WO2009027679A1 - Process for the preparation of guanidino substituted bi-and polyphenyls that are suitable as small molecule carriers - Google Patents

Abstract

A process for the production of a compound of formula (I), or a pharmaceutically acceptable salt thereof, Formula (I) which process comprises:(a) coupling a compound of formula (II) to a compound of formula (III) to form a compound of formula (IV).

Description

PROCESS FOR THE PREPARATION OF GUANIDINO SUBSTITUTED BI- AND POLYPHENYLS THAT

ARE SUITABLE AS SMALL MOLECULE CARRIERS

The present invention relates to a new synthetic route to small molecule carriers (SMCs) 5 and also to some new small molecule carriers. More specifically, the invention relates to new routes to SMCs that are useful for the in vitro and in vivo delivery of various cargo moieties into cells. It also relates to some new SMC products, which are particularly effective in achieving the delivery of cargo moieties into cells. 0 Over recent years, studies have shown that a variety of peptides, many of which are present in viral proteins, have the ability to cross biological membranes in various different cell types. These peptides, known as "protein transduction domains" (PTDs), can be linked to a wide variety of molecules with limited ability to cross membranes, (e.g., peptides, proteins, DNA), thereby enabling them to traverse biological membranes. Studies have shown that5 PTD fusion molecules introduced into mice exhibit delivery to all tissues, including the traversal of the blood-brain barrier [Schwarze, SR., Dowdy, SF., Trends Pharmacol. Sd, 2000, 21, 45]. Similar basic peptides are known to have anti-bacterial activity against MDR forms. 0 Most therapeutic drugs are limited to a relatively narrow range of physical properties. By way of example, they must be sufficiently polar for administration and distribution, but sufficiently non-polar so as to allow passive diffusion through the relatively non-polar bilayer of the cell. As a consequence, many promising drug candidates (including many peptide drugs) fail to advance clinically because they fall outside of this range, proving to5 be either too non-polar for administration and distribution, or too polar for passive cellular entry. A novel approach to circumvent this problem is to covalently tether these potential drugs to PTDs. However, it is very costly and time consuming to prepare such peptide- PTDs and their peptide structure often renders them susceptible to rapid degradation by cellular enzymes. 0 One solution to this problem is to use small molecule carriers (SMCs or "molecular tugs") that are more amenable than peptide-PTDs due to their in vivo stability by virtue of their resistance to cellular enzymes that degrade peptides.

WO 2005/123676, which is incorporated herein by reference, describes SMC compounds and a process for their production. However, the synthesis described is laborious and is not suitable for large-scale preparations.

The inventors have now found a new convergent synthesis for the compounds disclosed in WO 2005/123676. This new route enables an improvement in the overall yield and also allows easy synthesis of a range of different SMC compounds. The invention also relates to a number of new SMC compounds.

The present invention therefore provides a process for the of a compound of formula I, or a pharmaceutically acceptable salt thereof,

I wherein

Xl, X₂ and X₃ are each independently

NR₅ N NR₁ NR ₃R, where Y is an alkylene, alkenylene or alkynylene group, each of which may be optionally substituted with one or more substituents selected from alkyl, halo, CF₃, OH, alkoxy, NH₂, CN, NO₂ and COOH; W is absent or is O, S or NH;

R₁, R₂, R₃ and R₄ are each independently selected from H, alkyl, aryl and a protecting group

Pi ; R₇, Rg and R₉ are each independently selected from H, alkyl, halo, CF₃, OH, alkoxy, NH₂, CN, NO₂ and COOH; q and r are each independently 1, 2, 3 or 4; q¹ and r' are each independently 0, 1, 2 or 3, where q + q¹ and r + r' each equal 4; p is 1, 2, 3, 4 or 5, and p' is 0, 1, 2, 3 or 4, where p + p¹ is 5; n is 0, 1, 2, 3, 4, 5 or 6; and

L is (Z)_mNR₅R₆ wherein Z is a hydrocarbyl group and m is 0 or 1 wherein R₅ and R₆ are each independently H, CO(CH₂)JQ₁ or

where j and k are each independently 0, 1, 2, 3, 4 or 5, and Q₁ and Q₂ are each independently selected from COOH, a chromophore,

or R₅, R₆ and the nitrogen to which they are attached together form

which process comprises:

(a) coupling a compound of formula II to a compound of formula III to form a compound of formula IV

IV wherein: n, p, p', q, q', r and r' are defined as above; at least one X₁ ¹, X₂ ¹ and X₃ ¹ moiety is -W-Y-NR₁R₁₀ or -W-Y-NR₁ -C(=NR₂)-NR₃R₄ and the other X₁', X₂' and X₃' moieties are each independently selected from OH, SH, NH₂, -W-Y-NR₁R₁₀, and -W-Y-NR₁ -C(=NR₂)-NR₃R4 wherein R₁, W and Y are defined as above and R₁₀ is H or a protecting group P₂; one of Ji and J₂ is a leaving group LG₁, and the other is a boronic acid, a boronic ester, a borane group or a trihalogenoborate salt;

L"' is L, as defined above, a leaving group LG₂, -COR₁₈, -(Z)₁₁₁NHR₁₂, or a group which can be reduced to a moiety -(Z)_mNH₂, where Z and m are defined as above, R₁₂ is a protecting group P₃ and R₁₈ is hydrogen or a C₁-C₄ alkyl group; and

(b) if any OfX₁', X₂' and X₃ ¹ in the formula (IV) is other than -W-Y-NR₁ -C(=NR₂)- NR₃R₄, alkylating the X₁ ¹, X₂' and X₃' moieties so that they each represent a group of formula

where W, Y, R₁, R₂, R₃ and R₄ are defined as above, and, if necessary, converting the moiety L'" to a moiety L as defined above, to obtain a compound of formula I.

Also provided is a compound of formula (I), as defined above, or a pharmaceutically acceptable salt thereof, wherein r is 1 or 2 and the phenyl ring which carries L has no substituents ortho to the L moiety.

Further provided is a compound of formula Via, VIb, VIc, VId or Vie, or a pharmaceutically acceptable salt thereof,

VId VIe

wherein X₁, X₃ and L are as defined above.

Also provided is a compound of formula VII, or a pharmaceutically acceptable salt thereof,

VII wherein:

X₁, X₃, R₇, R₉ and L are as defined above; r is 1, 2, 3 or 4 and r' is 0, 1, 2 or 3, wherein r + r' is 4; p is 1, 2, 3, 4 or 5 and p' is 0, 1, 2, 3 or 4, where p + p' is 5; and p + r is 3.

As used herein, the term "hydrocarbyl" refers to a saturated or unsaturated, straight-chain, branched, or cyclic group comprising at least C and H that may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo, alkoxy, hydroxy, CF₃, CN, amino, COOH, nitro or a cyclic group. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain heteroatoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen, oxygen, phosphorus and silicon.

Preferably, the hydrocarbyl group is an aryl or alkyl group. Typically, the hydrocarbyl group is unsubstituted. More preferably, the hydrocarbyl group is an unsubstituted Ci_-6 alkyl group.

As used herein, the term "alkoxy" includes both straight chain and branched alkoxy groups which may be substituted (mono- or poly-) or unsubstituted. Preferably, the alkoxy group is a C_1-20 alkoxy group, more preferably a C_1-15 alkoxy group, more preferably still a C_1-I2 alkoxy group, more preferably still, a C_1-6 alkoxy group, more preferably a Cj_-3 alkoxy group. Particularly preferred alkoxy groups include, for example, methyoxy, ethyoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy and hexoxy. Suitable substituents include alkyl, halo, CF₃, OH, alkoxy, NH₂, CN, NO₂ and COOH. Preferably, the alkoxy group is unsubstituted. More preferably, the alkoxy group is an unsubstituted C_1-4 alkoxy group .

As used herein, the term "alkyl" includes both saturated straight chain and branched alkyl groups which may be substituted (mono- or poly-) or unsubstituted. Preferably, the alkyl group is a C_1-2O alkyl group, more preferably a C_1-I5, more preferably still a C_1-I2 alkyl group, more preferably still, a Ci_-6 alkyl group, more preferably a C_1-3 alkyl group.

Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl. Suitable substituents include halo, CF₃, OH, alkoxy, NH₂, CN, NO₂ and COOH. The term "alkylene" should be construed accordingly. Preferably, the alkyl group is unsubstituted. More preferably, the alkyl group is an unsubstituted C_1-4 alkyl group. As used herein, the term "aryl" refers to a substituted (mono- or poly-) or unsubstituted monoaromatic or polyaromatic system, wherein said polyaromatic system may be fused or unfused. Preferably, the term "aryl" includes groups having from 6 to 10 carbon atoms, e.g. phenyl, naphthyl etc. The term "aryl" is synonymous with the term "aromatic". Suitable substituents include alkyl, halo, CF₃, OH, alkoxy, NH₂, CN, NO₂ and COOH. Preferably, an aryl group is unsubstituted or is substituted with 1, 2 or 3 substituents selected from C_1-4 alkyl, halo, CF₃, OH, NH₂ groups. Typically, these substituents are themselves unsubstituted. Preferably, the aryl group is an optionally substituted phenyl group. More typically, the aryl group is an unsubstituted phenyl group.

As used herein, the term "alkenyl" refers to a group containing one or more carbon-carbon double bonds, which may be branched or unbranched, substituted (mono- or poly-) or unsubstituted. Preferably the alkenyl group is a C_2-20 alkenyl group, more preferably a C_2-15 alkenyl group, more preferably still a C_2-12 alkenyl group, or preferably a C_2-6 alkenyl group, more preferably a C_2-3 alkenyl group. Suitable substituents include alkyl, halo, CF₃, OH, alkoxy, NH₂, CN, NO₂ and COOH. The term "alkenylene" should be construed accordingly. Preferably, the alkenyl group is unsubstituted. More preferably, the alkenyl group is an unsubstituted C_2-4 alkenyl group.

As used herein, the term "alkynyl" refers to a carbon chain containing one or more triple bonds, which may be branched or unbranched, and substituted (mono- or poly-) or unsubstituted. Preferably the alkynyl group is a C_2-20 alkynyl group, more preferably a C_{2- 15} alkynyl group, more preferably still a C_2-12 alkynyl group, or preferably a C_2-6 alkynyl group or a C_2-3 alkynyl group. Suitable substituents include alkyl, halo, CF₃, OH, alkoxy, NH₂, CN, NO₂ and COOH. The term "alkynylene" should be construed accordingly. Preferably, the alkynyl group is unsubstituted. More preferably, the alkynyl group is an unsubstituted C_2-4 alkynyl group.

As used herein, the term "chromophore" refers to any functional group that absorbs light, giving rise to colour. Typically, the term refers to a group of associated atoms which can exist in at least two states of energy, a ground state of relatively low energy and an excited state to which it may be raised by the absorption of light energy from a specified region of the radiation spectrum. Often, the group of associated atoms contains delocalised electrons. The chromophore present in the compounds prepared by the process of the invention can be a conjugated Il system or a metal complex. Typically, a chromophore is a porphyrin, a polyene, a polyyne or a polyaryl. Preferably the chromophore is one of.

Protecting groups P₁, P₂ and P₃ are protecting groups suitable for protecting a nitrogen atom. Many examples of such protecting groups are known to the person skilled in the art, for example those protecting groups mentioned in "Protecting Group Chemistry" Jeremy Robertson, OUP, 2000, which is incorporated herein by reference. Preferably P₁, P₂ and P₃ are selected from benzyl, trityl, 9-phenylfluorenyl, benzydryl, fluorenyl, carbamate, benzylcarbamate (Cbz), t-butyl carbamate (Boc), 9-fluorenylmethyl carbamate (Fmoc), acetamide, p-toluenesulfonate (p-Ts), silyl and triisopropylsilyl (TIPS) groups.

