted at this dilution.

These results indicate that the compounds tested which are designed to inhibit the cellular DDX3X protein slow down virus replication considerably in a dose-dependent manner.

It is anticipated that in infected animals this reduction in virus production would give the immune system an advantage in combating infection and reducing clinical disease while allowing the establishment of long-term protective immune memory.

Example: MTT Toxicity Assay

All experiments shown were carried out in HEp2 cells. Cells were grown in 96 well plates overnight as previously described in 10% FCS medium to 95% confluence level. The medium was aspirated off and replaced with 200ul media with 2% FCS +/- compounds in DMSO carrier as indicated at an identical dilution series to that used in the plaque assays. 8 wells were used per compound/carrier alone dilution. An additional 2 rows of 8 cells were grown in medium alone for reference and subsequently 8 would be used as a cell death control as described below. Cells were incubated at 33 °C as indicated. If the incubation period exceeded 4 days the medium was replaced with the appropriate amount of fresh medium plus compound.

MTT assays were commenced 4 hours before the time period expired. Here 20 uL of MTT [(3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide)] solution (7.5 mg/ml in PBS) was added. One of the medium alone rows had 20 μΙ_, of SDS added (cell death control). The cells were placed back in the incubator at 33 °C. After 4 hours, 100 μΙ_, of a stop solution (50% formamide, 20% w/v SDS) was added and pipetted to dissolve the substrate. Plates were then left overnight to allow the bubbles to disappear and the OD was recorded at 595 nm. Following subtraction of background solution colour (cell death control), the optical density was expressed as a percentage compared to the DMSO control on each appropriate plate.

Results

Compounds 1, 4, 6, 35, and 37 were tested in an MTT assay which gives a measure of metabolic activity that can be inferred as an indication of cell viability. Solubility issues of compound 1 prevented the testing of the compound at ΙΟΟμΜ (Figures 11 to 15: Figure 11 - compound 1; Figure 12 - compound 4; Figure 13 - compound 6; Figure 14 - compound 35; Figure 15 - compound 37).

Compounds 4, 6 and 37 show inhibition of activity above 10 μΜ over the time course. Cell monolayers appeared intact and were not different from carrier controls as previously noted in the plaque assays. Compound 35 showed greater inhibition as previously observed with a reduction in viability above 3.2 μΜ after 4 days. Cell monolayers treated with 32 μΜ and above of compound 35 showed signs of death as the time course progressed. A rapid increase in cell growth on day 1 for compound 35 (Figure 14) could indicate an adverse cellular response (such as apoptosis). Compound 1 showed a similar level of inhibition to compounds 4, 6 and 37 with inhibition seen above 10 μΜ. However on days 3 and 4 compound 1 showed an increased sensitivity (Figure 11). This disappeared after 5 days (following a media change at day 4). Based on the solubility issues the increased toxicity seen on days 3 and 4 could indicate slow solubilisation of the compound. When the media was refreshed with insoluble compound cells could recover.

HCV core proteins, and alter their intracellular location. However, no link has previously been suggested between DDX3X and pneumovirus infection.

Pneumovirus replication requires translation of two open reading frames (ORF) from the M2 mRNA. ORF-2 overlaps with ORF-1. Host ribosomes access and translate the pneumovirus M2 ORF-2 by using one of two or three AUG codons located upstream of the ORF-1 termination codon. Expression from these initiation codons requires the prior termination of M2 ORF-1 translation. The hRSV M2-2 protein produced by coupled translation has been proposed to be involved in the switch between virus transcription and replication.

Summary of the present invention

The present inventors have identified that DDX3X binds to the hRSV M2 mRNA. The region to which DDX3X binds has a large degree of complex RNA secondary structure which is essential for coupled translation. Blocking binding of DDX3X to this region prevents initiation of hRSV M2-2 translation, thus preventing the switch between virus transcription and replication. The interaction between hRSV M2 mRNA and DDX3X provides a novel target to identify agents which can inhibit hRSV replication, and suggests the use of DDX3X inhibitors in the treatment of hRSV infection and other pneumovirus infections.

According to one aspect of the invention, there is provided a DDX3X inhibitor for use in the treatment of pneumovirus infection, such as for example RSV infection, in a mammal. According to a further aspect of the invention, there is provided a method of treating a pneumovirus infection, such as for example hRSV infection, in a subject, wherein the method comprises the administration to the said subject of an effective amount of a DDX3X inhibitor.

In some embodiments, the DDX3X inhibitor is a human DDX3X inhibitor, a bovine DDX3X inhibitor, an ovine DDX3X inhibitor, and/or a caprine DDX3X inhibitor.

In some embodiments, the DDX3X inhibitor is a compound of Formula (I):

2

(I)

wherein y is 0, 1, or 2;

each Z is independently selected from substituted or unsubstituted C _1-3 alkyl, substituted or unsubstituted C _1-3 acyl, substituted or unsubstituted C _1-3 alkoxy, substituted or

unsubstituted C _1-3 alkylthio, substituted or unsubstituted C _1-3 alkylsulfoxide, substituted or unsubstituted C _1-3 alkylsulfonyl, halo, nitro, and cyano;

each X is independently O or S;

L is a bond or is selected from:

where n is 0, 1, 2, 3 or 4, and the wavy lines indicate the points of attachment to the rest of the molecule;

each R ¹ is independently selected from H and substituted or unsubstituted C _1-3 alkyl;

each R ² is a bond or is independently selected from substituted or unsubstituted C _1-3 alkyl; each R ³ is selected from substituted or unsubstituted Ci- ₆ alkyl; and

either:

R ^a is selected from H and substituted or unsubstituted C _1-3 alkyl, and R ^b is selected from:

3 substituted or unsubstituted C _1-10 alkyl, substituted or unsubstituted C _2-10 alkenyl, substituted or unsubstituted C4-8 cycloalkyl, substituted or unsubstituted C4-8 cycloalkenyl, and

phenyl or 5- or 6-membered heteroaryl, each of which may be optionally substituted with up to 2 substituents Z,

or R ^a and R ^b , together with the nitrogen atom to which they are attached, form a 5- or 6- membered substituted or unsubstituted heterocyclyl ring;

or a tautomer or a pharmaceutically acceptable salt thereof.

In some further embodiments, each R ¹ is H.

In some further embodiments, y is 1.

In some further embodiments, each Z is independently selected from methyl, acetyl, methoxy, methylthio, methylsulfoxide, methylsulfonyl, bromo, nitro, cyano, chloro and substituted Ci alkyl. Preferably at least one Z is a meta or para substituent selected from methyl, methoxy, methylthio, cyano, nitro, bromo, chloro, and -CF ₃ . More preferably at least one Z is a meta or para substituent selected from methyl, methoxy, methylthio, cyano, chloro, and -CF ₃ .

In some further embodiments, each Z is independently selected from methyl, acetyl, methoxy, methylthio, methylsulfoxide, methylsulfonyl, bromo, nitro, and cyano.

Preferably at least one Z is a meta or para substituent selected from methyl, methoxy, methylthio, cyano, nitro and bromo.

In some further embodiments, L is a bond or is selected from:

In some further embodiments, n is 3.

In some further embodiments, R ^a is H.

4 In some further embodiments, R ^b is selected from unsubstituted C _1-10 alkyl and unsubstituted C4-8 cycloalkyl. Preferably, R ^b is selected from 2-methylcyclohexyl and n- octanyl.

In some further embodiments, R ^b is phenyl or 5- or 6-membered heteroaryl, each of which may be optionally substituted with up to 2 substituents Z. Preferably, R ^b is selected from phenyl, pyridinyl, and pyrimidinyl, each of which is substituted with one substituent Z.

In some further embodiments, y is 1;

each Z is independently selected from methyl, acetyl, methoxy, methylthio,

methylsulfoxide, methylsulfonyl, bromo, nitro, cyano, chloro and substituted Ci alkyl.; each X is independently O or S;

L is a bond or is selected from:

where n is 3, and the wavy lines indicate the points of attachment to the rest of the molecule;

R ^a is H; and

R ^b is selected from:

2-methylcyclohexyl, ft-octanyl,

phenyl substituted one substituent Z,

pyridinyl, and

pyrimidinyl.

In some further embodiments, y is 1;

each Z is independently selected from methyl, acetyl, methoxy, methylthio,

methylsulfoxide, methylsulfonyl, bromo, nitro, and cyano;

each X is independently O or S;

L is a bond or is selected from:

5

X

where n is 3, and the wavy lines indicate the points of attachment to the rest of the molecule;

R ^a is H; and

R ^b is selected from:

2-methylcyclohexyl, ft-octanyl,

phenyl substituted one substituent Z,

pyridinyl, and

pyrimidinyl.

In some further embodiments, the DDX3X inhibitor is a compound of Formula (II):

(Π)

or a tautomer or a pharmaceutically acceptable salt thereof.

Preferably, R ^a is H and R ^b is selected from 2-methylcyclohexyl and «-octanyl.

Preferably, R ^a is H, R ^b is selected from:

phenyl substituted with 1 substituent Z,

pyridinyl and

pyrimidinyl;

and at least one Z is a meta or para substituent selected from methoxy, methylthio, cyano, nitro, bromo, chloro and -CF ₃ .

In some further embodiments, the DDX3X inhibitor is a compound of Formula (II):

6

(Π)

or a tautomer or a pharmaceutically acceptable salt thereof.

Preferably, R ^a is H and R ^b is selected from 2-methylcyclohexyl and «-octanyl.

Preferably, R ^a is H, R ^b is selected from:

phenyl substituted with 1 substituent Z,

pyridinyl and

pyrimidinyl;

and at least one Z is a meta or para substituent selected from methoxy, methylthio, cyano, nitro and bromo.

In some further embodiments, the DDX3X inhibitor is a compound of Formula

(III):

(III)

wherein each Z is a meta or para substituent independently selected from methoxy, methylthio, cyano and nitro;

or a tautomer or pharmaceutically acceptable salt thereof.

Preferably, each Z is the same substituent, and both Z are meta substituents or both Z are para substituents.

In some further embodiments, the DDX3X inhibitor is a compound of Formula (III) where one Z is an ortho methyl substituent, and the other Z is a meta or para substituent selected from methoxy, methylthio, cyano and nitro;

or a tautomer or pharmaceutically acceptable salt thereof.

In some further embodiments, the DDX3X inhibitor is a compound of Formula

(IV):

7 wherein each Z is independently selected from methyl, methylthio and nitro;

or a tautomer or pharmaceutically acceptable salt thereof.

Preferably, each Z is the same substituent, and both Z are ortho substituents, both Z are meta substituents or both Z are para substituents.

Preferably, one Z is an ortho methyl substituent, and the other Z is a meta or para substituent selected from methylthio and nitro.

In some embodiments, the DDX3X inhibitor is selected from the group consisting of the compounds:

and tautomers and pharmaceutically acceptable salts thereof.

In other embodiments the DDX3X inhibitor is selected from the group consisting the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compounds:

and tautomers and pharmaceutically acceptable salts thereof.

9 More preferably, the DDX3X inhibitor is selected from the group consisting of the com ounds:

and tautomers and pharmaceutically acceptable salts thereof.

Alternatively the DDX3X inhibitor is selected from the group consisting of the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compounds:

10

and tautomers and pharmaceutically acceptable salts thereof.

Most preferably, the DDX3X inhibitor is selected from the group consisting of the com ounds:

and tautomers and pharmaceutically acceptable salts thereof.

Alternatively the DDX3X inhibitor is selected from the group consisting of the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compound:

and tautomers and pharmaceutically acceptable salts thereof.

In some embodiments, the mammal is selected from humans, cattle, sheep and goats.

According to a further aspect of the invention, there is provided a compound of

Formula (I):

(I)

11 wherein y, Z, X, L, n, R ¹ , R ² , R ³ , R ^a and R ^b are as defined above, or a tautomer or a pharmaceutically accept le salt thereof, provided that the compound is other than:

In some embodiments, the compound is selected from the group consisting of the com ounds:

12

and tautomers and pharmaceutically acceptable salts thereof.

In other embodiments, the compound is selected from the group consisting of the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compounds:

and tautomers and pharmaceutically acceptable salts thereof.

Preferably, the compound is selected from the group consisting of the compounds:

13

and tautomers and pharmaceutically acceptable salts thereof.

Alternatively, the compound is selected from the group consisting of the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compounds:

14

and tautomers and pharmaceutically acceptable salts thereof.

More preferably, the compound is selected from the group consisting of the com ounds:

and tautomers and pharmaceutically acceptable salts thereof.

Alternatively, the compound is selected from the group consisting of the compounds shown in the preceding paragraph and tautomers and pharmaceutically acceptable salts thereof and the following compound:

and tautomers and pharmaceutically acceptable salts thereof.

According to a further aspect of the invention, there is provided a compound of Formula (I):

(I)

wherein L is selected from

15

and y, Z, X, n, R ¹ , R ² , R ³ , R ^a and R ^b are as defined above, or a tautomer or a

pharmaceutically acceptable salt thereof.

According to a further aspect of the invention, there is provided a compound of Formula (I):

(I)

wherein X is S, and

y, Z, L, n, R ¹ , R ² , R ³ , R ^a and R ^b are as defined above, or a tautomer or a pharmaceutically acceptable salt thereof

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a compound as described above and a pharmaceutically- acceptable excipient.

According to a further aspect of the invention, there is provided a compound as described above for use in the treatment of the human or animal body.

Brief Description of the Figures

Fig. 1 shows two biological replicates carried out on different occasions for compound 1.

16 Fig. 2 shows the effect of 3 on plaque size (relative to a DMSO control) after 5 days. Fig. 3 shows the results of further tests carried out to screen inhibition over a number of days for compound 4.

Fig. 4 shows results for compound 6 tested using assays stopped after 5, 6 and 7 days. Fig. 5 shows results for compound 35 tested in a 5 day assay.

Fig. 6 shows results for compound 39 tested in a 4 day assay using BSC1 cells.

Fig. 7 shows results for compound 40 tested in a 4 day assay using BSC1 cells.

Fig. 8 shows results for compounds 1, 4 and 6 tested against Bovine RSV with a 6-day assay.

Fig. 9 shows results of a full plaque assay carried out for compound 4, tested against PVM with assays of 5 days and 6 days after incubation.

Fig. 10 shows the yield of virus from cells treated with compounds 1, 4, 6 and 35 at the various concentrations as shown.