Preferably, P₁ is a Boc group. <

Preferably, P₂ is a Cbz group.

Typically, P₂ and P₃ are different. More typically, P₁, P₂ and P₃ are all different. Preferably, P₂ and P₃ are orthogonal. More preferably, P₁ and P₂ are orthogonal to P₃.

Leaving group LG₁ is typically any group that can undergo oxidative addition with Pd(O). Those of skill in the art will easily be able to select appropriate leaving groups. LGj is preferably a halogen, triflate (OTf), tosylate (OTs), N-hydroxysuccinimide (OSu) or a mesylate (OMs) group. LGi is more preferably halogen, most preferably bromide or iodide. Leaving group LG₂ is typically a leaving group suitable for an aryl cyanation reaction. Those of skill in the art will easily be able to select appropriate such leaving groups. LG₂ is preferably a halogen, triflate (OTf), tosylate (OTs), N-hydroxysuccinimide (OSu) or a mesylate (OMs) group. LG₂ is more preferably halogen, most preferably bromide.

Leaving group LG₃ is typically a leaving group suitable for a nucleophilic substitution reaction at a saturated carbon centre. Those of skill in the art will easily be able to select appropriate leaving groups. LG₃ is preferably a halogen, triflate (OTf), tosylate (OTs), N- hydroxysuccinimide (OSu) or a mesylate (OMs) group. LG₃ is more preferably a OMs group.

Leaving group LG₄ can be any leaving group suitable for a guanidinylation reaction. The skilled reader will appreciate that LG₄ represents a moiety such that -NLG₄ is a leaving group in guanidinylation reaction. A skilled chemist can easily select appropriate leaving groups in this regard. Thus, preferred LG₄ groups include triflyl (Tf), tosyl (Ts) and mesyl (Ms) groups. LG₄ is most preferably a triflyl group, such that -NLG₄ represents -NTf.

Leaving group LG₄' can be any leaving group suitable for a guanidinylation reaction. A skilled chemist can easily select appropriate leaving groups in this regard. LG₄' is typically a halogen atom, triflate (OTf), tosylate (OTs), mesylate (OMs) or 1-pyrazole group, preferably a 1-pyrazole group.

Leaving group LG₅ can be any leaving group suitable for a nucleophilic substitution reaction at a carbonyl or thiocarbonyl group. Those of skill in the art will easily be able to select appropriate leaving groups. LG₅ is typically a halogen, triflate (OTf), tosylate (OTs), N-hydroxysuccinimide (OSu) or a mesylate (OMs) group. LG₅ is preferably a OSu group.

Leaving group LG₇ is typically a leaving group suitable for a nucleophilic substitution reaction at a saturated carbon centre. Those of skill in the art will easily be able to select appropriate leaving groups. LG₇ is preferably a halogen, triflate (OTf), tosylate (OTs), N- hydroxysuccinimide (OSu) or a mesylate (OMs) group. LG₇ is more preferably a OMs group.

As used herein, the term "guanidinylating" refers to the process of forming a guanidine moiety -NR₁-C(=NR₂)-NR₃R₄. Typical reagents and conditions for this process are described in US 6,380,358, and Katritsky, et al ARKIVOC 2005 (iv) 49-87 both of which are incorporated herein by reference.

US 6,380,358 describes reagents and methods for the synthesis of organic molecules containing a (protected) guanidine group, hi particular it describes processes for the guanidinylation of amines.

Katritsky, et al describes recent advances in the development of guanylating reagents, which are defined as compounds forming a guanidine structure by a chemical transformation. This document describes a wide rantge of possible reagents for the preparation of guanidines, including thioureas, isothioureas, carbodiimides and cyanamides, pyrazole-1-carboximidamides, triflyl guanidines, ammoiminomethane-sulfonic and -sulfinic acids and benzotriazole and imidazole-containing reagents.

The coupling reaction between the compounds of formulae (II) and (III) is typically an organometallic coupling, preferably a coupling mediated by Pd, more preferably Pd(O). The coupling is typically a Stille, Suzuki, Negishi, Hiyama or Kumada coupling. Preferably, the coupling is a Suzuki coupling.

More preferably, the reaction between the compounds of formulae (II) and (III) is a Suzuki coupling, performed using a Pd(O) catalyst in the presence of a base, preferably a nucleophilic base, more preferably triethylamine. The Pd(O) catalyst is typically used in a catalytic amount. The reaction is typically carried out at elevated temperature (i.e. above room temperature), preferably between 75 and 125⁰C, more preferably at 82°C or 100⁰C, most preferably at 82°C. A reaction time of 1 to 18 hours is typically employed, hi some cases a reaction time of 1, 3 or 18 hours may be used. The reaction is typically heated using microwave radiation or conventional heat sources. The reaction is typically carried out in an aqueous organic solvent, preferably aqueous toluene or aqueous isopropyl alcohol, more preferably aqueous isopropyl alcohol. The solvents used are preferably degassed prior to use. The reaction may be carried out by heating at 100⁰C a compound of formula II with a compound of formula III with PdCl₂dppf. CH₂Cl₂ and potassium phosphate in aqueous toluene. Preferably, the reaction is carried out by heating at 82°C a compound of formula II with a compound of formula III with Pd Cl₂dppf.CH₂Cl₂ and triethylamine in aqueous isopropyl alcohol for 18 hours.

In a preferred embodiment, X₃' is -W-Y-NR₁R₁₀ or -W-Y-NR₁-CC=NR₂)^R₃R₄. More preferably, all OfX₁ ¹, X₂ ¹ and X₃ ¹ are -W-Y-NR₁R₁₀ or -W-Y-NR₁-CC=NRs)-NR₃R₄.

In a more preferred embodiment, X₃ ¹ is -W-Y-NR₁R₁₀. More preferably, all OfX₁', X₂' and X₃' are -W-Y-NR₁R₁₀.

A boronic acid group is a group of formula -B(OH)₂.

Preferably, the boronic ester is -B(OR₁₃)(OR₁₄) or

where R₁₃ and Ri₄ are each independently selected from C₁-C₆ alkyl groups and R₁₅ is a C₁-C₆ alkyl or phenyl group. More preferably, the boronic ester is

where R₁₅ is a -CH(CH₃)₂CH(CH₃)₂- group.

Preferably, the borane is BR₁₆R₁₇ where R₁₆ and R₁₇ are each independently selected from Ci-C₆ alkyl groups. A trihalogenoborate salt group is a group of formula -(B(HaI)₃)TVI⁺, where each Hal group is the same or different and is independently chosen from halogen atoms. Halogen atoms are typically F, Cl, Br or I atoms, preferably F or Cl atoms, more preferably F atoms. In a preferred embodiment, each Hal group is the same, hi a more preferred embodiment, each Hal group is the same and is a fluorine atom.

The group M⁺ is any ionic group capable of acting as the counterion to the group - (B(HaI)₃)^", but is typically a monovalent metal ion, preferably a monovalent Group I metal ion. Monovalent Group I metal ions are typically Li⁺, Na⁺, K⁺ or Rb⁺, preferably Li⁺, Na⁺ or K⁺, more preferably K⁺.

Thus, in a particularly preferred embodiment, the group -(B(HaI)₃)TvI⁺ is -(BF₃)TC⁺.

hi a preferred embodiment, J₁ is a boronic acid, a boronic ester or a borane group and J₂ is a leaving group LG₁. More preferably, J₁ is a boronic acid or a boronic ester, more preferably a boronic acid or a compound of formula

O

-<o>⁵

wherein R₁₅ is as defined above, and J₂ is a halogen, for example bromine or iodine.

Most preferably, though, J, is a trihalogenoborate salt as defined above.

The compounds of formulae (II) and (III) are known compounds, or can be prepared by analogy with known methods.

For example, when J₁ is a boronic acid, boronic ester, or a borane, compounds of formula II can be prepared by treating a compound of formula IX with a boronating agent in the presence of a catalyst.

(IX) (ii) wherein X₁ ¹, R₇, p and p' are defined as above, LG is a leaving group, for example bromine, and B is a boronic acid, boronic ester or borane moiety. The boronating agent can be a diborane, for example, bispinacolactodiborane. The catalyst is preferably Pd. Preferably, the reaction is carried out between 50 and 100⁰C, more preferably at 8O⁰C. A long reaction time of several days is typically employed, for example 72 hours. The reaction is typically carried out in an organic solvent, preferably dimethyl sulphoxide (DMSO). KOAc is typically present.

Further, when J₁ is a trihalogenoborate salt group, compounds of formula II where Ji is a trihalogenoborate salt group can be prepared by treating compounds of formula II where J₁ is a boronic acid, boronic ester or borane group with a saturated solution OfMH(HaI)₂, where M and Hal are as defined above,

(H) (U) wherein Xi', R₇, p and p' are defined as above, B is a boronic acid, boronic ester or borane moiety and B' is a trihalogenoborate salt group. Preferably, the reaction is carried out at room temperature, which is typically between 18°C and 30°C, preferably 21°C. A reaction time of between 1 and 5 hours is typically employed, preferably 3 hours. The reaction is typically carried out in an organic solvent, preferably methanol. Compounds of formula II' may be prepared using similar methodology to that described in Skaff, et al, J.O.C., Vol. 70, No. 18, 2005, 7353-7363 or Eur. J. Org. Chem., 1999, 1875-1885, the entirety of which are incorporated herein by reference. The compound of formula IX can also be prepared by known methods. For example compounds of formula IX wherein each X₁' represents -W-Y-NR₁Ri₀ and W is O, S or NH can be obtained by reacting a compound of formula (X) in which each Xj" represents a hydroxy, thiol or amino group, with LG₃-Y-N-R₁Ri₀.

Compounds of formula IX wherein each X₁ ¹ represents -W-Y-NR₁ -C(=NR₂)-NR₃R₄ and W is O, S or NH can be obtained by reacting a compound of formula (X) in which each X₁" represents a hydroxy, thiol or amino group with LG₇-Y-NR₁ -C(=NR₂)-NR₃R₄. Alternatively, such compounds can be prepared from corresponding compounds of formula IX in which each X₁ ¹ represents -W-Y-NR₁R_1O by deprotecting any protected amine groups on -W-Y-NR₁ R₁₀ moieties at the Xi' position, and guanidinylating the deprotected moieties.

(X) (IX)

The compound of formula X in which each X₁" is hydroxy, thiol or amino can be generated by deprotecting a corresponding compound in which the or each X]" is a protected hydroxy, thiol or amino group. For example, a compound which carries an alkyl ether group at the or each X₁" position can be converted to a compound of formula X in which the or each Xj" is hydroxy by reaction with BBr₃.

Preferably, the reaction between the compound of formula X and LG₃-Y-N-RiRi₀ is carried out between 75 and 125⁰C, more preferably at 100°C. A reaction time of 1 to 5 hours can be employed, for example 2 hours. The reaction is typically carried out in an organic solvent, preferably dimethyl formamide (DMF).

Compounds of formula IX wherein each Xj' represents -W-Y-NRiR_1O and W is absent can be obtained by a Sonogashira coupling reaction between a suitably chosen alkyne, for example H(CsC)-Y-NR₁R_1O and a leaving group on the phenyl ring, followed by reduction of the C≡C group. Compounds of formula IX wherein each X₁' represents -W-Y-NR₁- C(=NR₂)-NR₃R₄ and W is absent can be obtained by a Sonogashira coupling reaction between a suitably chosen alkyne, for example H(CsC)-Y-NR₁ -C(=NR₂)-NR₃R₄ and a leaving group on the phenyl ring, followed by reduction of the C≡C group. Alternatively, compounds of formula IX wherein each X₁' represents -W-Y-NR₁ -C(=NR₂)-NR₃R₄ and W is absent can be obtained by a Sonogashira coupling reaction between a suitably chosen alkyne, for example H(CsC)-Y-NR₁R₁₀ and a leaving group on the phenyl ring, followed by reduction of the C≡C group, deprotecting any protected amine groups on -W-Y-NR₁R₁₀ moieties at the X₁' position and guanidinylating the deprotected moieties.

When J₂ is a leaving group LG (for example bromine) compounds of formula III in which each X₃ represents -Y-N-NR₁R₁₀ and W is O, S or NH can be prepared by reacting a compound of formula XI, in which each X₃" represents a hydroxy, thiol or amino group, with LG₃-Y-N-R₁R_1O- Compounds of formula El in which X₃' represents -Y-NR₁-

C(=NR₂)-NR₃R₄ and W is O, S or NH can be obtained by reacting a compound of formula (XI) in which each X₃" represents a hydroxy, thiol or amino group with LG₇-Y-NR₁- C(=NR₂)-NR₃R₄. Alternatively, such compounds can be prepared from corresponding compounds of formula (III) in which each X₃' represents -W-Y-NR₁R_1O by deprotecting any protected amine groups on -W-Y-NR₁R₁₀ moieties at the X₁' position, and guanidinylating the deprotected moieties.

(XI) m

The compound of formula XI in which each X₃" is hydroxy, thiol or amino can be generated by deprotecting a corresponding compound in which the or each X₃" is a protected hydroxy, thiol or amino group. For example, a compound which carries an alkyl ether group at the or each X₃" position can be converted to a compound of formula X in which the or each X₃" is hydroxy by reaction with BBr₃.

Preferably, the reaction between the compound of formula XI and LG₃-Y-N-R₁R_1O is carried out between 50 and 100⁰C, more preferably at 8O⁰C. A reaction time of 24 hours is typically employed. The reaction is typically carried out in an organic solvent, preferably dimethyl formamide (DMF).

Compounds of formula III wherein each X₃' represents -W-Y-NR₁R₁₀ and W is absent can be obtained by a Sonogashira coupling reaction between a suitably chosen alkyne, for example H(C≡C)- Y-NRj R₁₀ and a leaving group on the phenyl ring, followed by reduction of the C≡C group. Compounds of formula III wherein each X₃' represents -W-Y-NR₁- C(=NR₂)-NR₃R₄ and W is absent can be obtained by a Sonogashira coupling reaction between a suitably chosen alkyne, for example H(C=C)-Y-NR₁ -C(=NR₂)-NR₃R₄ and a leaving group on the phenyl ring, followed by reduction of the C≡C group. Alternatively, compounds of formula III wherein each X₃' represents -W-Y-NR₁ -C(=^rNR₂)-NR₃R₄ and W is absent can be obtained by a Sonogashira coupling reaction between a suitably chosen alkyne, for example H(CsC)-Y-NR₁R₁₀ and a leaving group on the phenyl ring, followed by reduction of the C≡C group, deprotecting any protected amine groups on -W-Y-NR₁Ri₀ moieties at the X₃' position and guanidinylating the deprotected moieties.

A compound of formular (III) in which J₂ is a trihalogenoborate salt group can be prepared by reacting a corresponding compound in which J₂ is a boronic acid, boronic ester or borane group with a compound of formula MH(HaI)₂, under the reaction conditions described above for the preparation of the compounds of formula (II).

A compound of formula (III) in which J₂ is a boronic acid, boronic ester or borane group can be prepared by reacting a corresponding compound in which J₂ is a leaving group with a boronating agent, under the reaction conditions described above for the preparation of the compounds of formula (II). A compound of formula (111) or (XI) in which L'" is CN can be prepared, for example, from a corresponding compound in which L'" is a leaving group, for example iodine, by reaction with CN^", preferably with zinc cyanide and Pd(H). Preferably, when J₂ is a leaving group, no more than 0.5 equivalents of CN^" are used in the reaction, to minimise formation of the dicyano compound.

Compounds of formula (IH) and (XI) in which L'" is -COR₁₈ are commercially available or can be prepared using known methods.

In a further embodiment of the invention, J₂ is a boronic acid, a boronic ester or a borane group or a trihalogenoborate salt group and J₁ is a leaving group LG₁.

In one embodiment, when any of the X₁', X₂' and X₃' moieties in the formula (IV) are OH, SH or NH₂, alkylation so that all X₁', X₂' and X₃' moieties represent a group of formula -W- Y-NRI-C(=NR₂)-NR₃PM is effected by either:

(i) alkylating any X₁', X₂' and X₃' moieties that are OH, SH or NH₂ so that they represent -W-Y-NR]R₁O, deprotecting any protected amine groups on -W-Y-NRiR₁₀ moieties present at the X₁', X₂' and X₃' positions and guanidinylating the deprotected moieties so that they each represent a group of formula -W-Y-NR₁ -C(=NR₂)-N NR₃R₄; or

(ii) alkylating any X₁', X₂' and X₃' moieties that are OH, SH or NH₂ so that they each represent a group of formula -W-Y-NRi -C(=NR₂)-NR₃R₄, deprotecting any protected amine groups on -W-Y-NRiRi₀ moieties present at the X]', X₂' and X₃' positions and guanidinylating the deprotected moieties so that they each represent a group of formula -W-Y-NR₁ -C(=NR₂)-NR₃R₄.

Preferably, the alkylation of hydroxy, thiol and amino groups at the X₁', X₂' and X₃' positions in the formula IV so that they represent -W-Y-NRiRi₀ is effected with a compound of formula LG₃-Y-NRiRi₀ where Ri, Y and LG₃ are defined as above and Rio is H or a protecting group P₂. Preferably, R_1O is a protecting group, for example Cbz. More preferably, R₁ is H and R₁₀ is a protecting group, for example Cbz.

Preferably, the alkylation of hydroxy, thiol and amino groups at the X₁', X₂' and X₃' positions in the formula IV so that they represent -W-Y-NR₁R₁₀ is carried out at elevated temperature (i.e. above room temperature). Preferably, the reaction is carried out between 50 and 100⁰C, more preferably at 8O⁰C. A reaction time of 1 to 4 hours is typically employed. In some cases, reaction times of 1, 3, 3.5 and 4 hours may be used. The reaction is typically carried out in an organic solvent, preferably dimethylformamide (DMF). CsCO₃ is typically added to the reaction mixture.

Preferably, the alkylation of any X₁', X₂' and X₃ ¹ moieties that are OH, SH or NH₂ so that they each represent a group of formula -W-Y-NR₁ -C(=NR₂)-NR₃R_( is effected by a compound of formula LG₇-Y-NR₁ -Q=NRz)-NR₃R₄.

Deprotection of any amine groups in the -W-Y-NR₁R_1O moieties present at the X₁', X₂' and X₃' positions can be carried out by standard techniques. The exact conditions employed will depend on the exact nature of the protecting group, hi the case where the amine groups in the -W-Y-NR₁R_1O moiety are protected with Cbz groups, the protecting groups can be removed with an acid, typically an anhydrous acid, preferably HBr and CH₃COOH in DCM.

In another embodiment, all of the X₁', X₂' and X₃' moieties are -W-Y-NR₁R₁₀ or -W-Y-NR₁- C(=NR₂)-NR₃R₄, and any necessary alkylation OfX₁', X₂' and X₃' moieties so that they each represent -W-Y-NR₁ -C(=NR₂)-NR₃R₄ is effected by deprotecting any protected amine groups on -W-Y-NR₁R₁₀ moieties present at the X₁', X₂' and X₃' positions and guanidinylating the deprotected moieties so that they represent a group of formula -W-Y- NR₁-C(^NRz)-NR₃R₄.

Preferably, said guanidinylation of deprotected -W-Y-NRiR₁₀ moieties at the X]', X₂' and X₃' positions is effected either by a compound of formula V, or a tautomer thereof, where R₂, R₃ and R₄ are as defined in claim 1 and LG₄ is a leaving group;

V

or by a compound of formula V, or a tautomer thereof,

R₄R₃N

^LG₄ ¹ R₂N

V

where R₂, R₃ and R₄ are as defined above and LG₄" is a leaving group.

More preferably, guanidinylation is effected with N,N-di-boc-N'-trifluoromethanesulfonyl guanidine or N_jN'-Di-Boc-lH-pyrazole-l-carboxamidine, most preferably N,N'-Di-Boc- 1 H-pyrazole- 1 -carboxamidine.

The term tautomer is well known to the person skilled in the art and refers to the result of a migration of a hydrogen atom accompanied by the switch of a single bond and adjacent double bond. The skilled person is aware that tautomers of certain molecules, such as those represented by formulae V and V above exist in, for example, aqueous media.

Preferred tautomers of the compounds of formula (V) are represented by the formula

R₄R₃N LG₄

R₂N Typically the guanidinylation is carried out at room temperature. Alternatively, the reaction mixture may be heated. The reaction is typically left to run overnight (i.e. a time in excess of 12 hours, for example, 18 hours). The reaction is typically carried out in an organic solvent, preferably DCM and triethylamine or diisopropylethylamine (DIEA), more preferably DCM and DIEA. In another embodiment at least one X₁', X₂' and X₃ ¹ moiety is -W-Y-NR₁R₁₀ and the other X₁', X₂' and X₃' moieties are each independently selected from OH, SH, NH₂ and -W-Y- NR₁R₁₀, wherein and R₁, W and Y are defined as above and R₁₀ is H or a protecting group, and in step (b) when any X₁', X₂' and X₃' moieties are OH, SH or NH₂, alkylation is effected so that all X₁', X₂' and X₃' moieties represent -W-Y-NR₁R₁₀, any protected amine groups on the X₁', X₂' and X₃ ¹ moieties are deprotected and the X₁', X₂' and X₃' moieties are guanidinylated so that they each represent a group of the formula -W-Y-NR₁ -C(=NR₂)- NR₃R₄, and, if necessary, the moiety L'" is converted to a moiety L, to obtain a compound of formula (I).

In this embodiment, in step (b) of the process of the invention, the alkylation and guanidinylation steps, and the conversion of L'" to a moiety L may be carried out in any order. Thus, for example, a first X₁', X₂' or X₃ ¹ moiety represented by -W-Y-NR₁R₁₀ can undergo guanidinylation so that it represents -W-Y-NRi-C(=NR₂)-NR₃R₄, followed by the alkylation of one or more OH, SH or NH₂ moieties at other X₁', X₂' or X₃ ¹ positions so that they represent -W-Y-NRiR_1O and further subsequent guanidinylation reactions. Preferably, though, when any OfX₁', X₂' and X₃' are OH, SH or NH₂, (i) alkylation is first effected so that all OfX₁', X₂' and X₃' represent -W-Y-NR₁R_1O, (ϋ) any protected amine groups on X₁', X₂' and X₃' moieties represented by -W-Y-NR₁R₁₀ are then deprotected and (iii) the Xj', X₂' and X₃' moieties are then guanidinylated so that they each represent a group of formula

where W, Y, Ri, R₂, R₃ and R₄ are defined as above.

Typically, in the present invention, when a moiety OH, SH or NH₂ is converted to a moiety -W-Y-NRiR₁₀, a catalyst is employed. Typically, this catalyst is an alkali metal carbonate, preferably sodium or cesium carbonate, more preferably cesium carbonate. Typically, in the present invention, when a moiety -W-Y-NR₁R₁₀ is converted to a moiety - W-Y-NR₁ -C(=NR₂)-NR₃R₄, the amine reacted with the compound of formula (V) or (V) is dried using known techniques before guanidinylation.