Fig. 11 shows results of an MTT assay for compound 1

Fig. 12 shows results of an MTT assay for compound 4

Fig. 13 shows results of an MTT assay for compound 6

Fig. 14 shows results of an MTT assay for compound 35

Fig. 15 shows results of an MTT assay for compound 37

Fig. 16 shows sequence (SEQ ID NO. 1) and coding capacity (SEQ ID NOs. 2 and 3) of the hRSV strain A2 M2 mRNA RSV strain A2 (subgroup A).

Fig. 17 shows sequence alignment of RSV M2 mRNAs (SEQ ID NOs. 4 to 7).

Fig. 18 shows amino acid sequence of human ATP-dependent RNA helicase DDX3X isoform 1. GenBank accession number NP 001347.3. (SEQ ID NO. 8).

Fig. 19 shows alignment of the amino acid sequences of mammalian ATP-dependent RNA helicase DDX3X (SEQ ID NOs. 9 to 13).

Description of the Sequence Listing

SEQ ID NO. 1 shows sequence of the hRSV strain A2 M2 mRNA RSV strain A 2

(subgroup A).

SEQ ID NOs. 2 and 3 show coding capacity of the hRSV strain A2 M2 mRNA RSV strain A2 (subgroup A).

SEQ ID NO. 4 shows sequence of human respiratory syncytial virus strain A2.

SEQ ID NO. 5 shows sequence of human respiratory syncytial virus strain Bl .

17 SEQ ID NO. 6 shows sequence of bovine respiratory syncytial virus strain A51908.

SEQ ID NO. 7 shows sequence of ovine respiratory syncytial virus.

SEQ ID NO. 8 shows amino acid sequence of human ATP-dependent RNA helicase DDX3X isoform 1.

SEQ ID NO. 9 shows sequence of ATP-dependent RNA helicase DDX3X isoform 1

[Homo sapiens].

SEQ ID NO. 10 shows sequence of Ddx3x protein [Mus musculus].

SEQ ID NO. 11 shows sequence of ATP-dependent RNA helicase DDX3X isoform 1

[Ovis aries].

SEQ ID NO. 12 shows sequence of ATP-dependent RNA helicase DDX3X isoform X2 [Bos taurus].

SEQ ID NO. 13 shows sequence of ATP-dependent RNA helicase DDX3X-like isoform X2 [Capra hircus]. Detailed description

The present invention enables the treatment of pneumovirus (such as hRSV) infection, involving the use of a DDX3X inhibitor. The invention further provides a number of compounds which are of interest for use as DDX3X inhibitors.

As used herein, the term "alkyl" refers to hydrocarbons, particularly saturated hydrocarbons, having the specified number of carbon atoms, and includes straight-chain and branched-chain groups and combinations thereof.

As used herein, the term "acyl" refers to hydrocarbons having the specified number of carbon atoms and including at least one carbonyl moiety, and includes straight-chain and branched-chain groups and combinations thereof. The carbonyl may be located at any position in the group, but is typically located adjacent to a point of attachment to the remainder of the molecule.

As used herein, the term "alkoxy" refers to hydrocarbons having the specified number of carbon atoms and including one or more oxygen atoms linked by single bonds to carbon, and includes straight-chain and branched-chain groups and combinations thereof. The oxygen atoms may be located at any position in the group, but one oxygen atom is typically located adjacent to a point of attachment to the remainder of the molecule.

As used herein, the term "alkylthio" refers to hydrocarbons having the specified number of carbon atoms and including one or more sulfur atoms linked by single bonds to

18 carbon, and includes straight-chain and branched-chain groups and combinations thereof. The sulfur atoms may be located at any position in the group, but one sulfur atom is typically located adjacent to a point of attachment to the remainder of the molecule.

As used herein, the term "alkylsulfoxide" refers to hydrocarbons having the specified number of carbon atoms and including at least one sulfinyl moiety, and includes straight-chain and branched-chain groups and combinations thereof. The sulfinyl moiety may be located at any position in the group, but is typically located adjacent to a point of attachment to the remainder of the molecule.

As used herein, the term "alkylsulfonyl" refers to hydrocarbons having the specified number of carbon atoms and including at least one sulfonyl moiety, and includes straight-chain and branched-chain groups and combinations thereof. The sulfonyl moiety may be located at any position in the group, but is typically located adjacent to a point of attachment to the remainder of the molecule.

As used herein, the term "halo" refers to an element found in Group 13 of the periodic table. Typical examples of halo substituents include fluoro, chloro and bromo. Another typical example is iodo.

As used herein, the term "cycloalkyl" refers to hydrocarbons having the specified number of carbon atoms and including at least one cyclic structure, and includes straight- chain and branched-chain groups, and combinations thereof.

As used herein, the term "alkenyl" refers to hydrocarbons having the specified number of carbon atoms, and including at least one carbon-carbon double bond, and includes straight-chain and branched-chain groups, and combinations thereof.

As used herein, the term "cycloalkenyl" refers to hydrocarbons having the specified number of carbon atoms, and including at least one carbon-carbon double bond and at least one cyclic structure. The double bond may be incorporated wholly or partially within the cyclic structure or may be distinct from the cyclic structure. The term includes straight- chain and branched-chain groups, and combinations thereof.

As used herein, the term "substituted" refers to a moiety in which one or more hydrogen atoms, typically 1, 2 or 3 hydrogen atoms, has each been replaced by a substituent independently selected from hydroxyl, methoxy, thio, methylthio, amino, methylamino, dimethylamino, fluoro, chloro, bromo, iodo, cyano, nitro, sulfonate (or sulfonic acid), sulfonamide, carboxylate (or carboxylic acid) and carbonamide.

The present invention also relates to a method of identifying an agent which modulates hRSV replication comprising contacting an agent with (a) an RNA construct

19 comprising the sequence of nucleotides 392 to 511 of the hRSV M2 transcript and (b) a protein comprising DDX3X, and determining whether the agent modulates the interaction between the construct and DDX3X.

The M2 mRNAs of pneumoviruses contain two open reading frames, referred to herein as M2-1 and M2-2. The region of nucleotides 392-511 of the hRSV M2 mRNA has strong secondary structure. The present inventors show that DDX3X interacts with this structure, to allow initiation of translation from the second ORF. The M2-2 protein translated from the M2 mRNA ORF-2 is required for the switch from transcription of the viral genome to replication, leading to virus particle assembly and release. Thus agents which interfere with this interaction are identified as useful in the treatment of infection by hRSV and other pneumoviruses.

The RNA constructs of the present invention comprise nucleotides 392 to 511 of the human M2 transcript, or an equivalent region in other pneumoviruses, including but not limited to bovine RSV, ovine RSV, caprine RSV. The sequence of the human ORF-1 region of the hRSV (strain A2) M2 transcript is shown in Figure 16 (SEQ ID NOs. 1-3). M2 transcripts from other pneumoviruses show a high degree of homology in this region seen in Figure 17 (SEQ ID NOs. 4-7). The construct may include additional sequences, for example flanking sequences from the hRSV M2 transcript. Assay methods which monitor for translation of the M2-2 ORF will include this ORF. Alternatively the construct may be provided with a reporter gene provided in frame with one of the AUG codons of the second ORF.

DDX3X is a DEAD box RNA helicase. The sequence of human DDX3X is provided in Figure 18 (SEQ ID NO. 8). DDX3X is highly conserved across mammalian species as shown in Figure 19 (SEQ ID NOs. 9-13). In view of the high degree of homology between pneumoviral M2 transcripts and DDX3X across species, the methods of the present invention can use pneumovirus constructs and DDX3X derived from other species, and can also be used to identify modulators which can be used to interfere with pneumovirus infection in a variety of mammalian species.

The compounds tested may be enhancers or inhibitors of the interaction between the construct and DDX3X, and thus act to enhance or inhibit pneumovirus replication, although preferably the method is used to identify an inhibitor of RSV replication. An inhibitor of pneumovirus replication interferes with the interaction between the construct and DDX3X.

20 Any compound(s) can be used in the method of the invention. The compound(s) may be any chemical compound(s) used in drug screening programmes. They may be natural or synthetic. Extracts of plants which contain several characterised or

uncharacterised components may also be used. Typically, organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 Daltons. Compounds can be biomolecules including peptide and peptide mimetics, oligonucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate compounds may be obtained from a wide variety of sources including libraries of synthetic or natural substances. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. The compound(s) may be the product(s) of a combinatorial library such as are now well known in the art (see e.g. Newton (1997) Expert Opinion Therapeutic Patents, 7(10): 1183-1194). Natural product libraries, such as display (e.g. phage display libraries), may also be used.

The methods of the invention allow the screening of one or more compounds for their ability to act as modulator of pneumovirus replication. The methods are preferably carried out in vitro or ex vivo.

Techniques for determining the effect of compound(s) on the interaction between the RNA construct and DDX3X protein are within the skill of those in the art. Any of those techniques may be used in accordance with the invention. The method may be carried out in vitro, such as in a cell-free system or alternatively in a cell-based system.

In accordance with the methods of the present invention, the activity of an agent may be determined by investigating directly the effect on the binding interaction between DDX3X and the RNA construct. Alternatively, the methods may be conducted to determine translation from the second open reading frame (ORF-2) of M2. Reporter systems may also be used, for example to provide a construct which comprises a reporter gene cloned in frame from the start codon of ORF-2. Expression of the reporter gene will only occur when DDX3X is able to interact with the construct. The agents which interfere with the interaction will prevent translation of the reporter gene.

Preferred in vitro translation systems utilise cell extracts which provide components necessary for the process of translation. These typically include macromolecular components such as ribosomes, tRNAs, aminoacyl tRNA synthetases, initiation, elongation and termination factors. The cell extracts may be of any origin provided they allow for

21 translation of the reporter coding sequence. Suitable cell extracts may be obtained from reticulocytes, such as rabbit reticulocytes, wheat germ and bacterial extracts, such as E. coli extracts. The cell extracts are suitably supplemented with additional components required for translation, such as amino acids, nucleotide triphosphate energy sources and other co-factors. The skilled person is familiar with the use of such systems.

In preferred embodiments, the RNA is added to the in vitro translation system directly in RNA form. Alternatively, a coupled or linked transcription/translation system may be utilised in which a DNA construct encoding the RNA is first transcribed prior to translation of the resulting RNA.

Techniques for measuring translation of a reporter coding sequence are well known in the art. Any suitable technique may be used. Preferred methods of measuring reporter coding sequence translation involve luminescence, fluorescence, or an immunoassay. For example, a reporter coding sequence may encode a luminescent or fluorescent protein such that the level of translation may be monitored through measurement of a luminescent or fluorescent signal. A suitable example of a luminescent reporter coding sequence is luciferase. A suitable example of a fluorescent reporter coding sequence is green fluorescent protein.

Measuring levels of translated protein using an immunoassay is also well known in the art. Any suitable immunoassay which allows for detection of a reporter coding sequence by an antibody may be used. Any suitable commercially available antibody for a given target may be used. An example of a suitable immunoassay is Enzyme-Linked Immunosorbent Assay (ELISA). In some embodiments, the ELISA assay may be performed in flat plates where wells are coated with binding proteins or antibodies which can bind and allow for detection of the translated reporter polypeptide. Other types of immunoassay include immunoprecipitation and Western blotting.

Modulators of pneumovirus replication can be formulated into pharmaceutical compositions. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

22 Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

For delayed release, the modulators of pneumovirus replication may be included in a pharmaceutical composition which is formulated for slow release, such as in

microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.

The dose of a modulator of pneumovirus replication may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated and the frequency and route of administration.

Preferably, daily dosage levels are from 5 mg to 2 g. That dose may be provided as a single dose or may be provided as multiple doses, for example taken at regular intervals, for example 2, 3 or 4 doses administered daily.

Embodiments of the invention are illustrated by the following examples.

23

Library 1 X = O

Library 2 X = S

Scheme 1 Synthesis of ligands in Ta

Library 3 X = O

Library 4 X = S

Scheme 2 Synthesis of ligands in Tables 3 and 4.

Table 1 : Library 1 diarylureas prepared and summary of activities in cellular RSV TCID ₅₀ assay. Solubility refers to a solution of ligand in DMSO (20mM) diluted in cell media to a working concentration of 100 μΜ and ranges from 'very poor' (almost insoluble) to 'very good' remaining freely soluble over extended periods at 4 °C.

24

25

Table 2 Library 2, diarylthioureas prepared and summary of activities in cellular RSV TCID ₅₀ assay. Solubility refers to a solution of ligand in DMSO (20mM) diluted in cell media to a working concentration of 100 μΜ and ranges from 'very poor' (almost insoluble) to 'very good' remaining freely soluble over extended periods at 4 °C.

26

Table 3 Library 3, alkylaryl ureas prepared and summary of activities in cellular RSV TCID ₅₀ assay. Solubility refers to a solution of ligand in DMSO (20mM) diluted in cell media to a working concentration of 100 μΜ and ranges from 'very low' (almost insoluble) to 'very good' remaining freely soluble over extended periods at 4 °C.

27

Table 4 Library 4 alkylaryl thioureas prepared and summary of activities in cellular RSV TCID ₅₀ . Solubility refers to a solution of ligand in DMSO (20mM) diluted in cell media to a working concentration of 100 μΜ and ranges from 'very low' (almost insoluble) to 'very good' remaining freely soluble over extended periods at 4 °C.

Library 5 X = O, X = S

Scheme S3 Synthesis of ligands in Table 5.

Table 5 Library 5, oligomeric ureas and thioureas prepared and summary of activities in cellular RSV TCID ₅₀ assay. Solubility refers to a solution of ligand in DMSO (20mM) diluted in cell media to a working concentration of 100 μΜ and ranges from 'very low' (almost insoluble) to 'very good' remaining freely soluble over extended periods at 4

°C.

28 2 H H H H good toxic at

100 μΜ 3 H H H H poor ++ toxic at

100 μΜ 4 poor ++ toxic at

100 μΜ 5 H H H H good toxic at

-^^ ^N y ^N ° o ° ^N γ- ^N Y^r' ^N ° ²

100 μΜ 6 good 7 good +++ toxic at

H ₃ CS ^A ^ ^J ° ° ^SCH ₃ 50 μΜ 8 I 1— 1 H H H good toxic at

50 μΜ

General Experimental. Reagents and chemicals were purchased from Sigma- Aldrich and Acros and were used as supplied; solvents were purchased from Fisher and were used as supplied. All reactions were carried out under an atmosphere of nitrogen. Silica flash chromatography was carried out under low pressure using Merck silica gel 60 H (230-400 mesh). MR spectra were acquired on Bruker DPX 300 or DPX 400 spectrometers with tetramethylsilane, δ = 0 ppm as an internal standard. Assignments were made using 2D methods including COSY, HMBC and HMQC NMR and coupling constants quoted to the nearest 1 Hz. Low-resolution mass spectra were measured on an Agilent 6130B ESI-quad instrument with an electrospray ionization (ESI) mass selective detector in positive ion or negative ion mode as indicated. MALDI mass spectra were acquired on a Bruker Ultraflex II instrument. High-resolution mass spectra (HRMS) were acquired on a Bruker MicroTOF instrument with an ESI mass selective detector in either positive or negative ion mode. Infrared spectra were acquired with a Bruker Alpha FT-IR spectrophotometer. Melting points (mps) were determined on a Stuart SMP10 melting point apparatus; mp > 250 °C were measured on an Olympus BH2 microscope equipped with a Linkam HFS 91 heating stage, or differential scanning calorimeter (DSC, Mettler

29 Toledo DSCl-400). Warwick Analytical Service Limited, performed CHN microanalysis using a CE440 Elemental Analyser.

Preparation of compounds

SAFETY Aromatic amines are highly toxic and considered as known or suspected human carcinogens. Isocyanates are sensitizers and toxic or highly toxic and their endpoint of hydrolysis is the parent amines. All procedures were carried out in an efficient fumehood, wearing standard laboratory protection. In addition, all reagents and reaction vessels were handled wearing a pair of thin nitrile gloves overgloved with medium weight nitrile gloves. All waste, including aqueous was disposed in specifically labeled containers.

General Procedure I -Addition of aromatic amines to isocyanates

Aromatic ureas or thioureas were prepared using a previously reported general method. ¹ To a solution of the amine (1 equiv., 5 mmol) in dry dichloromethane (5 mL) under dry N ₂ was added the isocyanate (1 equiv., 5 mmol) in dry dichloromethane (-10 mL) with stirring, and left overnight at room temperature. The precipitate was collected by filtration and washed with a little ethanol, or if no precipitate, volatiles were removed under reduced pressure on a rotary evaporator. The resulting mixture was then purified through either recrystallization or a flash silica column (details specified) to provide the desired product.

Preparation of 3-(2-methylphenyl)-l-(3-nitrophenyl)urea (1),

Compound 1 was prepared from o-toluidine and 3-nitrophenylisocyanate using General Procedure I on 20 mmol scale to give, after recrystallization from ethanol, the title product 1 as a cotton wool-like solid (3.782 g, 69%); mp 216 - 218 °C; 1H MR (400 MHz, DMSO-i ) δ 9.53 (br. s., 1H), 8.59 (t, J= 2 Hz, 1H), 8.08 (br. s., 1H), 7.83 (ddd, J= 8, 2, 1 Hz, 1H), 7.80 (dd, J= 8, 1 Hz, 1H), 7.71 (ddd, J= 8, 2, 1 Hz, 1H), 7.58 (t, J= 8 Hz, 1H), 7.15 - 7.23 (m, 2H), 7.01 (dt, J= 7, 1 Hz, 1H), 2.27 (s, 3H) ppm; mlz C ₁₄ H ₁₃ N ₃ O ₃ (ESI pos. mode) 294.0 (MNa ⁺ ); mlz (ESI neg. mode) 306.0 (M ³⁵ C1 ^" ), 270.1 (M-H ⁺ ).

In agreement with literature data: Radi, M.; Falchi, F.; Garbelli, A.; Samuele, A.; Bernardo, V.; Paolucci, S.; Baldanti, F.; Schenone, S.; Manetti, F.; Maga, G.; Botta, M. Bioorg. Med. Chem. Lett. 2012, 22, 2094-2098.

30 Preparation of l-(3-acetylphenyl)-3-(2-methylphenyl)urea (2)

o-Toluidine and 3-acetylphenylisocyanate were combined using General Procedure I on a 3.10 mmol scale to give, after recrystallization from ethanol, the title product 2 as off- white crystals (0.454 g, 55%); mp 198 - 199 °C; 1H NMR (300 MHz, DMSO- ) δ 9.26 (s, 1H), 8.07 (t, J = 1 Hz, 1H), 7.96 (s, 1H), 7.83 (d, J= 8 Hz, 1H), 7.70 (dd, J = 8, 1 Hz, 1H), 7.58 (d, J= 8 Hz, 1H), 7.44 (t, J = 8 Hz, 1H), 7.1 1 - 7.22 (m, 2H), 6.96 (dt, J = 7, 1 Hz, 1H), 2.57 (s, 3H), 2.25 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) δ 197.7, 152.6, 140.3, 137.4, 137.2, 130.2, 129.2, 127.7, 126.1, 122.9, 122.5, 121.7, 121.2, 1 17.1, 26.7, 17.8 ppm; Vmax (solid) 3282, 3080, 3026, 2974, 1670, 1641, 1586, 1560, 1433, 1360, 1274, 1239, 915, 795, 765, 662 cm ^"1 , mlz (ESI neg. mode) 267.1 (M-H) ^~ Elemental analysis calcd for Ci ₆ Hi ₆ N ₂ 0 ₂ : C, 71.62; H, 6.01 ; N, 10.44. Found: C, 71.41 ; H, 5.93, N, 10.28.

Preparation of 3-(3-cyanophenyl)-l-(2-methylphenyl)urea (3)

o-Toluidine and 3-cyanophenylisocyanate were combined using General Procedure I on a 3.47 mmol scale to give, after recrystallization from ethanol, the title product 3 as a cotton wool-like solid; mp 200 - 20FC; 1H NMR (300 MHz, DMS0 ) δ 9.35 (br. s., 1H), 8.08 (br. s., 1H), 8.01 (s, 1H), 7.78 (d, J = 8 Hz, 1H), 7.65 (d, J = 9 Hz, 1H), 7.50 (t, J= 8 Hz, 1H), 7.42 (d, J = 7 Hz, 1H), 7.12 - 7.22 (m, 2H), 6.98 (t, J = 7 Hz, 1H), 2.25 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) δ 152.5, 140.7, 136.9, 130.2 (2C), 128.2, 126.1, 125.1, 123.2, 122.6, 121.6, 120.5, 1 18.9, 1 1 1.6, 18.2 ppm; methyl peak missing but seen on

HMQC at 18.2 ppm. Peak at 130.2 ppm determined to be 2C (non identical) by HMQC; Vmax (solid) 3323, 3271, 2232, 1643, 1585, 1556, 1458, 1433, 1289, 1248, 795, 774, 753, 658 cm ^"1 ; mlz (ESI pos. mode) 274.1 (MNa ⁺ ); mlz 288.0 (ESI neg. mode) (M ³⁷ C1 ^" ), 286.0 (M ³⁵ C1 ^" ), 250.1 (M-H ⁺ ). Elemental analysis calcd for C ₁₅ H ₁₃ N ₃ O: C, 71.70; H, 5.21 ; N, 16.72. Found: C, 71.65; H, 5.16; N, 16.64.

Preparation of l-(2-methylphenyl)-3-[3-(methylsulfanyl)phenyl]urea (4)

o-Toluidine and 3-methylthiophenylisocyanate were combined using General Procedure I on a 3.03 mmol scale to give, after recrystallization from ethanol, the title product 4 as an amorphous solid (0.362 g, 44%); mp 175 - 176 °C; 1H NMR (400 MHz, DMSO- ) δ 9.06 (s, 1H), 7.93 (s, 1H), 7.82 (dd, J = 8, 1 Hz, 1H), 7.49 (t, J = 2 Hz, 1H), 7.23 (t, J = 8 Hz, 1H), 7.12 - 7.21 (m, 3H), 6.96 (dt, J= 7, 1 Hz, 1H), 6.87 (td, J = 8, 1 Hz, 1H), 2.47 (s, 3H), 2.25 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO-i ) δ 152.6, 140.4, 138.6, 137.2, 130.2, 129.3, 127.6, 126.1, 122.8, 121.1, 1 19.1, 1 14.9, 1 14.5, 17.8, 14.6 ppm; (neat) 3285, 1632, 1606, 1555, 1478, 1459, 1288, 1260, 1232, 926, 848, 772, 743, 665 cm ^"1 ; miz (ESI pos. mode) 295.0 (MNa ⁺ ); miz (ESI neg. mode) 307.0 (M ³⁵ C1 ^" ), 271.1 (M-H ⁺ ). HRMS (ESI pos. mode) miz Ci ₅ Hi ₆ N ₂ NaOS required 295.0876. Found miz 295.0877. Preparation of 3-(3-methanesulfinylphenyl)-l-(2-methylphenyl)urea (5)

To a stirred solution of l-(2-methylphenyl)-3-[3-(methylsulfanyl)phenyl]urea 4 (0.25 g, 0.92 mmol) in acetone - water (2: 1 v/v, 15 mL) at 0 °C was added dropwise a solution of sodium periodate (0.96 g, 0.92 mmol) in water (8 mL). A thick white precipitate of sodium iodate formed, which was removed by filtration after 4 hours and washed with a small portion of acetone. The filtrate was extracted with ethyl acetate (3 x 20 mL), with the addition of brine (15 mL) for the final extraction. The combined organics were washed with water, dried (MgS0 ₄ ) and evaporated under reduced pressure to give the crude product. Purification by flash chromatography (Si0 ₂ , 1% MeOH - EtOAc) gave a solid which was recrystallized from hot ethanol - water (1 :2 v/v) to give the title product 5 as a white powder (0.098 g, 35%); mp 139 - 144 °C; 1H NMR (300 MHz, DMSO- ) δ 9.35 (s, 1H), 7.97 (s, 1H), 7.87 (t, J= 2 Hz, 1H), 7.83 (d, J= 8 Hz, 1H), 7.51 - 7.57 (m, J= 8, 2 Hz, 1H), 7.47 (t, J= 8 Hz, 1H), 7.23 (td, J= 7, 1 Hz, 1H), 7.11 - 7.21 (m, 2H), 6.97 (dt, J= 7, 1 Hz, 1H), 2.73 (s, 3H), 2.25 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO- ) δ 152.5, 147.1, 140.8, 137.1, 130.2, 129.7, 127.7, 126.1, 122.9, 121.2, 119.7, 116.5, 112.3, 43.3, 17.8 ppm; Vmax (solid) 3438, 3292, 3056, 2917, 1640, 1585, 1457, 1425, 1242, 1019, 789, 761 cm ^"1 ; miz (ESI pos. mode) 311.0 (MNa ⁺ ); (ESI neg. mode) 323.0 (M ³⁵ C1 ^" ), 287.1 (M-H ⁺ ) ^" .

HRMS (ESI pos. mode) miz Ci ₅ Hi ₇ N ₂ 0 ₂ S (MH ⁺ ) required 289.1005. Found miz 289.1001 Preparation of l-(4-cyanophenyl)-3-(2-methylphenyl)urea (6)

o-Toluidine and 4-cyanophenylisocyanate were combined using General Procedure I on a 3.47 mmol scale to give, after recrystallization from ethanol (< 20 mL), the title product 6 as white needles (0.548 g, 63%); mp 207 - 210 °C; 1H NMR (400 MHz, DMSO-i ) δ 9.50 (br. s., 1H), 8.11 (br. s., 1H), 7.78 (dd, J= 8, 1 Hz, 1H), 7.74 (td, J= 9, 2 Hz, 2H), 7.65 (td, J= 9, 3 Hz, 2H), 7.15 - 7.23 (m, 2H), 7.00 (dt, J= 8, 1 Hz, 1H), 2.26 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO-i ) δ 152.2, 144.3, 136.8, 133.3, 130.2, 128.3, 126.2, 123.3, 121.6, 119.3, 117.8, 103.1, 17.8 ppm; v _max (solid) 3285, 3067, 3024, 2227, 1637, 1583, 1540, 1456, 1407, 1294, 1241, 1175, 847, 836, 755, 648, 552 cm ^"1 ; miz (ESI pos. mode) 274.0 (MNa ⁺ ), 252.1 (MH ⁺ ); miz (ESI neg. mode) 286.0 (M ³⁵ C1 ^" ), 250.1 (M-H ⁺ ) ^~ Elemental analysis calcd for C15H13N3O: C, 71.70; H, 5.21; N, 16.72. Found: C, 71.51; H, 5.16; N, 16.64.

Preparation of 3-(3-cyanophenyl)-l-(pyridin-3-yl)urea (7)

3-Aminopyridine and 3-cyanophenylisocyanate were combined using General Procedure I on a 3.47 mmol scale to give, after slow recrystallization from ethanol (- 30 mL), the product as red-tinged crystals (0.269 g, 33%). A second recrystallization from a minimum of hot ethanol gave the title product 7 as a white cotton wool-like mass of crystals (0.255 g, 31%); mp 197-198 °C (dec); 1H MR (400 MHz, DMSO-^ ₆ ) δ 9.17 (s, 1H), 9.03 (s, 1H), 8.63 (d, J= 3 Hz, 1H), 8.23 (dd, J= 5, 2 Hz, 1H), 7.99 (t, J= 2 Hz, 1H), 7.95 (ddd, J= 8, 3, 2 Hz, 1H), 7.71 (ddd, J= 8, 2, 1 Hz, 1H), 7.52 (t, J= 8 Hz, 1H), 7.45 (td, J= 8, 1 Hz, 1H), 7.34 (dd, J= 8, 5 Hz, 1H) ppm; ¹³ C NMR (101 MHz, DMS0 ) δ 152.5, 143.2, 140.4, 140.3, 136.0, 130.2, 125.6, 125.5, 123.6, 123.1, 121.0, 118.8, 111.6 ppm; Vmax (solid) 3285, 3249, 3068, 2230, 1712, 1582, 1537, 1475, 1424, 1326, 1278, 1247, 1199, 785, 739, 705, 679 cm ^"1 ; mlz (ESI pos. mode) 239.0 (MH ⁺ ); (ESI neg. mode) 283.0 (M ³⁵ C1 ^" ), 237.1 (M-H ⁺ ). Elemental analysis calcd for C ₁₃ H ₁₀ N ₄ O: C, 65.54; H, 4.23; N, 23.52. Found: C, 65.47; H, 4.18; N, 23.42.