Typically, in step (b) the alkylation of the X₁', X₂ ¹ and X₃' moieties so that they each represent a group of formula -W-Y-NR₁-C(=NR₂)-NR₃R₄is effected before the conversion of the moiety L'" to a moiety L.

Preferably, when L'" is a group which can be reduced to a moiety -(Z)_mNH₂, the conversion of the moiety L"' to a moiety L to obtain a compound of formula I comprises (i) reducing L'" and (ii) optionally reacting the thus obtained compound with a compound of formula LG₅CO(CH₂)_JQ₁ or LG₆C=S (NH)(CH₂)_kQ₂ where j, k, Q₁ and Q₂ are defined as above and LG₅ and LG₆ are leaving groups. In this embodiment, L'" is typically -CN or -CH₂NO₂, preferably -CN.

Typically, the reduction of L'" takes place under a hydrogen atmosphere, in the presence of a catalyst. Preferably, reduction is effected with Raney Nickel under a hydrogen atmosphere. The reaction typically takes place at room temperature. The reaction time is typically 16 hours. The reaction typically takes place in an organic solvent, preferably THF. NH₄OH may optionally be added.

Preferably, when L'" is a leaving group LG₂, the conversion of the moiety L'" to a moiety L in step (b) comprises (i) cyanating with a source of CN^" in the presence of a suitable catalyst, (ii) reducing the thus obtained CN moiety as described above and (iii) optionally reacting the thus obtained compound with a compound of formula LG₅CO(CH₂)_JQ₁ or LG₆C=S(NH)(CH₂)_IcQ₂ where j, k, Q₁, Q₂, LG₅ and LG₆ are defined as above.

Cyanation is typically effected with zinc cyanide and Pd(II). The reaction typically takes place at elevated temperature (i.e. greater than room temperature). The reaction preferably takes place at from 150 to 200⁰C, more preferably 16O⁰C. The reaction is preferably heated with microwave irradiation. A short reaction time of less than one hour is typically employed, preferably 10 minutes. The reaction typically takes place in an organic solvent, preferably DMA.

In a preferred embodiment of the invention, L'" is CN or a leaving group LG₂ which is cyanated in the process of the invention, and the CN moiety is reduced after the alkylation OfX₁', X₂' and X₃' moieties so that they each represent a group of formula -W-Y-NR₁- C(=NR₂)-NR₃R₄. This is advantageous because the amount of protection and deprotection necessary on functional groups is minimised. Further, it enables a wide range of different L moieties to be introduced easily.

Preferably, when L'" is -COR]₈, the conversion of the moiety L'" to a moiety L in step (b) comprises (i) reductively aminating the -COR₁₈ moiety and (ii) optionally reacting the thus obtained compound with a compound of formula LG₅CO(CH₂)JQ₁ or LG₆C=S(NH)(CH₂)_kQ₂.

Peferably, R₁₈ is hydrogen.

Typically, reductive amination of the -COR₁₈ moiety is effected by (a) reaction with NH₂ORi₉ or NHR₅R₆ and (b) reduction of carbon nitrogen double bond, wherein R^ is H or an alkyl group. Preferably, however, reductive amination is effected by NH₂ORi ₉, followed by reduction of the carbon nitrogen double bond.

Preferably R₁₉ is hydrogen.

Typically, the carbon nitrogen bond is reduced with H₂/Pd, SmZI₂, In/NEUCl, LiAH₄, Bu₃SnH with BF₃-OE₂, Bu₂SnClH in HMPA, Cl₃SiH and pyrrolidine carboxaldehyde, SmBr in HMPA, Z-propanol with a ruthenium catalyst, NaCN(BH₃) or trithylammonium formate with microwave irradiation. Appropriate reagents and conditions for such reductive amination processes are familiar to those of skill in the art. hi the case where Ri ₉ is hydrogen, reduction with H₂/Pd is preferred. Preferably, L'" is different from J₂. When L'" is different from J₂, issues of chemoselectivity in the coupling reaction may be avoided. More preferably, when L'" and J₂ are both leaving groups, J₂ is more reactive than L¹". More preferably, when L'" and J₂ are both leaving groups, J₂ is iodide and L¹" is Br. Oxidative addition between the compounds of formulae (JJ) and (UJ) with Pd(O) can then be effected preferably with iodide.

For compounds of formula I, p, q and r are each independently 1, 2, 3 or 4.

hi a preferred embodiment of the invention, Y is a C_1-1O alkylene group, a C_2-10 alkenylene group or a C_2-10 alkynylene group. More typically, Y is a C_1-12 alkylene group, preferably a C_1-1O alkylene group, even more preferably a C_1-6 alkylene group, and more preferably still, -CH₂CH₂-.

In a preferred embodiment, W is O, S or NH. More preferably, W is O.

Preferably, L'" is a leaving group LG₂, -COR₁₈ (in particular -CHO), or a group which can be reduced to a moiety -(Z)_1nNH₂. The group which can be reduced to a moiety -(Z)_mNH₂ is preferably -CN.

Preferably, m is 1 and Z is an alkylene group, more preferably, a C_1-12 alkylene group, more preferably still a C_1-1O alkylene group, even more preferably a C_1-6 or Ci_-4 alkylene group. More preferably, Z is a CH₂ group.

Preferably, one of R₅ and R₆ is H and the other is selected from H, CO(CH₂)_JQ₁ or C=S(NH)(CH₂)_IcQ₂, or R₅, R₆ and the nitrogen to which they are attached together form

In one preferred embodiment, L is selected from the following: -CH₂NH₂, -CH₂NHCOCH₂CH₂COOH,

Preferably, R], R₂, R₃ and R₄ are each independently selected from H and a protecting group P₁. More preferably, R₁ and R₃ are hydrogen and R₂ and R₄ represent H or Pi. Most preferably, Ri and R₃ are hydrogen and R₂ and R₄ are each independently selected from H and a butyloxycarbonyl (Boc) protecting group. Preferably, p, q and r are each independently 1 or 2.

In one preferred embodiment, p, q and r are all equal to 1.

In another preferred embodiment, p, q and r are all equal to 2.

In a further preferred embodiment, r is equal to 1 and p is equal to 2.

Preferably, R₇, R₈ and R₉ are all H.

hi one particularly preferred embodiment, X₁, X₂ and X₃ are the same and are all

where R₂ and R₃ are each independently H or a Boc protecting group.

Preferably, n is 0 or 1. More preferably, n is 0.

Typically, j and k are each independently 0, 1, 2 or 3, preferably 0, 1 or 2.

In a more preferred embodiment, the process of the invention is a process for the production of a compound of formula Ia or Ib

More preferably, X₁ and X₃ are the same and are both

where R₂ and R₃ are each independently H or a Boc protecting group.

More preferably, said compound of formula II is of formula Ha, lib, lie or Hd and/or said compound III is of formula Ilia, IEb, IIIc or IHd.

πa πb Hc πd

ffla mb me

Die πid

Most preferably, the compound of formula (I) is

1

or a compound obtainable by deprotecting any one of compounds 1 to 12 above. In a further especially preferred embodiment, the compound of formula (I) is

A further finding of the present invention is that compounds of formula (I) in which Z is para to an X₃ moiety have superior efficiency in delivering a cargo moiety inside a cell.

Thus, the present invention also provides a compound of formula (I), as defined above, or a pharmaceutically acceptable salt thereof, wherein r is 1 or 2 and the phenyl ring which carries L has no substituents ortho to the L moiety. Preferably, the phenyl ring which carries L has a hydrogen atom ortho to the moiety

More preferably, r is 1 and L is para to the X₃ moiety.

The present invention also provides a compound of formula Via, VIb, VIc, VId or Vie

VId Vie wherein X₁, X₃ and L are as defined above.

Compounds of formula VId and VIe are particularly preferred. These compounds, in which the linker position is para to the X₃ moiety, show particularly high efficacy.

Also provided is a compound selected from

The present invention also provides a compound of formula VII

VII wherein

X₁, X₃, R₇, Rg and L are as defined in any one of claims 1, 7, 8, 9 or 11; - r is 1, 2, 3 or 4 and r' is 0, 1, 2 or 3, wherein r + r¹ is 4; p is 1, 2, 3, 4 or 5 and p' is 0, 1, 2, 3 or 4, where p + p¹ is 5; and p + r equals 3.

Preferably, the compound of formula (VII) is

The present invention further provides a process for preparing a conjugate, which process comprises: (i) preparing a compound of formula I, or a pharmaceutically acceptable salt thereof, in which L is other than a moiety -(Z)_m-ρhthalimide, wherein Z and m are as defined in claim 1, by a process according to the invention; and (ii) reacting said compound of formula I with a cargo moiety selected from a protein, a peptide, an oligonucleotide, a nucleotide, a diagnostic agent, a biologically active compound, an antibody and a drug.

The cargo moiety can be an oligonucleotide, nucleotide, protein, peptide, biologically active compound, diagnostic agent, or a combination thereof.

The cargo moiety may be directly or indirectly linked to the carrier moiety, hi the embodiment wherein the cargo moiety is indirectly linked to the carrier, the linkage may be by an intermediary bonding group such as a sulphydryl or carboxyl group or any larger group, all such linking groups are herein referred to as linker moieties as discussed below. Preferably, the carrier and cargo moieties are linked directly.

The diagnostic agent can be nonbiological, for example a microbead. Appropriate processes for preparing a conjugate of a compound of formula I and a nonbiological diagnostic agent such as a microbead are familiar to those of skill in the art.

Examples of suitable oligonucleotide cargo moieties include genes, gene fragments, sequences of DNA, cDNA, RNA, nucleotides, nucleosides, heterocyclic bases, synthetic and non-synthetic, sense or anti-sense oligonucleotides including those with nuclease resistant backbones etc. or any of the above incorporating a radioactive label, that are desired to be delivered into a cell or alternatively to be delivered from a cell to its exterior. Preferably, the oligonucleotide cargo moiety is a gene or gene fragment.

Examples of suitable protein or peptide cargo moieties include; proteins, peptides, and their derivatives such as: antibodies and fragments thereof; cytokines and derivatives or fragments thereof, for example, the interleukins (IL) and especially the IL-I, IL-2, IL-3, IL-4, IL-5, IL- 6, IL-7, IL-8, IL-9, IL-10, IL-Il and IL- 12 subtypes thereof; colony stimulating factors, for example granulocyte-macrophage colony stimulating factor, granulocyte-colony stimulating factor (alpha and beta forms), macrophage colony stimulating factor (also known as CSF-I); haemopoietins, for example erythropoietin, haemopoietin-alpha and kit-ligand (also known as stem cell factor or Steel factor); interferons (IFNS), for example IFN-α, IFN- β and EFN-γ; growth factors and bifunctional growth modulators, for example epidermal growth factor, platelet derived growth factor, transforming growth factor (alpha and beta forms), amphiregulin, somatomedin-C, bone growth factor, fibroblast growth factors, insulin-like growth factors, heparin binding growth factors and tumour growth factors; differentiation factors and the like, for example macrophage differentiating factor, differentiation inducing factor (DEF) and leukaemia inhibitory factor; activating factors, for example platelet activating factor and macrophage activation factor; coagulation factors such as fibrinolytic/anticoagulant agents including heparin and proteases and their pro-factors, for example clotting factors VII, VEH, DC, X, XI and XII, antithrombin m, protein C, protein S, streptokinase, urokinase, prourokinase, tissue plasminogen activator, fibrinogen and hirudin; peptide hormones, for example insulin, growth hormone, gonadotrophins, follicle stimulating hormone, leutenising hormone, growth hormone releasing hormone and calcitonin; enzymes such as superoxide dismutase, glucocerebrosidase, asparaginase and adenosine deaminase; vaccines or vaccine antigens such as, for example hepatitis-B vaccine, malaria vaccine, melanoma vaccine and HIV-I vaccine; transcription factors and transcriptional modulators.