Preparation of 3-(4-cyanophenyl)-l-(pyridin-2-yl)urea (8)

2-Aminopyridine and 4-cyanophenylisocyanate were combined using General Procedure I on a 3.46 mmol scale to give the title product 8 as a white solid (0.20 g, 24%).

Recrystallization of 160 mg from a minimum of hot ethanol gave a colourless solid (98 mg, 12%); mp 212 - 213 °C (dec); 1H NMR (400 MHz, DMS0 ) δ 10.90 (br. s., 1H), 9.62 (s, 1H), 8.31 (ddd, J= 5, 2, 1 Hz, 1H), 7.76 - 7.81 (m, 3H), 7.73 (td, J= 9, 2 Hz, 2H), 7.54 (d, J= 9 Hz, 1H), 7.06 (ddd, J= 7, 5, 1 Hz, 1H); ¹³ C NMR (101 MHz,

DMSO-i ) 5 152.4, 151.9, 147.0, 143.5, 138.7, 133.3 (2C), 119.2, 118.7 (2C), 118.0, 112.1, 104.0; v _max (solid) 3203, 3060, 2973, 2221, 1702, 1581, 1476, 1414, 1305, 1243, 1155, 840, 770 cm ^"1 ; mlz (ESI pos. mode) 261.0 (MNa ⁺ ), 239.0 (MH ⁺ ); (ESI neg. mode) 273.0 (M ³⁵ C1 ^" ), 237.1 (M-H ⁺ ) ^~ Elemental analysis calcd for C ₁₃ H ₁₀ N ₄ O: C, 65.54; H, 4.23; N, 23.52. Found: C, 65.29; H, 4.13; N, 23.30.

Preparation of l-(2-methylphenyl)-3-(pyridin-2-yl)urea (9)

2-Aminopyridine and o-tolyl isocyanate were combined using General Procedure I on a

3.76 mmol scale to give, after rcrystallization from hot ethanol, the title product 9 as a

33 white solid (0.301 g, 35%); mp 212 - 213 °C; 1H NMR (400 MHz, OMSO-d ₆ ) δ 10.94 (br. s., 1H), 9.78 (s, 1H), 8.29 (ddd, J= 5, 2, 1 Hz, 1H), 8.05 (dd, J= 8, 1 Hz, 1H), 7.77 (ddd, J = 8, 7, 2 Hz, 1H), 7.30 (d, J = 8 Hz, 1H), 7.22 (d, J = 8 Hz, 1H), 7.18 (dt, J= 8, 2 Hz, 1H), 7.02 (ddd, J= 7, 5, 1, Hz, 1H), 6.98 (dt, J= 7, 1 Hz, 1H) , 2.34 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO- ) δ 153.1, 152.3, 146.3, 138.8, 137.3, 130.2, 126.9, 126.2, 122.7, 120.3, 117.2, 111.9, 18.1 ppm; Vmax (solid) 3199, 3057, 2982, 2915, 1686, 1605, 1478, 1458, 1415, 1289, 1240 cm ^"1 ; mlz (ESI pos. mode) 250.0 (MNa ⁺ ), 228.0 (MH ⁺ ); (ESI neg. mode): mlz 226.1 (M-H ⁺ ) ^" . Elemental analysis calcd for C ₁₃ H ₁₃ N ₃ O: C, 68.70; H, 5.77; N, 18.49. Found: C, 68.61; H, 5.78; N, 18.43.

Preparation of l-(2-methylphenyl)-3-(pyridin-3-yl)urea (10)

o-Tolyl isocyanate and 3-aminopyridine were combined using General Procedure I on a 3.76 mmol scale to give, after recrystallization from a minimum of hot ethanol, the title product 10 as white needles (0.278 g, 32%); mp 164 - 165°C; 1H NMR (400 MHz, DMSO-i ) δ 9.18 (s, 1H), 8.62 (d, J= 3 Hz, 1H), 8.20 (dd, J= 5, 2 Hz, 1H), 8.06 (s, 1H), 7.98 (ddd, J= , 8, 3, 2 Hz, 1H), 7.82 (dd, J= 8, 1 Hz, 1H), 7.32 (dd, J= 8, 5 Hz, 1H), 7.14 - 7.22 (m, 2H), 6.98 (dt, J= 7, 1 Hz, 1H), 2.26 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO- de) 6 152.7, 142.7, 139.8, 137.1, 136.6, 130.2, 127.9, 126.2, 124.9, 123.6, 123.0, 121.4, 17.8 ppm; v _max (solid) 3406, 2890, 1700, 1610, 1584, 1556, 1509, 1449, 1424, 1286, 1250, 802, 753, 703 cm ^"1 ; mlz (ESI pos. mode): 250.0 (MNa ⁺ ), 228.1 (MH ⁺ ); (ESI neg. mode): mlz 226.1 (M-H ⁺ ). Elemental analysis calcd for C ₁₃ H ₁₃ N ₃ O: C, 68.70; H, 5.77; N, 18.49. Found: 68.58; H, 5.78; N, 18.37.

Preparation of 3-(3-cyanophenyl)-l-(pyridin-2-yl)urea (11)

2-Aminopyridine and 3-cyanophenyl isocyanate were combined using General Procedure I on a 3.46 mmol scale to give a white solid (220 mg). Recrystallization from hot ethanol, gave the title product 11 as white cotton wool-like crystals (0.140 g, 17%); mp 164 - 165°C; 1H NMR (400 MHz, DMSO-d ₆ ) δ 10.93 (br. s., 1H), 9.63 (s, 1H), 8.31 (ddd, J= 5, 2, 1 Hz, 1H), 8.08 (t, J= 2 Hz, 1H), 7.75 - 7.82 (m, 2H), 7.54 (t, J= 8 Hz, 1H), 7.46 - 7.50 (m, 2H), 7.05 (ddd, J= 7, 5, 1 Hz, 1H) ppm; ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 152.5,

152.2, 146.9, 139.9, 138.7, 130.2, 126.0, 123.5, 121.4, 118.7, 117.8, 112.0, 111.7 ppm;

Vmax (solid) 3207, 3059, 2982, 2228, 1707, 1609, 1582, 1562, 1479, 1417, 1317, 1150, 771,

725 cm ^"1 ; mlz (ESI pos. mode) 487.2 (M ₂ Na ⁺ ). Elemental analysis calcd for C ₁₃ H ₁₀ N ₄ O: C,

65.54; H, 4.23; N, 23.52. Found: C, 65.48; H, 4.21; N, 23.61.

34 Preparation of 3-(3-cyanophenyl)-l-(pyrimidin-2-yl)urea (12)

2-Aminopyrimidine and 3-cyanophenyl isocyanate were combined using General

Procedure I on a 3.47 mmol scale to give a white solid (-100 mg). Recrystallization from hot ethanol (poor solubility, 150 mL), gave the title product 12 as a white powder (0.077 g, 9%); mp 260 - 266 °C; 1H MR (400 MHz, DMSO-d ₆ ) δ 11.80 (br. s., 1H), 10.37 (br. s., 1H), 8.70 (d, J= 5 Hz, 2H), 8.14 (t, J= 2 Hz, 1H), 7.95 (td, J= 8, 2 Hz, 1H), 7.57 (t, J= 8 Hz, 1H), 7.53 (td, J= 8, 2, Hz, 1H), 7.18 (t, J= 5 Hz, 1H) ppm. Insufficient solubility for carbon MR. v _max (solid) 3140, 3065, 2980, 2916, 2223, 1693, 1615, 1582, 1561, 1515, 1407, 1300, 1240, 1225, 785, 725 cm ^"1 ; mlz (ESI neg. mode) 274.0 (M ³⁵ C1 ^" ), 238.1 (M- H ⁺ ) ^" ; HRMS (ESI pos. mode) mlz Ci ₂ H ₉ N ₅ ONa required 262.0699 (MNa ⁺ ). Found mlz 262.0701. Barely soluble in CH ₂ C1 ₂ , insoluble in 80:20 MeOH:H ₂ 0. Elemental analysis calcd for Ci ₂ H ₉ N ₅ 0: C, 60.25; H, 3.79; N, 29.27. Found: C, 60.10; H, 3.670; N, 29.23. Preparation of 3-(3-methoxyphenyl)-l-(2-methylphenyl)urea (13)

o-Toluidine and 3-methoxyphenylisocyanate using General Procedure I on a 3.35 mmol scale to give, after recrystallization from hot ethanol, the title product 13 as white needles (0.327 g, 39%); mp 162 - 162 °C; 1H NMR (300 MHz, DMS0 ) δ 9.03 (s, 1H), 7.91 (s, 1H), 7.84 (d, J= 8 Hz, 1H), 7.10 - 7.22 (m, 4H), 6.91 - 6.98 (m, 2H), 6.55 (dd, J= 8, 2 Hz, 1H), 3.73 (s, 3H), 2.24 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) δ 159.7, 152.5, 141.1, 137.3, 130.1, 129.5, 127.4, 126.1, 122.6, 121.0, 110.2, 107.1, 103.6, 54.8, 17.8 ppm; v _max (solid) 3274, 2923, 2826, 1633, 1590, 1545, 1455, 1293, 1225, 1160, 1055, 759 cm ^"1 ; mlz (ESI pos. mode) 279.0 (MNa ⁺ ), 257.0 (MH ⁺ ); (ESI neg. mode) 291.0 (M ³⁵ C1 ^" ), 255.1 (M- H ⁺ ) ^~ Elemental analysis calcd for Ci ₅ Hi ₆ N ₂ 0 ₂ : C, 70.29; H, 6.29; N, 10.93. Found: 70.12; H, 6.23; N, 10.88.

Preparation of 3-(2-methylphenyl)-l-[4-(methylsulfanyl)phenyl]urea (14)

o-Toluidine and 4-methylthiophenylisocyanate were combined using General Procedure I on a 3.46 mmol scale to give, after recrystallization from hot ethanol, the title product 14 as a white powder (0.315 g, 38%); mp 205 - 206 °C; 1H NMR (250 MHz, DMS0 ) δ

8.98 (s, 1H), 7.86 (s, 1H), 7.80 (d, J = 8 Hz, 1H), 7.38 - 7.45 (m, J = 9 Hz, 2H), 7.18 - 7.25

(m, J= 8 Hz, 2H), 7.08 - 7.18 (m, 2H), 6.93 (dt, J= 7, 1 Hz, 1H), 2.42 (s, 3H), 2.23 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO- ,) δ 152.6, 137.6, 137.3, 130.2, 129.7, 127.8, 127.5,

126.1, 122.6, 121.0, 118.7, 17.9, 16.0 ppm; v _max (solid) 3283, 2977, 2919, 1637, 1581,

35 1543, 1489, 1282, 1236, 826, 796, 760, 740 cm ^"1 ; mlz (ESI pos. mode) 295.0 (MNa ⁺ ); (ESI neg. mode) 307.0 (M ³⁵ C1) ^" , 271.0 (M-H) ^~ Elemental analysis calcd for Ci ₅ Hi ₆ N ₂ OS: C, 66.15; H, 5.92; N, 10.29. Found: C, 65.96; H, 5.84; N, 10.17. Preparation of l-(4-methoxyphenyl)-3-(2-methylphenyl)urea (15)

o-Toluidine and 4-methoxyphenylisocyanate were combined using General Procedure I on a 3.35 mmol scale to give, after recrystallization from hot ethanol, the title product 15 as a white powder (0.300 g, 35%); mp 200 - 201°C; 1H NMR (400 MHz, DMSO- ) δ 8.83 (s, 1H), 7.86 (d, J= 8 Hz, 1H), 7.82 (s, 1H), 7.35 - 7.40 (m, 2H), 7.11 - 7.19 (m, 2H), 6.94 (dt, J= 8, 1 Hz, 1H), 6.86 - 6.91 (m, J= 9 Hz, 2H), 3.73 (s, 3H), 2.25 (s, 3H) ppm; ¹³ C NMR (101 MHz, DMSO-i ) δ 154.3, 152.8, 137.6, 132.9, 130.1, 127.2, 126.1, 122.4, 120.8, 119.7, 114.0, 55.1, 17.9 ppm; v _max (solid) 3292, 3042, 2837, 1638, 1598, 1555, 1509, 1455, 1239, 1032, 828, 799 cm ^"1 ; mlz (ESI pos. mode) 279.0 (MNa ⁺ ), 257.0 (MH ⁺ ); (ESI neg. mode) 291.0 (M ³⁵ C1) ^" , 255.1 (M-H ⁺ ) ^~ Elemental analysis calcd for Ci ₅ Hi ₆ N ₂ 0 ₂ : C, 70.29; H, 6.29; N, 10.93. Found: C, 70.02; H, 6.13; N, 10.80.

Preparation of l-(3-bromophenyl)-3-(2-methylphenyl)urea (16).

o-Tolyl isocyanate (666 mg, 5 mmol) and 3-bromoaniline (860 mg, 5 mmol) were combined using General Procedure I to produce the title compound 16 as a white solid (1.011 g, 66%); mp 215-218 °C; 1H NMR (300 MHz, DMSO-i ) δ 9.24 (s, 1H), 8.02 (s, 1H), 7.90 (t, J= 2 Hz, 1H), 7.78 (d, J= 8 Hz, 1H), 7.10 (m, 5H), 6.97 (t, J= 8 Hz, 1H), 2.24 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO) δ 176.63, 147.86, 111.54, 77.57, 70.33, 40.29, 40.01, 39.74, 39.61, 39.46, 39.18, 38.90, 38.62 ppm; v _max (solid) 3280, 1635, 1542 cm ^"1 ; m/z (ESI pos. mode) 383.981 (MHBr ⁺ , 100%), 405.956 (MHNaBr ⁺ , 93%); HRMS (ESI pos. mode) m/z Ci ₄ Hi ₃ BrN ₂ ONa required: 327.0103. Found: 327.0167. Elemental analysis calcd for Ci ₄ Hi ₃ BrN ₂ 0: C 55.10%, H 4.29%, N 9.18%. Found: C 54.88%, H 4.23%, N 9.01%.

Preparation of 3-(2-methylphenyl)-l-(3-nitrophenyl)thiourea (17)

o-Toluidine (0.54 g, 5 mmol) and 3-nitrophenylisothiocyanate (0.90 g, 5 mmol) were combined with General Procedure I to give, after recrystallization from hot methanol, the title product 17 as white crystals (0.41 g, 45 %); mp 171 - 173 °C; 1H NMR (300 MHz, chloroform-ύ δ 8.17 (t, J= 2 Hz, 1H), 8.01 - 7.91 (m, 1H), 7.92 - 7.79 (m, 1H), 7.71 (s, 1H), 7.42 (t, J = 8 Hz, 1H), 7.35 - 7.20 (m, 4H), 7.16 (s, 1H), 2.29 (s, 3H) ppm; ¹³ C (75

36 MHz, chloroform-ύ 180.5, 147.4, 141.0, 137.3 132.2, 134.9, 130.5, 129.6, 127.9, 126.8, 126.3, 118.5, 117.6, 17.8 ppm; Vmax (solid) 3340, 3149, 2956, 1534, 1497, 1459, 1339, 1283, 1258, 1237, 1208, 1155, 1108, 1089, 987, 945, 853, 805, 751, 741, 709, 687 cm ^"1 ; mlz = 286.0, (M ⁺ ), 310.0 (MNa ⁺ ); HRMS (ESI pos. mode) m/z Ci ₄ Hi ₃ N ₃ Na0 ₂ S (MNa ⁺ ) required 310.0621. Found: 310.0615.

Preparation of l-(3-cyanophenyl)-3-(2-methylphenyl)thiourea (18)

o-Toluidine (0.54 g, 5 mmol) and 3-cyanophenylisothiocyanate (0.90 g, 5 mmol) were combined with General Procedure I to give, after recrystallization from hot ethanol, the title product 18 as pale yellow crystals (0.42 g, 31%); mp 166 - 168 °C; 1H MR (300 MHz, chloroform-ύ δ 7.45 - 7.30 (m, 3H), 7.35 - 7.20 (m, 6H), 7.17 (s, 1H), 2.29 (s, 3H) ppm; ¹³ C (75.5 MHz, DMSO- ) δ 180.5, 140.6, 137.4, 134.9, 130.4, 129.7, 128.4, 127.9, 127.7, 126.8, 126.6, 126.2, 118.6, 111.0 17.8 ppm; v _max (solid) 3217, 3145, 2979, 2240, 1586, 1543, 1519, 1481, 1455, 1429, 1358, 1320, 1297, 1283, 1270 cm ^"1 ; mlz (ESI pos. mode) 268.0, (MH ⁺ ), 290.0 (MHNa ⁺ ); HRMS (ESI pos. mode) m/z Ci ₅ Hi ₃ N ₃ NaS (MNa ⁺ ) required 290.0722. Found: 290.0729.

Preparation of l-(2-methoxyphenyl)-3-(2-methylphenyl)thiourea (19)

o-Toluidine (0.54 g, 5 mmol) and 4-methylthiophenylisothiocyanate (0.91 g, 5 mmol) were combined with General Procedure I to give, after recrystallization from hot ethanol the title product 19 as white crystals (0.52 g, 38%); mp 140 - 142 °C; 1H NMR (300 MHz, chloroform-ύ δ 8.18 (d, J= 8 Hz, 1H), 7.75 (s, 1H), 7.53 (s, 1H), 7.35 - 7.18 (m, 4H), 7.09 (t, J= 8, 2 Hz, 1H), 6.93 (t, J= 8, 1 Hz, 1H), 6.81 (d, 1H), 3.68 (s, 3H), 2.28 (s, 3H); ¹³ C (75.5 MHz - DMSO-i ) δ 180.0, 151.6, 137.5, 134.9, 130.3, 128.2, 127.7, 126.6, 126.1, 125.8, 125.3, 119.8, 111.4, 55.6, 17.7 ppm; (solid) 3336, 3125, 2936, 1598, 1533, 1505, 1484, 1459, 1434, 1276, 1248, 1202, 1179, 1159, 1044, 1026, 745, 725, 699 cm ^"1 ; mlz 273.0 (MH ⁺ ), 295.0 (MHNa ⁺ ); HRMS (ESI pos. mode) m/z Ci ₅ Hi ₆ N ₂ NaOS (MHNa ⁺ ) required 295.0876. Found: 295.0874. Preparation of 3-(2-methylphenyl)-l-[4-(methylsulfanyl)phenyl]thiourea, (20) o-Toluidine (0.54 g, 5 mmol) and 4-methylthiophenylisothiocyanate (0.91 g, 5 mmol) were combined with General Procedure I to give, after recrystallization from hot ethanol, the title product 20 as white crystals (0.93 g, 62%); mp 149 - 151°C; 1H NMR (300 MHz,

DMSO-i ) δ 9.64 (s, 1H), 9.33 (s, 1H), 7.48 - 7.37 (m, 2H), 7.30 - 7.10 (m, 6H), 2.46 (s,

37 3H), 2.24 (s, 3H); ^li C (75.5 MHz, DMSO- ,) δ 180.3, 164.0, 137.7, 136.7, 134.8, 133.5, 130.3, 127.9, 126.4, 126.4, 126.1, 124.6, 124.4, 17.9, 15.2 ppm; Vmax (solid) 3223, 1580 (w), 1498 (s), 1347, 1255, 1201, 1089, 1016, 853, 790, 751, 718, 627 cm ^"1 ; mlz 31 1.0 (MHNa ⁺ ); HRMS (ESI pos. mode) m/z Ci ₅ Hi ₆ N ₂ NaS ₂ (MHNa ⁺ ) required 31 1.0647.

Found: 31 1.0637.

Preparation of 3-(2-methylcyclohexyl)-l-(3-nitrophenyl)urea 21

2-Methylcyclohexylamine and 3-nitrophenylisocyanate were combined using General Procedure I on a 3.05 mmol scale, to give after recrystallization from hot ethanol, the title product 21 as yellow needles (0.202 g, 24%); mp 173 - 175 °C; 1H NMR (400 MHz,

DMSO-i ) δ 8.85 (s, 1H), 8.53 (t, J = 2 Hz, 1H), 7.74 (dd, J= 2, 8 Hz, 1H), 7.61 (d, J = 9 Hz, 1H), 7.50 (t, 7= 8 Hz, 1H), 6.18 (d, 7 = 9 Hz, 1H), 3.16 (ddt, 7 = 4, 9, 1 1 Hz, 1H), 1.86 (dd, 7= 3, 13 Hz, 1H), 1.72 (dt, 7 = 2, 13 Hz, 2H), 1.62 (d, 7= 12 Hz, 1H), 0.98 - 1.37 (m, 5H), 0.91 (d, 7 = 7 Hz, 3H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) δ 154.4, 148.1, 141.9, 129.8, 123.5, 1 15.3, 1 1 1.3, 53.9, 37.6, 34.0, 33.5, 25.3, 25.2, 19.2 ppm; v _max (solid) 2195, 3072, 2927, 2856, 1655, 1638, 1519, 1346 cm ^"1 ; mlz (ESI pos. mode) 300.0 (MNa ⁺ ); (ESI neg. mode) 312.1 (M ³⁵ C1 ^" ), 276.1 (M-H ⁺ ). Elemental analysis calcd for C14H19N3O3 : C, 60.63; H, 6.91 ; N, 15.15. Found: C, 60.53; H, 6.85; N, 15.04. Preparation of l-(4-cyanophenyl)-3-(2-methylcyclohexyl)urea (22)

4-Cyanophenyl isocyanate (721 mg, 5 mmol) and l-amino-2-methyl cyclohexane (566 mg, 5 mmol) were combined with General Procedure I to produce a white solid. This product was recrystallized from hot ethanol and a small volume of water to give the title compound 11 as a white powder (529 mg, 49%), mp 190-192 °C; 1H NMR (300 MHz, chloroform-i ) δ 7.63 (s, 1H), 7.56 - 7.42 (dd, 7 = 9 Hz, 5 Hz, 4H), 5.24 (d, 7 = 9 Hz, 1H), 3.26 (dq,

7 = 1 1, 4 Hz, 1H), 1.98 (dd J = 14, 3 Hz) 1.76 - 1.61 (m, 4H), 1.16 - 0.96 (m, 4H) 0.94 (d, 7 = 7 Hz, 3H) ppm; ¹³ C NMR (75.5 MHz, chloroform-i ) δ 154.8, 144.0, 133.3, 1 19.4, 1 18.3, 104.5, 58.6, 55.1, 38.7, 34.3, 25.6, 25.4, 19.2 ppm; (solid) 3302, 2924, 2226, 1634, 1558, 1516, 1310, 1228, 1 175, 835 cm ^"1 ; mlz (ESI pos. mode) 258.1 (MH ⁺ , 36%), 280.1 (MNa ⁺ , 100%); HRMS mlz (ESI pos. mode) C15H20N3O required 258.1601. Found 258.1600. Elemental analysis calcd for C15H19N3O: C 70.01%, H 7.44%, N 16.33%. Found C 69.98%, H 7.44%, N 16.20%.

Preparation of l-(3-cyanophenyl)-3-(2-methylcyclohexyl)urea (23)

38 To a stirred solution of 2-methylcyclohexylamine (0.57 g, 5 mmol) in dichloromethane (5 mL) at 0 °C under nitrogen was added a solution of 3-cyanophenyl isocyanate (0.72 g, 5 mmol) in dichloromethane (5 mL) from a pressure equalized dropping funnel. The reaction was stirred overnight and the crude product was isolated by evaporation under reduced pressure. Trituration with ethanol - water and brief contact with dry ice / acetone gave the title product 23 as a white solid (0.38 g, 30%); mp 168 - 174°C; 1H (300 MHz, DMSO-i ) δ 8.65 (s, lH), 7.94 (s, 1H), 7.55 (d, J= 9 Hz, 1H), 7.41 (t, J= 8 Hz, 1H), 7.30 (J= 10 Hz, 1H), 6.18 (d, J= 9 Hz, 1H) 3.14 (dq, J= 10, 4 Hz, 1H), 1.85 (m, 1H), 1.78 - 1.51 (m, 4H), 1.35 - 1.00 (m, 6H), 0.90 (d, J= 6 Hz, 3H) ppm; ¹³ C (75.5 MHz, DMS0 ) δ 154.5, 141.5, 130.0, 124.2, 122.0, 119.9, 119.0, 111.4, 53.8, 37.7, 34.0, 33.5, 25.3, 25.2, 19.2 ppm; v _max (solid) 3395, 3300, 2928, 2850, 1653, 1547, 1481, 1411, 1310, 1232, 1029, 880, 809, 686, 591, 475 cm ^"1 ; mlz (ESI pos. mode) 280.1 (MNa ⁺ ), 258.1 (MH ⁺ ). HRMS (ESI pos. mode) mlz C ₁ 5H ₂ 0N3 ₀ (MH ⁺ ) required 258.1601. Found 258.1602. Preparation of l-(4-cyanophenyl)-3-octylurea (24).

4-Cyanophenyl isocyanate (544 mg, 5 mmol) and octylamine (642 mg, 5 mmol) were combined using General Procedure I to produce a white solid. This product was purified using flash column chromatography (Si0 ₂ , 30:70 EtOAc : petroleum ether 40-60°) to give the title compound 24 as a white powder (887 mg, 76%); mp 115-120 °C; 1H MR (300 MHz, chloroform-ύ δ 7.64 (s, 1H), 7.56 - 7.41 (m, 4H), 5.47 (t, J= 6 Hz, 1H), 3.29 - 3.16 (q, J= 7 Hz, 2H), 1.50 (m, 2H), 1.37 - 1.17 (m, 10H), 0.86 (t, J= 7 Hz, 3H) ppm; ¹³ C NMR (75 MHz, chloroform-i ) δ 164.4, 132.7, 117.9, 42.7, 31.2, 29.4, 28.6, 26.3, 22.2 ppm; Vmax (solid) 3374, 2923, 2219, 1685, 1657, 1592, 1535, 1507, 1320, 1231, 1172, 835 cm ^"1 ; m/z (ESI pos. mode) 272.1 (M-H ⁺ , 100%). Elemental analysis calcd for Ci ₆ H ₂ 3N ₃ 0: C 70.30%, H 8.48%, N 15.37%. Found: C 70.39%, H 8.46%, N 15.63%.

Preparation of l-(2-methylphenyl)-3-octylurea, (25)

To a stirred solution of octylamine (0.65 g, 5 mmol) in dichloromethane (5 mL) at 0 °C under dry nitrogen was added a solution of 2-methylphenyl isocyanate (0.67 g, 5 mmol) in dichloromethane (5 mL) from a pressure equalized dropping funnel. The reaction was stirred overnight and the white solid was collected by filtration under reduced pressure on a sintered glass funnel. Recrystallization from hot ethanol, with addition of a small quantity of water, gave the title product 25 as a white solid (0.46 g, 36%); mp 113 °C; 1H (300 MHz, chloroform-ύ δ 7.35 (1H, d, J= 8 Hz), 7.20-7.05 (4H, m), 5.85 (1H, br. s.), 4.50 (1H, br with app. t structure), 3.20 (2H, dd, J= 10, 7 Hz), 2.25 (3H, s), 1.60 (2H, s, bound H ₂ 0), 1.48 (2H, m), 1.30 - 1.20 (10H, m), 0.85 (3H, t, J= 7 Hz) ppm; ¹³ C (75.5 MHz, chloroform-ύ δ 164.6, 155.9, 135.5, 130.4, 126.5, 125.4, 125.0, 39.8, 31.2, 29.6, 28.7, 28.6, 26.2, 22.0, 17.3, 13.5 ppm; v _max (solid) 3336, 3300, 2960, 2926, 2853, 1585, 1552, 1481, 1458, 1303, 1290, 1260, 1236, 1213, 1190, 1106, 1082, 1043, 758, 734 cm ^"1 ; mlz (ESI neg. mode) 261 (M-H ⁺ ). HRMS (ESI pos. mode) mlz Ci ₆ H ₂ 6N ₂ NaO required

285.1937, found 285.1932.