Examples of a suitable non-nucleotide/proteinaceous biologically active cargo moieties are drug moieties selected from cytotoxic agents, anti-neoplastic agents, anti-hypertensives, cardioprotective agents, anti-arrhythmics, ACE inhibitors, anti-inflammatory's, diuretics, muscle relaxants, local anaesthetics, hormones, cholesterol lowering drugs, anti-coagulants, anti-depressants, tranquilizers, neuroleptics, analgesics such as a narcotic or anti-pyretic analgesics, anti-virals, anti-bacterials, anti-fungals, bacteriostats, CNS active agents, anticonvulsants, anxiolytics, antacids, narcotics, antibiotics, respiratory agents, antihistamines, immunosuppressants, immunoactivating agents, nutritional additives, anti-tussives, diagnostic agents, emetics and anti-emetics, carbohydrates, glycosoaminoglycans, glycoproteins and polysaccharides; lipids, for example phosphatidyl-ethanolamine, phosphtidylserine and derivatives thereof; sphingosine; steroids; vitamins; antibiotics including lantibiotics; bacteristatic and bactericidal agents; antifungal, anthelminthic and other agents effective against infective agents including unicellular pathogens; small effect or molecules such as noradrenalin, alpha adrenergic receptor ligands, dopamine receptor ligands, histamine receptor ligands, GABA/benzodiazepine receptor ligands, serotonin receptor ligands, leukotrienes and triodothyronine; cytotoxic agents such as doxorubicin, methotrexate and derivatives thereof.

In one preferred embodiment, the cargo moiety is selected from a protein, a peptide, an antibody and a drug.

In another preferred embodiment the cargo moiety is protein A, a bacterially derived protein that binds strongly to conventional antibodies.

Previous studies have demonstrated that a fusion protein containing the protein transduction domain of HIV-I TAT and the B domain of staphylococcal protein A can be used to internalise antibodies into mammalian cells [Mie et al, Biochemical and Biophysical Research Communications 310 (2003); 730-734].

Preferably, the compound of formula I is linked to commercially available (natural) protein A via a lysine NH₂ group of protein A.

In one particularly preferred embodiment of the invention, the conjugate is the.reaction product of a protein (such as for example, protein A) and a compound of formula (I) as shown above wherein L¹ is (Z)_mNR₅R₆ where Z is a hydrocarbyl group, as defined above, and m is 0 or 1; where R₅ and R₆ are each independently H, CO(CH₂)jQj or C=S(NH)(CH₂)_kQ₂ where j and k are defined above and Q₁ and Q₂ are each independently

hi an alternative preferred embodiment, a cysteine residue may be engineered into the protein to allow conjugation to said compound of formula I. Further details on the preparation of cysteine modified proteins may be found in Neisler et al [Bioconjugate Chem. 2002, 13, 729-736]. Preferably, the cargo moiety is covalently attached to the L group of the compound of formula I. Thus, in step (ii), typically, a reactive group in the cargo moiety reacts with a reactive group in the L moiety of the compound of formula (I).

Typically, in step (ii) a nucleophilic group on the cargo moiety (for example an amine, thiol or hydroxy group) displaces a leaving group in the moiety L in the formula (I). For example, a thiol-containing protein (eg geminin) can react with a compound of formula (I) in which Q₁ or Q₂ is -S-S-(2-pyridyl) via thiol exchange. Similarly, a nucleophilic moiety such as the 2'-hydroxy group on the taxol or docetaxel molecule can displace a succinimyl, -S-S-(2-pyridyl), iodine, -S-S(O)₂-OMe or -CO-O-N-succininyl group in the moiety L in formula (I).

Alternatively, when L is nucleophilic (eg when L is -NH₂), it can react in step (iii) with an electrophilic site in the cargo moiety.

In one preferred embodiment, the cargo moiety is directly linked to the carrier moiety.

In another preferred embodiment, the cargo moiety is indirectly linked to the carrier moiety by means of a linker moiety. In this embodiment, the cargo moiety comprises a protein, a peptide, an oligonucleotide, a nucleotide, a diagnostic agent, a biologically active compound or a drug which is attached to a linker moiety. Typically, a reactive site on the linker group reacts with the moiety L in the formula (I) as explained above.

Direct linkage may occur through any convenient functional group on the cargo moiety, such as a hydroxy, carboxy or amino group. Indirect linkage will occur through a linking moiety. Suitable linking moieties include bi- and multi-functional alkyl, aryl, aralkyl or peptidic moieties, alkyl, aryl or aralkyl aldehydes acids esters and anhydrides, sulphydryl or carboxyl groups, such as maleimido benzoic acid derivatives, maleimido proprionic acid derivatives and succinimido derivatives or may be derived from cyanuric bromide or chloride, carbonyldiimidazole, succinimidyl esters or sulphonic halides and the like. The functional group on the linker moiety used to form covalent bonds between the compound of formula I and the cargo moiety may be, for example, amino, hydrazino, hydroxyl, thiol, maleimido, carbonyl, and carboxyl groups, etc. The linker moiety may include a short sequence of from 1 to 4 amino acid residues that optionally includes a cysteine residue through which the linker moiety bonds to the compound of formula I. Alternatively, the compound of formula I and the cargo moiety may be linked by leucine zippers, dimerisation domains, or an avidin/biotin linker.

In one preferred embodiment, the cargo moiety is selected from a recombinant antibody, a Fab fragment, a F(ab')₂ fragment, a single chain Fv, a diabody, a disulfide linked Fv, a single antibody domain and a CDR.

As used herein, the term "CDR" or "complementary determining region" refers to the hypervariable regions of an antibody molecule, consisting of three loops from the heavy chain and three from the light chain, that together form the antigen-binding site.

By way of example, the antibody may be selected from Herceptin, Rituxan, Theragyn (Pemtumomab), Infliximab, Zenapex, Panorex, Vitaxin, Protovir, EGFRl or MFE-23.

In one preferred embodiment, the cargo moiety is a genetically engineered fragment selected from a Fab fragment, a F(ab')₂ fragment, a single chain Fv, or any other antibody- derived format.

Conventionally, the term "Fab fragment" refers to a protein fragment obtained (together with Fc and Fc¹ fragments) by papain hydrolysis of an immunoglobulin molecule. It consists of one intact light chain linked by a disulfide bond to the N-terminal part of the contiguous heavy chain (the Fd fragment). Two Fab fragments are obtained from each immunoglobulin molecule, each fragment containing one binding site. In the context of the present invention, the Fab fragment may be prepared by gene expression of the relevant DNA sequences.

Conventionally, the term "F(ab')₂" fragment refers to a protein fragment obtained (together with the pFc' fragment) by pepsin hydrolysis of an immunoglobulin molecule. It consists of that part of the immunoglobulin molecule N-terminal to the site of pepsin attack and contains both Fab fragments held together by disulfide bonds in a short section of the Fc fragment (the hinge region). One F(ab')₂ fragment is obtained from_each immunoglobulin molecule; it contains two antigen binding sites, but not the site for complement fixation. In the context of the present invention, the F(ab')₂ fragment may be prepared by gene expression of the relevant DNA sequences.

As used herein, the term "Fv fragment" refers to the N-terminal part of the Fab fragment of an immunoglobulin molecule, consisting of the variable portions of one light chain and one heavy chain. Single-chain Fvs (about 30 KDa) are artificial binding molecules derived from whole antibodies, but which contain the minimal part required to recognise antigen.

hi another preferred embodiment, the cargo moiety is a synthetic or natural peptide, a growth factor, a hormone, a peptide ligand, a carbohydrate or a lipid.

The cargo moiety can be designed or selected from a combinatorial library to bind with high affinity and specificity to a target antigen. Typical affinities are in the 10^"6 to 10^~15 M K_d range. Functional amino acid residues present in the cargo moiety may be altered by site-directed mutagenesis where possible, without altering the properties of the cargo moiety. Examples of such changes include mutating any free surface thiol-containing residues (cysteine) to serines or alanines, altering lysines and arginines to asparagines and histidines, and altering serines to alanines.

In a further embodiment of the invention, the cargo moiety is a drug. In this embodiment, the conjugate can be described as a delivery system. Preferably, the delivery system is therapeutically active in its intact state.

More preferably, the drug moiety is derived from a cytotoxic drug.

More preferably, the drug moiety is selected from DNA damaging agents, anti-metabolites, anti-tumour antibiotics, natural products and their analogues, dihydrofolate reductase inhibitors, pyrimidine analogues, purine analogues, cyclin-dependent kinase inhibitors, thymidylate synthase inhibitors, DNA intercalators, DNA cleavers, topoisomerase inhibitors, anthracyclines, vinca drugs, mitomycins, bleomycins, cytotoxic nucleosides, pteridine drugs, diynenes, podophyllotoxins, platinum containing drugs, differentiation inducers and taxanes.

Even more preferably, the drug moiety is selected from methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, tri-substituted purines such as olomoucine, roscovitine and bohemine, flavopiridol, staurosporin, cytosine arabinoside, melphalan, leurosine, actinomycin, daunorubicin, doxorubicin, mitomycin D, mitomycin A, carninomycin, aminopterin, tallysomycin, podophyllotoxin (and derivatives thereof), etoposide, cisplatinum, carboplatinum, vinblastine, vincristine, vindesin, paclitaxel, docetaxel, taxotere retinoic acid, butyric acid, acetyl spermidine, tamoxifen, irinotecan and camptothecin.

hi one preferred embodiment, the drug moiety is directly linked to the carrier moiety.

hi another preferred embodiment, the drug moiety is indirectly linked to the carrier moiety by means of a linker moiety.

In another preferred embodiment, each carrier moiety bears more than one drug moiety.

hi one preferred embodiment, where each carrier moiety bears more than one drug moiety, the drug moieties are different.

hi one preferred embodiment, where each carrier moiety bears more than one drug moiety, each drug moiety is linked to the carrier moiety by way of a linker moiety, hi one particularly preferred embodiment, each drug moiety is linked to the carrier moiety by an identical linker moiety. In an alternative embodiment, each drug moiety is linked to the carrier moiety by a different linker moiety.

hi a further preferred embodiment of the invention, the delivery system may further comprise a targeting moiety. The targeting moiety is capable of directing the delivery system to the specific cell type to which it is preferable for the drug moiety to function. Thus, the targeting moiety acts as an address system biasing the body's natural distribution of drugs or the delivery system to a particular cell type. The targeting moiety may be attached to the drug moiety or alternatively to the carrier moiety.

In one preferred embodiment, the targeting moiety is directly linked to the carrier moiety.

hi another preferred embodiment, the targeting moiety is indirectly linked to the carrier moiety by means of a linker moiety.

Direct linkage may occur through any convenient functional group on the targeting moiety, such as a hydroxy, carboxy or amino group. Indirect linkage will occur through a linking moiety. Suitable linking moieties include bi- and multi-functional alkyl, aryl, aralkyl or peptidic moieties, alkyl, aryl or aralkyl aldehydes acids esters and anhydrides, sulphydryl or carboxyl groups, such as maleimido benzoic acid derivatives, maleimido proprionic acid derivatives and succinimido derivatives or may be derived from cyanuric bromide or chloride, carbonyldiimidazole, succinimidyl esters or sulphonic halides and the like. The functional groups on the linker moiety used to form covalent bonds to the targeting moiety may be two or more of, e.g., amino, hydrazino, hydroxyl, thiol, maleimido, carbonyl, and carboxyl groups, etc. The linker moiety may include a short sequence of from 1 to 4 amino acid residues that optionally includes a cysteine residue through which the linker moiety bonds to the targeting moiety. Alternatively, the targeting moiety may be linked by leucine zippers, dimerisation domains, or an avidin/biotin linker.