Preparation of 3-[2-(dimethylamino)ethyl]-l-(2-methylphenyl)urea (26)

To a stirred solution of N,N-dimethylethylamine (0.44 g, 5 mmol) in dichloromethane (5 mL) at 0 °C under dry nitrogen was added a solution of 2-methylphenyl isocyanate (0.67 g, 5 mmol) in dichloromethane (5 mL) dropwise from a pressure equalized dropping funnel. The reaction was stirred overnight then evaporated under reduced pressure. The solid product was recrystallized from warm diethyl ether with a few drops of dichloromethane, which gave after 4 days, the title product 26 as a white solid (0.72 g, 65%); mp 120 -

123°C; 1H (300 MHz, chloroform-i ) δ 7.50 (dd, J= 8, 1 Hz, 1H), 7.20 - 7.10 (m, 3H) 6.95 (dd, J= 7, 6 Hz, 1H), 5.65 - 5.58 (1H, m), 3.30 (2H, q, J= 5.5 Hz), 2.40 (t, J= 5.8 Hz, 2H), 2.24 (s, 3H), 2.17 (s, 6H) ppm; ¹³ C (75.5 MHz, chloroform-i ) δ 156.4, 136.2, 130.6, 130.1, 126.2, 124.3, 123.9, 58.4, 44.6, 37.4, 17.3 ppm; v _max (solid) 3300, 2900, 2760, 1630, 1562, 1458, 1292, 1243, 1096, 1057, 850, 754, 665 cm ^"1 ; mlz (ESI pos. mode) 222 (MH ⁺ ). HRMS (ESI pos. mode) mlz Ci ₂ H ₂₀ N ₃ O (MH ⁺ ) required 222.1601. Found 222.1605.

Preparation of N-(2-methylphenyl)morpholine-4-carboxamide (27)

To a stirred solution of morpholine (0.45 g, 5 mmol) in dichloromethane (5 mL) at 0 °C under nitrogen, was added o-tolyl isocyanate (0.67 g, 5 mmol) in a solution of

dichloromethane (5 mL) dropwise from a pressure equalized dropping funnel. The reaction was stirred overnight and the white precipitate collected by filtration under reduced pressure (0.38 g, 38%), estimated >95% pure by MR and thin layer

chromatography. This solid was recrystallized with some difficulty from ethanol - water to give the title product 27 as colourless crystals (0.06 g, 5%); mp 160 - 163°C; 1H (300 MHz, chloroform-ύ δ 7.60 (d, J 8.1 = Hz, 1H), 7.20 - 7.10 (m, 2H), 7.00 (1H, dd, J= 8 Hz), 6.10 (br. S, 1H), 3.72 (dd, J= 5, 4 Hz, 4H), 3.48 (4H, dd, J= 5, 4 Hz), 2.22 (s, 3H) ppm; ¹³ C (75.5 MHz, chloroform-i ) δ 155.5, 136.7, 130.4, 129.2, 126.8, 124.4, 123.1, 66.5, 44.4, 17.8 ppm; v _max (solid) 3286, 3000, 2850, 1630, 1510, 1456, 1379, 1300, 1255, 1112, 996, 888, 744, 714, 565 cm ^"1 ; mlz (ESI pos. mode) 243. 1, (MNa ⁺ ), 463.2 (M ₂ Na ⁺ ); HRMS (ESI pos. mode) mlz Ci ₂ Hi ₆ N ₂ Na0 ₂ (MNa ⁺ ) required 243.1104. Found 243.1108.

Preparation of l-(2-methylphenyl)-3-octylthiourea (28)

General Procedure I was used with o-tolyl isothiocyanate (0.75 g, 5 mmol) and octylamine (0.65 g, 5 mmol) to give, after purification by flash column chromatography (Si0 ₂ , 15 :85 EtOAc : 40-60° petroleum ether) the title product 28 as a colourless oil (0.12 g, 9 %); mp 95 - 97 °C; ¹ H MR (300 MHz, chloroform-i ) δ 7.36 - 7.12 (m, 5H), 5.59 (s, 1H), 3.57 (dd, J= 8, 6 Hz, 2H), 2.26 (s, 3H), 1.32 - 1.18 (m, 10H), 0.85 (t, J = 7 Hz, 3H) ppm; ¹³ C (75.5 MHz, chloroform-ύ δ 180.3, 135.3, 131.2, 128.0, 127.0, 126.9, 45.0, 28.5, 31.1,

28.6, 28.5 26.2, 22.0, 17.2, 13.5 ppm; v _max (oil) 3157, 2953, 2925, 2849, 1536, 1504, 1456, 1282, 1249, 1214, 1175, 1152, 1115, 1086, 1036, 740, 707 cm ^"1 , mlz (ESI pos. mode) 279.1 (MH ⁺ ), 301.1 (MHNa ⁺ ); HRMS (ESI pos. mode) mlz Ci ₆ H ₂₆ N ₂ NaS (MNaH ⁺ ) required 301.1709. Found 301.1695.

Preparation of 3-[2-(dimethylamino)ethyl]-l-(2-methylphenyl)thiourea (29)

General Procedure I was used with o-tolyl isothiocyanate (0.75 g, 5 mmol) and NJT- dimethylenediamine (0.44 g, 5 mmol) to give, after purification by flash column chromatography (Si0 ₂ , 15:85 EtOAc : 40-60° petroleum ether) the title product 29 as a colourless oil (0.80 g, 67 %); 1H MR (300 MHz, chloroform-i ) δ 7.75 (s, 1H), 7.29 - 7.12 (m, 4H), 6.57 (s, 1H), 3.64 - 3.55 (m, 2H), 2.38 (t, J= 6 Hz, 2H), 2.25 (s, 3H), 2.04 (s, 6H) ppm; ¹³ C NMR (75.5 MHz, chloroform-i ) δ 181.3, 134.5, 130.6, 129.7, 127.2, 122.9, 120.1, 43.6, 40.3, 26.5, 17.4 ppm; mlz (ESI pos mode) 238.1 (M ⁺ H). HRMS (ESI pos. mode) mlz Ci ₂ H ₂₀ N ₃ S (MH ⁺ ) required 238.1372, found 238.1362.

Preparation of N-(2-methylphenyl)morpholine-4-carbothioamide, (30)

General Procedure I was used with o-tolyl isothiocyanate (0.75 g, 5 mmol) and morpholine

(0.44 g, 5 mmol) to give, after recrystallization from hot ethanol the title product 30 as white crystals (1.08 g, 92 %); mp 145 - 147 °C; 1H NMR (300 MHz, chloroform-i ) δ 7.26 - 7.03 (m, 4H), 6.95 (s, 1H), 3.81 - 3.64 (m, 8H), 2.24 (s, 3H) ppm; ¹³ C (75.5 MHz, chloroform-i δ 183.7, 138.0 131.6, 130.5, 126.3, 125.8, 124.2, 65.5, 49.0, 17.4 ppm;

Vmax (oil) 3159, 2854, 1518, 1492, 1461, 1401, 1318, 1295, 1281, 1264, 1218, 1204, 1114,

1064, 1027, 942, 910, 864, 751, 719, 693 cm ^"1 ; mlz 237.0 (MH ⁺ ), 259.0 (MNaH ⁺ ). HRMS

(ESI pos. mode) mlz Ci ₂ Hi ₆ N ₂ NaOS (MNaH ⁺ ) required 259.0876. Found 259.0863.

41 General Procedure II - addition of isocyanate to bifunctional amines.

Aromatic bis-ureas and -thioureas were prepared using a previously reported method (Radi, M.; Falchi, F.; Garbelli, A.; Samuele, A.; Bernardo, V.; Paolucci, S.; Baldanti, F.; Schenone, S.; Manetti, F.; Maga, G.; Botta, M., Discovery of the first small molecule inhibitor of human DDX3X specifically designed to target the RNA binding site: Towards the next generation HIV-1 inhibitors. Bioorg. Med. Chem. Lett. 2012, 22 (5), 2094-2098). To a solution of the amine (1 equiv., 5 mmol) in dry dichloromethane (5 mL) under dry N ₂ was added the isocyanate (2 equiv., 10 mmol) in dry dichloromethane (-10 mL) with stirring, and left overnight at room temperature. The precipitate was collected by filtration and washed with hot ethanol on a fluted filter paper. The resulting solid was insoluble in most solvents except dimethylformamide and dimethylsulfoxide.

Preparation of l,l'-(l ₉ 3-phenylene)bis(3-(3-nitrophenyl)urea) (31).

Compound 31 was prepared using General Procedure II with w-phenylenediamine

(540 mg, 5 mmol) and nitrophenyl isocyanate (1.64 g, 10 mmol) to produce the title compound 31 as a white-yellow solid (1.948 g, 85%); mp 289 ± 1 °C; 1H NMR (300 MHz, DMSO-i ) δ 9.15 (s, 2H), 8.93 (s, 2H), 8.60 (t, J= 2 Hz, 2H), 7.88 - 7.75 (m, 3H), 7.57 (t, J = 8 Hz, 2H) 7.20 (dd, J = 9, 7 Hz 1H), 7.10 (dd, J= 7, 2 Hz, 2H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) 5 152.3, 148.1, 141.0, 139.7, 130.0, 129.1, 124.3, 116.2, 112.4, 112.1, 108.5 ppm; v _max (solid) 3291, 1651, 1595, 1551 cm ^"1 ; m/z (ESI pos. mode) 459.124 (MNa ⁺ , 41%), 461.140 (MNaH ₂ ⁺ , 100%), 463.159 (MNaH ₄ ⁺ , 25%), 465.227 (MNaH ₆ ⁺ , 13%). HRMS (ESI pos. mode) mlz C ₂₀ Hi ₆ N ₆ NaO ₆ required 459.1024. Found 459.0812.

Elemental analysis calcd for C ₂₀ Hi ₆ N ₆ O6: C 55.05%, H 3.70%, N 19.26%. Found: C 54.31%, H 3.67%, N 19.13%.

Preparation of l,l'-(l ₉ 3-phenylene)bis(3-(3-nitrophenyl)thiourea), (32)

General Procedure II was used with w-phenylenediamine (0.38 g, 3.5 mmol) and 3- nitrophenylisothiocyanate (1.27 g, 5 mmol) to give after recrystallisation from hot ethanol the title product as yellow crystals (0.97 g, 60%); mp 190 - 193 °C; 1H NMR (300 MHz,

DMSO-i ) δ 10.29 - 10.22 (m, 2H), 10.08 (s, 2H), 8.45 (t, J= 2 Hz, 2H), 7.97 - 7.80 (m,

4H), 7.68 (t, J= 2 Hz, 1H), 7.54 (t, J= 8 Hz, 2H), 7.37 (dd, J= 9, 7 Hz, 1H), 7.31 - 7.21

(m, 2H) ppm; ¹³ C (75.5 MHz, DMSO- ,) δ 179.5, 164.0, 147.3, 140.8, 139.1, 129.8, 129.4,

129.0, 120.0, 118.6, 117.8, ppm; v _max (oil) 3016, 2364, 1523, 1455, 1438, 1347, 1319,

42 1281, 1234, 1091, 1000, 970, 935, 878, 862, 831, 787, 742, 730, 682, 665 cm ^"1 . HRMS (ESI pos. mode) mlz C ₂ oHi ₆ N6Na0 ₄ S2 required 491.0567. Found 491.0569.

Preparation of l,l'-(l ₉ 3-phenylene)bis(3-(3-(methylthio)phenyl)urea) (33).

Compound 33 was prepared using general procedure II with w-phenylenediamine (600 mg, 5 mmol) and 3-(methylthio)phenyl isocyanate (1.76 g, 10 mmol) to give the title compound 33 as a white powder (1.54 g, 65%); mp 305 ± 1 °C; 1H NMR (300 MHz, DMSO-^ ₆ ) δ 8.76 (br. s, 1H), 8.66 (br. s, 1H), 7.67 (d, J= 2 Hz, 1H), 7.49 (t, J= 2 Hz, 1H), 7.20 - 7.01 (m, 5H), 6.86 (d, J = 8 Hz, 1H), 5.76 (s, 4H), 2.46 (s, 6H) ppm; ¹³ C NMR (75 MHz, DMSO-i ) δ 164.0, 152.3, 140.2, 138.6, 129.2, 129.1, 119.1, 115.0, 114.6, 111.8, 107.9, 54.9 ppm; v _max (solid) 3276, 1633, 1553, 1479, 1395, 1281, 1222, 681, 647 cm ^"1 ; m/z (ESI pos. mode) 439.180 (MH ⁺ , 12%), 461.159 (MNa ⁺ , 100%); HRMS (ESI pos. mode) mlz C ₂₂ H ₂₂ N ₄ Na0 ₂ S2 required 461.1076. Found 461.1232. Elemental analysis calcd for C22H22N4O2S2: C 60.25%, H 5.06%, N 12.78%. Found: C 60.10%, H 5.02%, N 12.77%.

Preparation of l,l'-(l ₉ 3-phenylene)bis(3-(3-methoxyphenyl)urea) (34).

Compound 34 was prepared using general procedure II using w-phenylenediamine

(541 mg, 5 mmol) and 3-methoxyphenylisocyanate (1.49 g, 10 mmol) to produce the title compound M as a white solid (440 mg, 22%); mp 294-297 °C; 1H NMR (300 MHz, DMSO-i ) δ 8.61 (s, 2H), 8.41 (s, 2H), 7.62 (t, J= 2 Hz, 1H), 7.35 (d, J= 7 Hz, 4H), 7.12 (m, 1H), 7.00 (d, J= 7 Hz, 1H), 6.86 (d, J = 7 Hz, 4H), 3.71 (s, 6H) ppm; ¹³ C NMR (75 MHz, DMSO-i¾) 5 163.8 154.4, 129.0, 119.9, 113.9, 111.4, 55.1 ppm; (solid) 3305, 1639, 1599, 1562, 1512, 1493, 1300, 1242, 1035, 879, 648 cm ^"1 ; m/z (ESI pos. mode) 429.214 (MNa ⁺ , 100%); HRMS (ESI pos. mode) mlz C ₂₂ H ₂₂ N ₄ Na0 ₄ required 429.1533. Found 429.1669. Elemental analysis calcd for C ₂₂ H ₂₂ N ₄ 0 ₄ : C 65.01%, H 5.46%, N

14.78%. Found: C 64.71%, H 5.56%, N 14.78%.