The present invention also provides a conjugate obtainable by reacting a compound of formula (I), as defined above, in which r is 1 or 2 and the phenyl ring which carries L has no substituents ortho to the L moiety, or a compound of formula Via, VIb, VIc, VId, VIe or VII, as defined above, with a cargo moiety as defined above, or a pharmaceutically acceptable salt of said conjugate.

Another aspect of the invention relates to a pharmaceutical composition comprising a compound or conjugate of the invention admixed with one or more pharmaceutically acceptable diluents, excipients or carriers. Even though the compounds and conjugates of the present invention (including their pharmaceutically acceptable salts, esters and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.

Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the "Handbook of Pharmaceutical Excipients, 2^nd Edition, (1994), Edited by A Wade and PJ Weller.

Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).

Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.

The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).

Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.

Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

The compounds of the invention can be present as salts or esters, in particular pharmaceutically acceptable salts or esters.

Pharmaceutically acceptable salts of the compounds of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. sulphuric acid, phosphoric acid or hydrohalic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C₁-C₄)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid.

Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterifϊed. Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (d-C₄)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid.

Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcohols of 1- 12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).

In all aspects of the present invention previously discussed, the invention includes, where appropriate all enantiomers and tautomers of compounds of the invention. The man skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.

Some of the compounds of the invention may exist as stereoisomers and/or geometric isomers — e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all the individual stereoisomers and geometric isomers of those compounds, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).

The present invention also includes all suitable isotopic variations of the compound or pharmaceutically acceptable salt thereof. An isotopic variation of a compound of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁷0, ¹⁸0, ³¹P, ³²P, ³⁵S, ¹⁸F and ³⁶Cl, respectively. Certain isotopic variations of the agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as ³H or ¹⁴C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., ²H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.

The present invention also includes the use of solvate forms of the compounds of the present invention. The terms used in the claims encompass these forms.

The invention furthermore relates to the compounds and/or conjugates of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.

The invention further includes the compounds of the present invention in prodrug form. Such prodrugs are generally compounds of the invention wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Such reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out be an esterase etc. Other such systems will be well known to those skilled in the art.

The pharmaceutical compositions of the present invention may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration. For oral administration, particular use is made of compressed tablets, pills, tablets, gellules, drops, and capsules. Preferably, these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose.

Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.

An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.

Injectable forms may contain between 10-1000 mg, preferably between 10-250 mg, of active ingredient per dose.

Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.

A person of ordinary skill in the art can easily determine an appropriate dose of one of the compositions of the invention to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

Depending upon the need, the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight, hi an exemplary embodiment, one or more doses of 10 to 150 mg/day will be administered to the patient.

In a particularly preferred embodiment, the one or more compounds and/or conjugates of the invention are administered in combination with one or more other therapeutically active agents, for example, existing drugs available on the market. In such cases, the compounds of the invention may be administered consecutively, simultaneously or sequentially with the one or more other therapeutically active agents.

Drugs in general are more effective when used in combination. In particular, combination therapy is desirable in order to avoid an overlap of major toxicities, mechanism of action and resistance mechanism(s). Furthermore, it is also desirable to administer most drugs at their maximum tolerated doses with minimum time intervals between such doses. The major advantages of combining chemotherapeutic drugs are that it may promote additive or possible synergistic effects through biochemical interactions and also may decrease the emergence of resistance in cells which would have been otherwise responsive to initial chemotherapy with a single agent.

By way of example, numerous combinations are used in current treatments of cancer and leukaemia. A more extensive review of medical practices may be found in "Oncologic Therapies" edited by E. E. Vokes and H. M. Golomb, published by Springer.

The present invention also provides use of a compound or conjugate of the invention in the manufacture of a medicament for use delivering a drug to a patient transdermally. The present invention also provides a skin patch which comprises a compound or conjugate of the invention and a pharmaceutically acceptable carrier or diluent.

Figure 1 shows the cellular uptake, via live microscopy, of three compounds of the invention conjugated to Geminin labelled with AlexFluor 488 (11-g^*, 33-g^* and 35-g^*) in two cell lines (Wl -38 human diploid fibroblast and human U2OS osteosarcoma cells).

Figure 2 shows the results of cellular uptake monitored by FACS analysis of eight compounds of the invention conjugated to Geminin labelled with AlexFluor 488 (23-g , 26- g^*, 18-g^*, 20-g\ 33-g^*, 31-g^*, 35-g^* and 11-g^*) in two cell lines (Wl-38 HDF and U2OS).

Figure 3 shows the results of a cell proliferation assay monitored via confocal fluorescence microscopy of 3 compounds of the invention coupled to 15 Geminin in Wl-38 and U2OS cell lines.

The following Examples illustrate the invention.

Examples

All starting materials were either commercially available or synthesised by methods reported in the literature. ¹H and ¹³C spectra were recorded on a Bruker AMX-300 spectrometer. Chemical shifts are reported as ppm relative to TMS as internal standard. Mass spectra were recorded on either a VG ZAB SE spectrometer (EI, FAB) or a Gilson- Finningan AQA LC-mass spectrometer using C-18 column (Hypersil BDS 100 x 4.6 mm, 5 μm). Microanalysis was carried out by the Analytical Services Section, Department of Chemistry, University College London. Purification was by reverse-phase HPLC (Gilson) using preparative C-18 columns (Hypersil PEP 100 x 21 mm, 5μm). Melting points were determined on a Gallenkamp melting point apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer Spectrum One series FT-IR spectrophotometer. The microwave experiments were run on a Biotage Initiator 60 microwave.

Examples 1 to 3: Synthesis of SMOC carrying 4 guanidine moieties

l,4-Dibromo-2,3-dimethoxy-benzene

M. Albrecht, Synthesis - Stuttgart 1996, 230

n-BuLi 1.6M in hexane (75 ml, 120 mmol) was added dropwise at 0 ⁰C to a solution of veratrole (3.3 g, 24 mmol) and TMEDA (22.5 ml, 150 mmol) in anhydrous ether (250 ml) under nitrogen and stirred at room temperature for 3 days. The reaction mixture was cooled to -78 ⁰C and Br₂ (7.5mL, 147 mmol) was added, after stirring for further day at room temperature. The reaction mixture was diluted with ether (150 mL) and washed with water (150 mL), IN HCl (150 mLχ2) and brine (150 mL), dried over Na₂SO_4. The solvent was removed under vacuum and purified by silica gel flash chromatography using cyclohexane/ AcOEt as eluent (5/1) to afford l,4-dibromo-2,3-dimethoxy-benzene as a colourless oil (1.15 g, 16% yield). ¹H-NMR (CDCl₃): 53.83 (s, 6H), 7.18 (s, 2H).

1, 2-Dimethoxy-3-bromo-6-iodobenzene

To a solution of veratrole (12.7 mL, 0.1 mol) in dry ether (200 mL) was added, under nitrogen at 0 ⁰C, TMEDA (16.4 mL, 0.12 mol) followed by BuLi (1.6M in hexane, 75 mL, 0.12 mol). The mixture was allowed to warm to room temperature and stirred for 1 h. After cooling to 0 °C, TMSCl (15.3 mL, 0.12 mol) was added. The reaction mixture was stirred at 0 ⁰C for 30 minutes. After stirring overnight at room temperature, the reaction was cooled again to 0 ⁰C and TMEDA (16.4 mL, 0.12 mol) and BuLi (1.6M in hexane, 75 mL, 0.12 mol) were added. After stirring for 2 h at room temperature, the reaction mixture was cooled to - 40 °C and a solution of (CBrCl₂)₂ (32 g, 0.1 mol) in dry ether (50 mL) was added. After stirring for 1 h at minus 30 °C, the reaction mixture was allowed to warm to 0 ⁰C and water (100 mL) was added slowly. The organic layer was separated and washed with water (2 x 100 mL), dried over Na₂SO₄ and filtered. The solvent was removed under vacuum and the crude product purified by flash chromatography using DCM/Et₂O (85/15) as eluent to afford l,2-dimethoxy-3-bromo-6-trimethylsilylbenzene (25.7 g, 89% yield) as a yellow oil. 1H-NMR (CDCl₃): .50.29 (s, 9H), 3.85 (s, 3H), 3.91 (s, 3H), 6.98 (d, J= 7.9 Hz, IH), 7.27 (d, J= 8 Hz, IH).

¹³C-NMR (CDCl₃): δ-0.63, 60.2, 60.4, 119.7, 127.9, 130.4, 149.4, 158.6.

Theoretical Mass: (M + H) 288.01811. Measured Mass: (M + H) 288.01863

To a solution of l,2-dimethoxy-3-bromo-6-trimethylsilylbenzene (5.8 g, 20 mmol) in DCM

(60 mL) was added dropwise at - 50 °C a solution of iodine chloride (3.6 g, 22 mmol) in DCM (20 mL) keeping the temperature below - 40 ⁰C. After stirring for 2 h at - 50 ⁰C, the reaction mixture was quenched with Na₂S₂O₃ 1 M (100 mL). The layers were separated and the aqueous layer was extracted with DCM (100 mL). The combined organic layers were dried over Na₂SO₄, filtered and concentrated under vacuum. The residue was then crystallized from MeOH (30 mL) to afford the title compound (1.82 g, 26% yield) as a white solid.

¹H-NMR (CDCl₃): .53.86 (s, 6H), 7.02 (d, J- 8.7 Hz, IH), 7.35 (d, J= 8.6 Hz, IH). 1³C-NMR (CDCl₃): 560.8, 60.8, 91.3, 118.4, 129.7, 134.4, 150.7, 154.1. Theoretical Mass: (M + H) 342.88306. Measured Mass: (M + H) 342.88334 M.p. 30-32°C

2-(Benzyloxycarbonylamino)ethyl methanesulfonate

To a solution of 2-(benzyloxycarbonylamino)ethanol (25 g, 0.128 mol) and Et₃N (70 mL,

0.51 mol) in DCM (250 mL) was added methanesulfonyl chloride (25 mL, 0.32 mol) dropwise at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred overnight. The organic layer was washed with HCl IM (2 x 50 mL), sat. aq.

NaHCO₃ (2 x 50 mL), brine (2 x 50 mL), and dried over MgSO₄ and filtered. The solvent was removed under vacuum to afford the title compound (35 g, 100% yield) as an orange solid which was used in the next step without further purification.

¹H-NMR (CDCl₃): 52.92 (s, 3H), 3.54 (q, J= 5 Hz, 2H), 4.39 (t, J= 5 Hz, 2H), 5.08 (s,

2H), 5.84(bs, 1 H), 7.3 l(m, 5H).