Preparation of l-(2-nitrophenyl)-3-(3-{2-[2-(3-{[(2-nitrophenyl)carbamoyl]a mino}- propoxy)ethoxy] ethoxy} propyl)ur ea (35)

To a stirred solution of 4,7, 10-trioxa-l, 13-tridecanediamine (0.82 g, 10 mmol) in dichloromethane (10 mL) at 0 °C under nitrogen was added a solution of 3-nitrophenyl isocyanate (3.28 g, 20 mmol) in dichloromethane (10 mL) from a pressure equalized dropping funnel. The reaction was stirred overnight and the crude product was isolated a by removal of volatiles on a rotary evaporator in an efficient fumehood. Purification by

43 flash column chromatography (Si0 ₂ , 1% MeOH - EtOAC gave the title product 35 as a white solid (3.703 g, 68%) mp 99 - 101 °C; 1H (300 MHz, DMSO-i ) δ 9.00 (s, 1H), 8.50 (t, J= 2 Hz, 1H), 7.72 (dd, J= 7, 1 Hz, 1H), 7.52 (dd, J 8, 1 = Hz, 1H), 7.49 (t, J= 8 Hz, 1H), 6.32 (t, J = 6 Hz, 1H), 3.60 - 3.50 (m, 4H), 3.48 (m, 4H), 3.15 (q, J = 7 Hz, 2H), 1.70 (q, J= 7 Hz, 2H) ppm; ¹³ C (75.5 MHz, DMSO-i ) δ 154.9, 148.1, 141.9, 129.8, 123.6, 123.6, 115.3, 111.4, 69.7, 69.5, 68.1, 36.6, 29.8 ppm; Vmax (solid) 3326, 3300, 2950, 2900, 1634, 1553, 1481, 1347, 1259, 1102, 968, 874, 832, 801, 728, 690, 640 cm ^"1 ; mlz (ESI pos. mode) 571.1 (MNa ⁺ ); ESI (neg. mode) 547.1 (M-H) ^" , 583.1 (M ³⁵ C1) ^" , 585.1 (M ³⁷ C1) ^~ . HRMS (ESI pos. mode) mlz C24H33N6O9 (MNa ⁺ ) required 549.2304, found 549.2309.

Preparation of l-[3-(methylsulfanyl)phenyl]-3-[3-(2-{2-[3-({[3-(methylsulfa nyl)- phenyl] carbamoyl}amino)propoxy] ethoxy}ethoxy)propyl] urea, (36)

To a solution of 4,7, 10-trioxa-l, 13-tridecanediamine (1.13 g, 5.1 mmol) in

dichloromethane (10 mL) at 0 °C was added dropwise 3-(methylthio)phenyl isocyanate (1.67 g, 10.1 mmol) in dichloromethane (10 mL). The solution was allowed to warm to room temperature and stirred for 3 hrs, then evaporated under reduced pressure. The reaction mixture was purified by flash column chromatography (Si0 ₂ , 5:95 MeOH:EtOAc) to give the title product 36 as a white solid (1.91 g, 3.47 mmol, 68.7%); mp 106-108 °C; 1H MR (300 MHz, CDC1 ₃ ) δ 7.40 (t, J= 2 Hz, 2H), 7.33 (s, 2H), 7.15 (t, J= 8 Hz, 2H), 7.05 (dd, J= 7, 2 Hz, 2H), 6.84 (dd, J= 8, 1 Hz, 2H), 5.62 (t, J= 6 Hz, 2H), 3.70-3.65 (m, 4H), 3.63-3.58 (m, 4H), 3.57 (t, J= 6 Hz, 4H), 3.30 (quartet, J= 6 Hz, 4H), 2.42 (s, 6H), 1.73 (quintet, J= 6 Hz, 4H); ¹³ C NMR (101 MHz, CDC1 ₃ ) δ 156.1, 140.2, 139.3, 129.2, 120.4, 116.7, 115.7, 70.3, 69.9, 69.3, 38.4, 28.9, 15.6 ppm; v _max (solid) 3303, 2865, 1628, 1562, 1481, 1117, 613 cm ^"1 ; mlz (ESI pos. mode) 573.1 (MNa ⁺ ); Elemental analysis calcd for C ₂₆ H ₃₈ N ₄ 0 ₅ S ₂ : C 56.70% H 6.69% N 10.17%. Found: C 56.70% H 6.70% N 10.20%.

Preparation of l-[4-(methylsulfanyl)phenyl]-3-[3-(2-{2-[3-({[4-(methylsulfa nyl)- phenyl] carbamoyl} amino)propoxy] ethoxy}ethoxy)propyl] urea, (37)

To a solution of 4,7, 10-trioxa-l, 13-tridecanediamine (1.23 g, 5.58 mmol) in

dichloromethane (10 mL) at 0 °C was added dropwise a solution of 4-(methylthio)phenyl isocyanate (1.68 g, 10.2 mmol) in dichloromethane (10 mL). The solution was allowed to warm to room temperature and stirred for 24 hrs, then evaporated under reduced pressure.

The reaction mixture was purified by flash column chromatography (Si0 ₂ , 10:90

MeOH:EtOAc), followed by recrystallization from hot ethanol to give the title product 37

44 as a white solid (0.464 g, 0.84 mmol, 16.5%); mp 137-139 °C; 1H MR (400 MHz, CDC1 ₃ ) δ 7.32 (d, J= 9 Hz, 4H), 7.22 - 7.18 (br. s, 2H), 7.20 (d, J= 9 Hz, 4H), 5.55 (s, 2H), 3.73 - 3.68 (m, 4H), 3.65 - 3.62 (m, 4H), 3.60 (t, J= 6 Hz, 4H), 3.35 (quartet, J= 6 Hz, 4H), 2.44 (s, 6H), 1.76 (quintet, J= 6 Hz, 4H) ppm; ¹³ C MR (101 MHz, CDCI ₃ ) δ 156.1, 137.4, 131.2, 128.7, 120.0, 70.3, 69.9, 69.2, 38.5, 28.9, 17.2 ppm; v _max (solid) 3299, 2868, 1634, 1594, 1558, 1136, 817, 506 cm ^"1 ; mlz (ESI pos. mode) 573.1 (MNa ⁺ ). Elemental analysis calcd for C ₂₆ H ₃₈ N ₄ O ₅ S ₂ C 56.70% H 6.96% N 10.17%. Found C 56.30% H 6.92% N 10.13%. Preparation of l-(2-methylphenyl)-3-(3-{2-[2-(3-{[(2-methylphenyl)carbamoyl ]- amino}propoxy)ethoxy]ethoxy}propyl)urea, (38)

To a solution of 4,7, 10-trioxa-l, 13-tridecanediamine (1.12 g, 5.08 mmol) in

dichloromethane (10 mL) at 0 °C, was added dropwise a solution of o-tolyl isocyanate (1.45 g, 10.9 mmol) in dichloromethane (10 mL). The solution was allowed to warm to room temperature and stirred for 24 hrs, then evaporated under reduced pressure. The reaction mixture was then purified by flash column chromatography (S1O ₂ , 10:90

MeOH:EtOAc) to give the title product 38 as a white solid (1.83 g, 3.76 mmol, 74.0%); mp 78-82 °C; 1H NMR (400 MHz, CDC1 ₃ ) δ 7.53 (d, J= 8 Hz, 2H), 7.16 (t, J= 7 Hz, 4H), 7.02 (t, J= 6 Hz, 2H), 6.40 (s, NH, 2H), 5.41 (t, J= 6 Hz, NH, 2H), 3.60-3.51 (m, 12H), 3.32 (quartet, J = 6 Hz, 4H), 2.24 (s, 6H), 1.73 (quintet, J = 6 Hz, 4H) ppm; ¹³ C NMR

(101 MHz, CDCI ₃ ) δ 156.7, 136.9, 130.6, 126.7, 124.5, 124.0, 70.4, 69.7, 69.5, 38.1, 29.5, 17.9 ppm; v _max (solid) 3306.5, 1629.4, 1561.9, 1243.0, 1104.4, 753.3, 652.2 cm ^"1 , mlz (ESI pos mode) 509.2 (MNa ⁺ ). Elemental analysis calcd for C ₂₆ H ₃₈ N ₄ O ₅ C 64.16% H 7.88% N 11.52%. Found C 64.03% H 7.88% N 11.48%.

Preparation of l-(3-chlorophenyl)-3-(o-tolyl)urea, (39)

o-Toluidine and 3-chlorophenylisocyanate were combined using General Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 39 (1.02 g, 78%); mp 201-202 °C; 1H NMR (300 MHz, DMS0 ) δ 9.21(s, 1H), 7.99 (s, 1H), 7.80 (d, J=8 Hz, 1H), 7.76-7.72 (m, 1H), 7.34-7.09 (m, 4H), 7.04-6.92 (m, 2H), 2.24 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.0, 152.5, 141.4, 137.0, 133.2, 130.4, 130.2, 127.9, 126.1, 123.0, 121.3, 117.3, 116.4, 17.8 ppm; v _max (solid) 3287, 1633, 1585, 1546, 1480, 1457, 1269, 1259, 1235, 760 cm ^"1 ; mlz Ci ₄ Hi ₃ ClN ₂ 0 (ESI pos. mode) Calcd for [MNa] ⁺ = 283.06 Found: 283.0. Preparation of l-(o-tolyl)-3-(3-(trifluoromethyl)phenyl)urea, (40)

o-Toluidine and 3-trifluoromethylphenylisocyanate were combined using General

Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 40 (1.27 g, 86%); mp 185 °C; 1H MR (300 MHz, DMSO): δ 9.37(s, 1H), 8.03 (s, 2H), 7.78 (d, J=8 Hz, 1H), 7.60-7.48 (m, 2H), 7.31 (d, J=7 Hz, 1H), 7.22-7.08 (m, 2H), 6.97 (t, J=8 Hz, 1H), 2.25 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.0, 152.6, 140.7, 136.9, 130.2, 129.9, 129.7, 129.3, 128.1, 126.1, 123.1, 121.5, 117.9, 113.8, 17.8 ppm; Vmax (solid) 3298, 1637, 1560, 1331, 1155, 1124, 1067 cm ^"1 ; mlz C ₁₅ H ₁₃ F ₃ N ₂ O (ESI pos. mode) Calcd for [MNa] ⁺ = 317.09 Found: 317.1.

Preparation of l-(4-bromophenyl)-3-(i>-tolyl)urea (41)

o-Toluidine and 3-bromophenylisocyanate were combined using General Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 41 (1.19 g, 78%); mp 240 °C; 1H NMR (300 MHz, DMSO): δ 9.14(s, 1H), 7.95 (s, 1H), 7.81 (d, J=8 Hz, 1H), 7.44 (s, 4H), 7.21-7.09 (m, 2H), 6.95 (t, J=8 Hz, 1H), 2.23 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.1, 152.5, 139.3, 137.1, 131.5, 130.2, 127.7, 126.1, 122.9, 121.2, 119.9, 113.0, 17.8 ppm; v _max (solid) 3288, 1637, 1584, 1546, 1478, 1457, 1238, 1071, 1009, 835, 796 cm ^"1 ; mlz Ci ₄ Hi ₃ BrN ₂ 0 (ESI pos. mode) Calcd for [MNa] ⁺ = 329.01 Found: 329.0.

Preparation of l-(o-tolyl)-3-(4-((trifluoromethyl)thio)phenyl)urea (42)

o-Toluidine and 4-thiotrifluoromethylphenylisocyanate were combined using General Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 42 (1.24 g, 76%) mp 222-224 °C; 1H NMR (300 MHz, DMSO): δ 9.39(s, 1H), 8.05 (s,

1H), 7.81 (d, J=8 Hz, 1H), 7.62 (s, 4H), 7.21-7.11 (m, 2H), 6.97 (m, 1H), 2.24 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.0, 152.3, 143.1, 137.5, 136.9, 131.7, 130.2, 128.0, 126.2, 123.1, 121.4, 119.1, 118.7, 113.7, 17.8 ppm; v _max (solid) 3349, 3251, 3180, 3115, 3069, 1655, 1587, 1540, 1489, 1452, 1148, 1107, 1086 cm ^"1 ; mlz Ci ₅ Hi ₃ F ₃ N ₂ OS (ESI pos. mode) Calcd for [MNa] ⁺ = 349.06 Found: 349.1.

Preparation of l-(o-tolyl)-3-(4-(trifluoromethoxy)phenyl)urea (43)

o-Toluidine and 4-trifluoromethoxyphenylisocyanate were combined using General

Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product

46 43 (1.10 g, 71%); mp 190-191 °C; 1H NMR (300 MHz, DMSO): δ 9.21(s, 1H), 7.97 (s, 1H), 7.81 (d, J=8 Hz, 1H), 7.59-7.50 (m, 2H), 7.30 (d, J=9 Hz, 2H), 7.20-7.10 (m, 2H), 7.00-6.91 (m, lH), 2.24 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.1, 164.1, 154.41,

152.6, 139.1, 137.1, 130.2, 128.7, 127.8, 126.2, 122.9, 121.8, 121.2, 119.1, 17.8 ppm; Vmax (solid): 3279, 1640, 1551, 1263, 1202, 1152 cm ^"1 ; mlz C ₁₅ H ₁₃ F ₃ N ₂ O ₂ (ESI pos. mode)

Calcd for [MNa] ⁺ = 333.08 Found: 333.1.

Preparation of l-(4-iodophenyl)-3-(o-tolyl)urea (44)

o-Toluidine and 4-iodophenylisocyanate were combined using General Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 44 (1.43 g,

81%); mp 240-242 °C; 1H NMR (300 MHz, DMSO): δ 9.12(s, 1H), 7.94 (s, 1H), 7.81 (d, J=8 Hz, 1H), 7.63-7.56 (m, 2H), 7.35-7.28 (m, 2H), 7.21-7.09 (m, 2H), 6.99-6.91 (m, 1H), 2.23 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.3, 152.4, 139.7, 137.3, 137.1, 130.2,

127.7, 126.1, 122.8, 121.1, 120.2, 17.8 ppm; (solid) 3275, 1637, 1583, 1548, 1280, 1239, 1006, 833 cm ^"1 ; mlz Ci ₄ Hi ₃ IN ₂ 0 (ESI pos. mode) Calcd for [MNa] ⁺ = 375.0 Found: 375.0

Preparation of l-(3,4-dimethoxyphenyl)-3-(o-tolyl)urea (45).

o-Toluidine and 4-iodophenylisocyanate were combined using General Procedure I on a 5 mmol scale to give, after recrystallization from ethanol, the title product 45 (1.24 g, 87%); mp 192-194 °C; 1H NMR (300 MHz, DMSO): δ 8.87 (s, 1H), 7.87-7.79 (m, 2H), 7.21-7.08 (m, 3H), 6.97-6.83 (m, 3H), 3.76 (s, 3H), 3.72 (s, 3H), 2.23 (s, 3H) ppm; ¹³ C NMR (75 MHz, DMSO): δ 164.3, 152.7, 148.8, 143.8, 137.5, 133.5, 130.1, 127.2, 126.1, 122.4, 120.8, 112.5, 109.7, 103.5, 55.8, 17.8 ppm; 3296, 1631, 1549, 1512, 1269, 1230, 1137, 1025 cm ^"1 ; MS: mlz Ci ₆ Hi ₈ IN ₂ 0 ₃ (ESI pos. mode) Calcd for [MNa] ⁺ = 309.12 Found: 309.1.