¹³C-NMR (CDCl₃): 537.2, 40.3, 66.9, 68.6, 128.1, 128.6, 136.3, 156.5. Theoretical Mass: (M + H) 274.07491. Measured Mass: (M + H) 274.07510

Microanalysis: %C 47.86 (48.34), %H 5.66 (5.66), %N 5.08 (5.12)

M.p. 44-46°C

Dibenzyl 2,2'-(3-bromo-6-iodo-l,2-phenyIene)bis(oxy)bis(ethane-2,l-diyl)dicarbamate

To a solution of 1, 2-dimethoxy-3-bromo-6-iodobenzene (5.7 g, 16.5 mmol) in DCM (50 mL) was added a 1.0 M DCM solution OfBBr₃ (16.5 mL, 16.5 mmol) dropwise at 0 °C. The reaction mixture was allowed to warm to room temperature. After stirring overnight, the reaction mixture was cooled to 0 °C and MeOH (2 mL) was added dropwise. After stirring for 10 minutes at room temperature, the organic layer was washed with IN HCl (2 x 20 mL), water (20 mL) and brine (20 mL), dried over MgSO₄, filtered and concentrated under vacuum. The resulting 3-bromo-6-iodocatechol was then dissolved in DMF (70 mL) and Na₂CO₃ (5.25 g, 49.5 mmol) and 2-(benzyloxycarbonylamino)ethyl methanesulfonate (13.5 g, 49.5 mmol) were added. After stirring at 80°C under nitrogen for 24 h, the reaction mixture was poured onto water (200 mL) and extracted with EtOAc (2 x 100 mL). The organic layer was washed with water (100 mL), dried over Na₂SO₄ and filtered. The solvent was then removed under vacuum and the crude compound was purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to afford the title compound (6.5 g, 59% yield) as a white solid.

ESMS; m/z 669, 671 (M+l), 686,688 (M+18)

¹H-NMR (CDCl₃): .53.53 (q, J= 5 Hz, 4H), 4.05 (m, 4H), 5.12 (s, 4H), 5.63 (bs, IH), 5.65 (bs, IH), 7.03 (d, J= 8.5 Hz, IH), 7.34 (m, HH).

¹³C-NMR (CDCl₃): 541-1, 41.2, 66.8, 72.8, 72.9, 91.4, 118.5, 128.1, 128.5, 130.2, 134.9, 136.6, 149.4, 152.8, 156.5.

Theoretical Mass: (M + Na) 690.99166. Measured Mass: (M + Na) 690.98946 Microanalysis: %C 47.01 (46.66), %H 4.01 (3.92), %N 4.13 (4.19) M.p. 79°C

Dibenzyl 2,2'-(3-bromo-6-cyano-l,2-phenylene)bis(oxy)bis(ethane-2,l- diyl)dicarb amate

A mixture of dibenzyl 2,2'-(3-bromo-6-iodo-l,2-phenylene)bis(oxy)bis(ethane-2,l- diyl)dicarbamate (1.34 g, 2 mmol), zinc cyanide (0.12 g, 1 mmol), Pd₂dba₃ (37 mg, 0.04 mmol), dppf (44 mg, 0.08 mmol), zinc acetate (75 mg, 0.4 mmol) and zinc (26 mg, 0.4 mmol) in dimethylacetamide (10 mL) was heated at 140⁰C for 10 minutes under microwave irradiation. The reaction mixture was diluted in EtOAc (25 mL) and water (100 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (7/3) as eluent to afford the title compound (0.69 g, 61% yield) as a white solid.

ESMS; m/z 568-570 (M+l), 585-587 (M+18). 1H-NMR (CDCl₃): 53.54 (bs, 4H), 4.08 (bs, 2H), 4.25 (bs, 2H), 5.10 (s, 2H), 5.11 (s, 2H),

5.52 (bs, 2H), 7.21-7.38 (m, 12H).

¹³C-NMR (CDCl₃): 541-1, 66.9, 73.0, 74.3, 107.4, 115.4, 124.5, 128.1, 128.2, 128.5, 128.6,

128.8, 129.2, 136.4, 150.0, 155.0, 156.4.

Theoretical Mass: (M + Na) 590.09026. Measured Mass: (M + Na) 590.09091 IR v _Max KBR/cm^" 3322 (br NH, NH₂), 2231 (CN), 1694 (C=ON)

M.p. 91-92⁰C

l-Bromo-2,3-dimethoxy-benzene

n-BuLi 1.6M in hexane (70 mL, 112 mmol) was added dropwise at 0 ⁰C to a solution of veratrole (10 g, 72.3 mmol) and TMEDA (10.9 mL, 72.3 mmol) in anhydrous ether (50 mL) under nitrogen and stirred at room temperature for 2 h. The reaction mixture was cooled to -78 ⁰C and (CBrCl₂)₂ (31.2 g, 112 mmol) was added, after stirring for a further 10 min, the cooling bath was removed and the reaction vessel allowed to warm to room temperature. The reaction mixture was diluted with ether (50 mL), washed with water (50 mL), IN HCl (2 x 50 mL), brine (50 mL) and dried over Na₂SO_4. The solvent was removed under vacuum and the crude product was purified by silica gel flash chromatography using hexane/DCM as eluent (5/1) to afford l-bromo-2,3-dimethoxy-benzene as a colourless oil (12.1 g, 77% yield). 1H-NMR (CDCl₃): 3.81 (s, 3H), 3.83 (s, 3H), 6.85 (d, J= 8.3 Hz, IH), 6.61-6.93 (m, IH), 6.96 (d, J= 8.3 Hz, IH).

¹³C-NMR (CDCl₃): 60.3, 60.6, 109.6, 111.7, 117.4, 132.1, 149.4, 151.5.

Dibenzyl 2,2'-(3-bromo-l,2-phenylene)bis(oxy)bis(ethane-2,l-diyl)dicarbamate

The l-bromo-2,3-dimethoxybenzene (2.17 g, 10 mmol) was dissolved in DCM (60 ml) and treated with 1.0 M DCM solution OfBBr₃ (15 mL, 15 mmol) at 0 °C and then allowed to warm to room temperature. After stirring overnight, the reaction mixture was cooled to 0 ⁰C, 2 mL of MeOH was added, and the solvent was removed under vacuum. The residue was dissolved in 20 mL of EtOAc and washed with IN HCl (2 x 50 mL), water, brine and dried over Na₂SO₄. The solvent was removed under vacuum. The crude residue was then dissolved in DMF (40 mL) and Cs₂CO₃ (9.75 g, 30 mmol) and 2- (benzyloxycarbonylamino)ethyl methanesulfonate (6.8 g, 25 mmol) were added. After stirring at 100 ⁰C under nitrogen for 2 h, a further amount OfCs₂CO₃ (9.75 g, 30 mmol) and 2-(benzyloxycarbonylamino)ethyl methanesulfonate (6.8 g, 25 mmol) were added. After stirring at 100 ⁰C for 2 h, the reaction mixture was poured onto water (900 mL) and extracted with EtOAc (3 x 100 mL). The organic layers were combined, washed with water (50 mL) and dried over Na₂SO₄. The solvent was removed under vacuum and purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to afford the title compound (2.45 g, 45% yield) as a white solid. ESMS; m/z 543, 545 (M+l), 560, 562 (M+18)

¹H-NMR (CDCl₃): 63.54 (m, 4H), 4.06 (m, 4H), 5.09 (s, 4H), 5.62(bs, IH), 5.75(bs, IH), 6.82 (d, J= 8 Hz, IH), 6.91 (t, J= 8 Hz, IH), 7.13 (dd, J= 8 Hz, J= 1.3 Hz, IH), 7.32 (m, 10H). 1³C-NMR (CDCl₃): 540.5, 41.3, 66.8, 68.2, 71.8, 112.9, 118.0, 125.4, 128.1, 128.2, 128.5, 136.4, 152.6, 156.5.

Theoretical Mass: (M + Na) 565.09501. Measured Mass: (M + Na) 565.09508 Microanalysis: %C 57.58 (57.47), %H 5.10 (5.01), %N 5.16 (5.04) M.p. 54°C

Dibenzyl 2,2'-(3-(4,4,5,5-tetramethyI-l,3,2-dioxaborolan-2-yl)-l,2- phenylene)bis(oxy)bis(ethane-2,l-diyl)dicarbamate

Pd(dppf)Cl₂ (1.5 g, 1.9 mmol), potassium acetate (5.4 g, 56 mmol) and bispinacolatodiboran (10 g, 41 mmol) were weighted in a three necked round bottom flask and DMSO (100 ml) was added. After stirring for 2 minutes, dibenzyl 2,2'-(3-bromo-l,2- phenylene)bis(oxy)bis(ethane-2,l-diyl)dicarbamate (20 g, 37 mmol) was added. The reaction mixture was heated to 80°C for 72 h. After cooling the mixture at room temperature, 1 L of water and 0.5 L of toluene were added. Toluene was separated and washed with brine, dried over MgSO₄, filtered and evaporated to dryness. The resulting oil was purified by silica chromatography using DCM to DCM/EtOAc (9/1) as eluent yielding

(6.4g, 30%) as a white solid.

ESMS; m/z 591(M+1)

¹H-NMR (CDCl₃): .61.30 (s 12H), 3.54 (t, J = 4.6 Hz, 4H), 4.04 (m, 2H), 4.14 (t, J= 4.4 Hz, 2H), 5.11 (s, 4H), 5.12 (bs, IH), 5.25 (bs, IH), 6.49 (bs, IH), 6.98 (m, 2H), 7.33 (bs,

10H).

¹³C-NMR (CDCl₃): 624.6, 40.7, 41.5, 66.7, 66.8, 72.7, 83.9, 117.8, 124.2, 128.0, 128.1,

128.4, 128.5, 129.2, 136.5, 136.7, 151.1, 153.1, 156.5, 156.6 Theoretical Mass: (M + Na) 613.26970. Measured Mass: (M + Na) 613.26890 Microanalysis: %C 65.12 (65.09), %H 6.76 (6.66), %N 4.74 (4.74) M.p. 59°C

Potassium (2,3-bis(2-(benzyIoxycarbonylamino)ethoxy)phenyl) trifluoroborate

To a stirred solution of Benzyl 2,2'-(3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l,2- phenylene)bis(oxy)bis(ethane-2,l-diyl)dicarbamate (5.66 g, 9.6 mmol) in MeOH (50 ml) was added dropwise a solution of KHF₂ (6.0 g, 76.72 mmol) in H₂O (17.0 ml). The clear yellow solution forms a white precipitate after 5 minutes. The resulting mixture is stirred at room temperature for 3 hrs. The mixture is then filtered and the white solid residue washed with DCM (3 x 20 ml), cold acetone (2 x 20 ml) and hot acetone (2 x 20 ml). The washings and the filtrate are combined and the solvent removed in vacuo to leave a brown solid which is further purified by washing several times with diethyl ether to afford the title compound (4.85 g, 8.40 mmol, 88%) as a white solid. 1H-NMR (500 MHz) (CDCl₃): δ_H 3.07- 3.35 (m, 4H, 2 x CH₂NH), 3.77 (bs, 4H, 2 x CH₂O), 5.19 (s, 4Η, 2 x ArCH₂O), 5.70 (bs, 1Η, NH), 5.77 (bs, 1Η, NH), 6.57 (d, J= 6.57 Hz, IH, ArH), 6.73 (t, J= 7.2 Hz, IH, ArH), 6.97 (d, J= 5.5, 1Η, ArH), 7.34 (m, 11Η). 1³C-NMR (500 MHz) ( CDCl₃): δ_c 40.7, 40.8, 66.7, 67.6, 124.4, 127.9, 128.0, 128.1, 128.4, 128.5, 136.6, 150.7, 156.8, 157.3. Microanalysis (C₂₆H₂₇BF₃KN₂O₆): C 54.75 %, H 4.77%, N 4.91%, B 1.90%, F 9.99%, K 6.85 %; Found: C 54.19%, H 4.80%, N 4.77%, B 1.92%, F 10.22%, K 6.36%. Tetrabenzyl 2,2',2^M,2'"-(4-cyanobiphenyl-2,2',3,3'- tetrayltetrakis(oxy))tetrakis(ethane-2,l-diyl)tetracarbamate

A degassed mixture of dibenzyl 2,2'-(3-bromo-6-cyano-l,2-phenylene) bis(oxy)bis(ethane- 2,l-diyl)dicarbamate (0.3 g, 0.5 mmol), dibenzyl 2,2^I-(3-(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)-l,2-phenylene)bis(oxy)bis(ethane-2,l-diyl)dicarbamate (0.35 g, 0.6 mmol), PdCl₂dppf. CH₂Cl₂ (20 mg, 0.025 mmol), potassium phosphate (0.21 g, 1 mmol), toluene (1 mL) and water (0.1 mL) was heated at 100°C for 18 h. The reaction mixture was diluted in EtOAc (25 mL) and water (25 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (1/1) as eluent to afford the title compound (0.48 g, 100% yield) as a yellow oil. ESMS; m/z 952 (M+l), 969 (M+18) 1H-NMR (CDCl₃): .53.20 (m, 4H), 3.50 (m, 4H), 3.80 (m, 4H), 3.93 (m, 2H), 4.26 (m, 2H), 4.85 (bs, IH), 4.98 (s, 2H), 5.01 (s, 2H), 5.08 (s, 4H), 5.31(bs, IH), 5.56 (bs, IH), 5.68 (bs, IH), 6.86 (m, 2H), 7.05 (m, 3H), 7.30 (m, 20H).