Example: Plaque inhibition assay

Viruses

An identical assay procedure was carried out for hRSV (strain A2) bovine RS

(strain Snook) and PVM (strain 15). Virus stocks were diluted 10 fold in GMEM

(Invitrogen) so that the wells containing cells received sufficient virus to generate approximately 2000, 200 and 20 plaques per well.

47 Growth medium

GMEM plus 10% FCS (Biosera), penicillin (4 units/ml) and streptomycin (4 μ§/ιη1) (Invitrogen), 3 mM glutamine (Invitrogen).

Overlay medium

GMEM containing 2% (v/v) FCS, 1% (w/v) carboxymethylellulose (CMC) (Sigma), penicillin (4 units/ml), streptomycin (4 μg/ml), 3 mM glutamine.

The overlay medium also contained the compound to be tested at appropriate concentrations. Typically the 6 concentrations of the test compounds used were: 100 μΜ, 31.6 μΜ, 10 μΜ, 3.16 μΜ, 1 μΜ and 0.316 μΜ.

Test compounds

Stocks of the test compounds were prepared in DMSO (Sigma) (carrier). Each stock compound was diluted 1 in 200 in overlay medium to give dilutions stated above.

Plaque Assay

Cells of a standard cell line known to support pneumovirus replication (typically HEp2 or BSC1 cells) were grown in standard 12 well plates at 37 °C in an atmosphere of 5% (v/v) C0 ₂ until confluent. Medium was aspirated from the cells and replaced with 200 μΐ aliquots of virus. Aliquots were prepared by diluting virus stocks 10-fold in GMEM (Invitrogen) as described above with no FCS. A mock (no virus) was also set up to test cell viability. The virus was incubated with the cells for 1 hour at 33 °C with gentle rocking every 5-10 minutes. The inocula were removed and 2 ml of overlay medium was added. Overlay medium was either test (plus compound) or control (medium alone or medium containing DMSO). The plates were incubated at 33 °C for 4-8 days to allow plaques to develop as indicated. A volume of 1 ml of additional overlay medium

(containing the test compound as appropriate) was added every 3 days. All virus dilutions were carried out with each concentration of test compound, or treated as controls, and each was carried out in triplicate.

Following incubation, cells were fixed by addition of an equal volume of 4% (v/v) glutaraldehyde (Sigma) in PBS with further incubation for a minimum of 2 hours. The fixing solution was washed off under a slow flowing stream of water and 1 ml of crystal violet (0.075% w/v: Sigma) was added and left for 20 minutes at room temperature. The

48 crystal violet was removed and the fixed cell monolayers washed. Plates were air dried. Plaques counts were recorded from wells with >20 and <200 in number. These numbers provide the most robust statistical reliability for viable plaque counts.

This experimental approach screens the ability of compounds to block the formation of plaques. A lower number of plaques than the DMSO carrier control shows that the compound is inhibiting virus growth.

96 well plate TCID ₅₀

This method provides a rapid approach to initially screen compounds and was developed as an increased number of compounds were synthesised. Cells of a standard cell line known to support pneumovirus replication (typically HEp2 or BSC1 cells) were grown to 95% confluence in a 96 well plate. Virus was then added over 5 different half log dilutions starting at a multiplicity of infection of 0.02 plus mock control. Three dilutions of compound plus a DMSO control were screened per plate. Cells were fixed once the highest dilution of virus showed signification destruction or cell death in the DMSO control. Identical plate repeats for each compound dilution were fixed in subsequent days as indicated.

Stained plates were read at 595 nM, higher optical density (OD) readings indicate more viable cells remain. Therefore this application not only shows if virus growth is blocked but if the compound is detrimental to cells (reading the mock control).

Compound 1

Figure 1 shows two biological replicates carried out on different occasions for compound 1 using HEp2 cells.

We observed that plaques that were present in the treated wells at concentrations of compound 1 above 1 μΜ were smaller than in the DMSO control. The level of inhibition seen between the repeats clearly differs between the repeats. This may be because the plaques that counted are only just developing, making biological variables more pronounced. Both assays were stopped after 5 days but a small difference in host cell viability can delay viral growth and give the observed results. Compound 1 is relatively insoluble and a concentration between 5-10 μΜ represents an upper limit, consistent with no increase in activity seen at > 10 μΜ in the TCID50 assay. Note the required quantity of 1 gave the appearance of insolublity in cell medium at 100 μΜ and a virus inhibition experiment was not performed.

49 Following these initial results, further plaque assays were carried out with compounds 3, 4, 6, 35, 37, 39 and 40.

Compound 3

Figure 2 shows the effect of 3 on plaque size (relative to a DMSO control) after 5 days using F£Ep2 cells. Where plaques were present, their size was much smaller than those seen with the DMSO control. In particular, plaques observed at 31.6 μΜ and to some extent 10 μΜ were very small.

As before, the total number of plaques counted from 6 different wells in the presence of DMSO alone was set at 100% (standard deviation of the mean of DMSO control 5.4%). Plaques counted in the presence of 3 at the concentrations indicated are given as a percentage of the DMSO control. The IC ₅₀ was estimated to be 50 μΜ, although further experiments at concentrations of 30-80 μΜ are required to confirm this.

Compound 4

It was hypothesised that these compounds are delaying plaque formation, so that, at lower dilutions, virus plaques will appear. Further tests using HEp2 cells were carried out to screen inhibition over a number of days for compound 4. The results are shown in Figure 3. This confirmed the delayed plaque formation hypothesis.

IC ₅₀ values were calculated as 4.1 μΜ (Day 5), 8.5 μΜ (Day 6), and 15 μΜ

(Day 7). Thus, the data show an increasing IC ₅₀ value with time of incubation of the assay, as expected. This emphasises that the compound does not inhibit virus replication completely, but significantly limits it. This is consistent with our understanding of the role of the M2-2 protein in the virus replication cycle.

Compound 4 showed toxicity at 100 μΜ and to some degree at 31.6 μΜ. No obvious toxicity was seen at 10 μΜ, although this concentration was also less effective.

Compound 6

This compound was tested using assays stopped after 5, 6 and 7 days using HEp2 cells. The results are shown in Figure 4. The IC ₅₀ on day 5 was 10 μΜ. A 50% reduction was not reached on days 6 and 7

Compound 35

50 This compound was tested in a 5 day assay using HEp2 cells. The results are shown in Figure 5. The IC ₅₀ was 12.5 μΜ. At 100 μΜ, compound 35 showed toxicity and showed some signs of precipitating out of solution. Subsequent dilutions also showed a reduction in plaque numbers (as shown) and size.

Compound 37

This compound was tested in a 5 day assay using F£Ep2 cells. The IC ₅₀ was 17.5 μΜ. At 100 μΜ, compound 37 showed toxicity and showed some signs of precipitating out of solution.

Compound 39

This compound was tested in a 4 day assay using BSC1 cells. The results are shown in Figure 6. The IC ₅₀ was 16.5 μΜ. At 100 μΜ, compound 39 showed some toxicity and was moderately soluble in cell medium. Subsequent dilutions also showed a reduction in plaque numbers (as shown) and size.

Compound 40

This compound was tested in a 4 day assay using BSC1 cells. The results are shown in Figure 7. This compound showed moderate inhibition of plaque formation. 100 μΜ, compound 40 showed low toxicity.

Example: activity against other pneumoviruses

Compounds 1, 4 and 6 were also tested against bovine RSV and pneumonia virus of mice (PVM).

Bovine RSV

The compounds were tested with a 6-day assay using F£Ep2 cells. The results are shown in Figure 8. IC ₅₀ values of 80 μΜ (compound 4) and 15 μΜ (compound 6) were calculated. Compound 1 again precipitated out before 50% reduction was seen.

Pneumonia virus of mice (PVM)

A full plaque assay was carried out for compound 4 using F£Ep2 cells, with assays of 5 days and 6 days after incubation. The results are shown in Figure 9. ICsovalues of

51 32 μΜ (day 5) and 100 μΜ (day 6) were obtained. Compounds 1 and 6 were not tested fully, but appear to have similar IC ₅₀ values to compound 4 for the dilutions tested.

The compounds (1, 4, 6 and 35) also significantly reduce the size of plaques of hRSV, bRSV and PVM. These smaller size plaques are still counted in the assay, but indicate a significant reduction in virus replication and spread.

Example: Effect on virus yield

The initial studies on compounds assessed their ability to reduce the number of plaques detected in a conventional plaque assay. When the plaques were observed it was noted that the plaque size was significantly reduced in the presence of some compounds. These data suggested that the compounds reduced the amount of virus produced from infected cells and hence slowed the rate of plaque development. A reduction in virus yield is a desirable attribute for antiviral compounds and this was therefore tested directly.

A cell monolayer was infected at a low multiplicity of infection (a small amount of virus relative to the total number of cells) such that only a few cells were infected. CMC was not added to the overlay medium, so that virus particles released from cells could spread throughout the dish and infect new cells. If left, this process will continue until eventually the whole cell monolayer is destroyed. The supernatant which contains the released virus from cells can be collected and the virus titre can be measured using a standard plaque assay. This gives an indication of released (circulating) virus.

The compounds were therefore tested to determine whether they affected the amount of infection virus particles released into the supernatant. Method

A monolayer of cells of a standard cell line known to support pneumovirus replication (typically HEp2 or BSC 1 cells) were infected at a moi of 0.02 for an hour at

33 °C in 200 μΐ of medium lacking serum. The inoculum was removed and 2 ml of overlay medium (GMEM 2% FCS) was added with either carrier or compounds as indicated. After a designated period, 1.5 ml of supernatant was carefully collected without disturbing the cells and stored at -80 °C. Individual plates were used for each time point.

The virus titre in the medium was calculated by carrying out a standard plaque assay with the exception of the following change. During the initial 1 hour step where the virus is first added to the cells the volume of inoculum added was 2 ml instead of 200 μΐ. Thus the

52 supernatant was diluted at least 1 in 20 taking the final concentration of any compound present to a level below its active range. Following virus adsorption period (1 hour) the inoculum was removed and replaced with 2% CMC overlay medium (without compounds).

Four compounds were tested. Compound 1 was tested at concentrations of 32 μΜ and 10 μΜ. Compounds 4, 6 and 35 were each tested at 100 μΜ, 32 μΜ and 10 μΜ. The carrier DMSO alone was used as a control. Supernatants were collected on day 4, day 5 and day 6 post infection. Data were processed by establishing the yield of virus in cells treated with DMSO carrier alone. This was set at 100% and the yield of virus from cells treated with the four compounds at the various concentrations were calculated relative to this value. The data are shown in Figure 10.

Conclusion

The data show a clear dose-dependent effect of all four compounds with yield of virus reduced as the concentration increased. All compounds show a statistically significant reduction in released virus at 32μΜ (p<0.05, compared to the carrier treated control, assessed using a Mann- Witney test and indicated by an asterisk in Figure 10). It is anticipated that in infected animals this reduction in virus production would give the immune system an advantage in combating infection and reducing clinical disease while allowing the establishment of long-term protective immune memory. Compound 35 at ΙΟΟμΜ appears to be the most active compound with no released virus detected when infected cells were treated with 100 μΜ of compound. This represents a reduction in virus yield of at least 2.3 x 10 ³ -fold. However a high level of cellular toxicity of this compound was noted at this dilution.

These results indicate that the compounds tested which are designed to inhibit the cellular DDX3X protein slow down virus replication considerably in a dose-dependent manner.

It is anticipated that in infected animals this reduction in virus production would give the immune system an advantage in combating infection and reducing clinical disease while allowing the establishment of long-term protective immune memory.

Example: MTT Toxicity Assay

All experiments shown were carried out in HEp2 cells. Cells were grown in 96 well plates overnight as previously described in 10% FCS medium to 95% confluence level. The

53 medium was aspirated off and replaced with 200ul media with 2% FCS +/- compounds in DMSO carrier as indicated at an identical dilution series to that used in the plaque assays. 8 wells were used per compound/carrier alone dilution. An additional 2 rows of 8 cells were grown in medium alone for reference and subsequently 8 would be used as a cell death control as described below. Cells were incubated at 33 °C as indicated. If the incubation period exceeded 4 days the medium was replaced with the appropriate amount of fresh medium plus compound.

MTT assays were commenced 4 hours before the time period expired. Here 20 uL of MTT [(3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide)] solution (7.5 mg/ml in PBS) was added. One of the medium alone rows had 20 μΙ_, of SDS added (cell death control). The cells were placed back in the incubator at 33 °C. After 4 hours, 100 μΙ_, of a stop solution (50% formamide, 20% w/v SDS) was added and pipetted to dissolve the substrate. Plates were then left overnight to allow the bubbles to disappear and the OD was recorded at 595 nm. Following subtraction of background solution colour (cell death control), the optical density was expressed as a percentage compared to the DMSO control on each appropriate plate.

Results

Compounds 1, 4, 6, 35, and 37 were tested in an MTT assay which gives a measure of metabolic activity that can be inferred as an indication of cell viability. Solubility issues of compound 1 prevented the testing of the compound at ΙΟΟμΜ (Figures 11 to 15: Figure 11 - compound 1; Figure 12 - compound 4; Figure 13 - compound 6; Figure 14 - compound 35; Figure 15 - compound 37).

Compounds 4, 6 and 37 show inhibition of activity above 10 μΜ over the time course. Cell monolayers appeared intact and were not different from carrier controls as previously noted in the plaque assays. Compound 35 showed greater inhibition as previously observed with a reduction in viability above 3.2 μΜ after 4 days. Cell monolayers treated with 32 μΜ and above of compound 35 showed signs of death as the time course progressed. A rapid increase in cell growth on day 1 for compound 35 (Figure 14) could indicate an adverse cellular response (such as apoptosis).

54 Compound 1 showed a similar level of inhibition to compounds 4, 6 and 37 with inhibition seen above 10 μΜ. However on days 3 and 4 compound 1 showed an increased sensitivity (Figure 11). This disappeared after 5 days (following a media change at day 4). Based on the solubility issues the increased toxicity seen on days 3 and 4 could indicate slow solubilisation of the compound. When the media was refreshed with insoluble compound cells could recover.

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