¹³C-NMR (CDCl₃): 540.6, 41.1, 66.8, 68.0, 70.9, 72.7, 73.9, 75.0, 112.0, 114.1, 116.0, 122.6, 124.5, 127.0, 128.0, 128.2, 128.5, 131.1, 136.4, 136.5, 139.3, 145.8, 149.5, 151.6, 154.2, 156.2, 156.5.

Theoretical Mass: (M + Na) 974.35882. Measured Mass: (M + Na) 974.360673 Microanalysis

%C 64.92 (64.63), %H 5.51 (5.81), %N 6.81(7.09) IR v _Max KBR/cm- 3418 (br NH), 3335 (br NH), 2230 (CN), 1713 (C=O) Mp 72-74°C Tetrabenzyl 2,2',2",2'"-(4-cyanobiphenyl-2,2',3,3'- tetrayltetrakis(oxy))tetrakis(ethane-2,l-diyl)tetracarbamate (Alternative protocol)

To a degassed solution of potassium (2,3-bis(2-(benzyloxycarbonylamino)ethoxy)phenyl) trifluoroborate (800 mg, 1.40 mmol) in isopropanol/water (2:1) (26 mL) was added PdCl₂dppf. CH₂Cl₂ (40 mg, 0.049 mmol) and triethylamine (600 μL, 4.2 mmol). The mixture was stirred at room temperature for 2 minutes then dibenzyl 2,2'-(3-bromo-6- cyano-l,2-phenylene) bis(oxy)bis(ethane-2,l-diyl)dicarbamate (400 mg, 0.70 mmol) was added. The reaction mixture was stirred at 82 °C for 18 h. The reaction mixture was diluted with IM HCl (aq) (50 mL) and the product extracted with DCM (3 x 200 mL). The combined organic fractions were dried over MgSO₄, filtered and concentrated under reduced pressure. The product was purified by flash chromatography using a gradient of 0- 50% EtOAc in cyclohexane over 10 CV to afford the title product (530 mg, 79%) as a white foam.

2, 3, 2', 3'-Tetra{2-[iV, iV'-bis(tert-butoxycarbonyl)guaiiidino]-ethyloxy}-4- cyanobiphenyl

Benzyl 2,2',2",2"'-(4-cyanobiphenyl-2,2',3,3^I-tetrayl)tetrakis(oxy)tetrakis(ethane-2,l- diyl)tetracarbamate (2 g, 2.1 mmol) was dissolved in DCM (100 mL) and HBr (30% in acetic acid, 20 mL) was added dropwise. After stirring at room temperature for 1.5 h, water (25 mL) was added to the mixture, the layers were separated and the aqueous layer was washed with DCM (2 x 25 mL). The water was then removed under vacuum and the crude tetra-amine was carefully dried under vacuum for several hours.

The resulting solid was suspended in DCM (10 mL) and Et₃N (2.9 mL, 21 mmol) and N, N- di-boc-ΛT-trifluoromethanesulfonyl-guanidine (3.3 g, 8.4 mmol) were added. The mixture was stirred overnight at room temperature, diluted with DCM (40 mL) then washed with 2M NaHSO₄ 2M (25 mL) followed by sat. aq. NaHCO₃ (25 mL) and brine (25 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to afford the title compound (1.87 g, 65% yield) as a white solid. ESMS; m/z 1385 (M+l), 693 (M+2), 462 (M+3) 1H-NMR (CDCl₃): .81.47 (m, 72H), 3.47 (m, 2H), 3.54 (m, 2H), 3.90 (m, 8H), 4.16 (t, J= 5 Hz, 2H), 4.38 (t, J= 5 Hz, 2H), 7.00 (m, 3H), 7.18 (d, J= 8 Hz, IH), 7.29 (d, J= 8 Hz, IH), 8.45 (m, 2H), 8.79 (m, 2H), 11.40 (s, IH), 11.42 (s, IH), 11.48 (bs, 2H). 1³C-NMR (CDCl₃): 526.8, 28.0, 28.0, 28.2, 39.9, 40.6, 40.8, 41.3, 73.2, 78.8, 78.9, 79.0, 79.2, 82.9, 82.9, 83.1, 107.1, 113.7, 115.7, 123.2, 124.0, 127.6, 127.7, 130.8, 138.4, 144.8, 149.6, 151.6, 152.8, 152.9, 153.0, 153.0, 154.2, 156.0, 156.3, 163.3, 163.4. Theoretical Mass: (M + Na) 1406.71832. Measured Mass: (M + Na) 1406.71565 IR v _Max KBR/cm- 3335 (br, NH), 2231 (CN), 1722 (C=O), 1641 (C=N) Mp 101-103⁰C

2, 3, 2', 3'-Tetra{2-[iV, Λ^-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-4- cyanobiphenyl (Alternative protocol)

To a solution of benzyl 2,2',2",2'"-(4-cyanobiphenyl-2,2',3,3^l- tetrayl)tetrakis(oxy)tetrakis(ethane-2,l- diyl)tetracarbamate (550 mg, 0.58 mmol) in DCM (25 mL) was added a 30% solution of HBr in acetic acid (5.5 mL) dropwise. The reaction mixture was stirred at room temperature for 1 h then water (20 mL) was added and the layers separated. The organic fraction was extracted with water (20 mL). The combined aqueous fractions were concentrated under reduced pressure and the crude product was dried under vacuum for 8 hours. The resulting solid was suspended in DCM (25 mL) and DBEA (1 mL, 5.84 mmol) was added. After stirring for 10 min N,N'-Di-Boc-lH-pyrazo Ie-I- carboxamidine (790 mg, 2.54 mmol) was added and the reaction mixture stirred at room temperature for 18 h. The reaction mixture was diluted with DCM (50 mL) then washed with 10% citric acid (aq) (25 mL), followed by sat. aq. NaHCO₃ (25 mL) and brine (25 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The product was purified by flash chromatography using a gradient of 0-50% EtOAc in cyclohexane over 12 CV to afford the title product (570 mg, 71%) as white foam.

2, 3, 2', 3'-Tetra{2-[iV, iV'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-4- (aminomethyl)biphenyl (Example 1)

Tert-butyl (2,2^I,2",2'"-(4-cyanobiρhenyl-2,2',3 ,3 '-tetrayl)tetrakis(oxy)tetrakis (ethane-2, 1 - diyl))tetrakis(azanediyl)tetrakis((tert-butoxycarbonylamino)methan- 1 -yl- 1 - ylidene)tetracarbamateylidene)tetracarbamate (0.47 g, 0.34 mmol) was dissolved in THF (7 mL) and NH₄OH (1.4 mL) was added followed by Raney Nickel (0.5 g). The mixture was stirred vigorously under H₂ atmosphere for 16 h. The catalyst was filtered off and washed several times with MeOH. Diethylenetriamine (leq) was added and the mixture stirred for 30 minutes then concentrated under vacuum. The residue was dissolved in EtOAc (10 mL) and washed with sat. aq. NaHCO₃ (10 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was purified by flash chromatography using DCM/MeOH (97/3) as eluent to afford the title compound (0.21Og, 44% yield) as a white solid. ESMS; m/z 1388 (M+l), 695 (M+2), 464 (M+3)

¹H-NMR (CDCl₃): δl.41 (m, 72H), 3.44 (m, 4H), 3.78 (m, 10H), 4.13 (m, 4H), 6.93 (m,

5H), 8.43 (bt, IH), 8.49 (bt, IH), 8.78 (m, 2H), 11.38 (m, 2H), 11.44 (bs, IH), 11.48 (bs,

IH).

¹³C-NMR (CDCl₃): 628.1, 28.3, 40.0, 40.9, 41.2, 41.4, 41.7, 67.2, 70.6, 71.0, 71.4, 78.8, 78.9, 79.0, 79.1, 82.6, 82.8, 83.0, 112.8, 123.3, 123.8, 126.8, 131.4, 132.5, 136.9, 145.2, 149.2, 149.4, 151.7, 152.8, 152.9, 153.0, 156.0, 156.2, 156.3, 163.4, 163.5. IR v Max KBR/cm^' 3335 (br NH, NH₂), 1722 (C=O), 1641 (C=N) Mp 105-107⁰C iV-{2, 3, 2', 3'-Tetra{2-[iV, iV-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-biphenyl- 4-ylmethyl}-3-[2-pyridyI)dithio]propionamide (Example 2)

To a solution of 2, 3, 2', 3 '-terra {2- [N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-4-

(aminomethyl)biphenyl (0.10 g, 0.072 mmol) and Z-Pr₂EtN (20 μL, 0.10 mmol) in DCM

(1.5 mL) was added N-succinimidyl-3-(2-pyridyldithio)propionate ( 22 mg, 1 eq) and the mixture was stirred in the dark for 24 h. The solvent was removed under vacuum and the crude oil was purified by preparative TLC (ethyl acetate/hexane 1/1) to afford the title compound (13 mg, 11% yield).

ESMS; m/z 1587 (M+l), 794 (M+2), 530 (M+3)

¹H-NMR (CDCl₃): δ 1.43-1.51 (m, 72H), 2.70 (t, J= 7.2 Hz, 2H), 3.11 (t, J= 7.2 Hz, 2H),

3.45-3.50 (m, 4H), 3.84-3.93 (m, 8H), 4.15-4.21 (m, 4H), 4.46 (d, J= 5.6 Hz, 2H), 6.89-

6.92 (m, 2H), 6.99-7.08 (m, 4H), 7.57-7.68 (m, 2H), 8.36 (m, IH), 8.46 (br t, IH), 8.55 (br t, IH), 8.80 (br t, 2H), 11.35 (bs, IH), 11.41 (bs, IH), 11.47 (bs, IH), 11.53 (bs, IH).

IR v _Max KBR/cm^" 3336 (NH), 1722 (C=O), 1641 (C=N)

Theoretical Mass: (M + H) 1585.76459. Measured Mass: (M + H) 1585.05043

iV-[2, 3, T, 3'-Tetra(2-guanidino-ethyloxy)-biphenyl-4-yImethyl]-3-[2- pyridyl)dithio]propionamide, trifluoroacetate salt (Example 3)

N'-Bis(tert-butoxycarbonyl)guanidino] -ethyloxy } -biphenyl-4-ylmethyl } -3 - [2- pyridyl)dithio]propionamide (15 mg, 0.0094 mmol) was dissolved in a mixture of TFA/

H₂O/triisopropylsilane 95/2.5/2.5 (1 mL) and stirred at room temperature for 3 h. The solvent was removed under vacuum to give the title compound as colourless oil (15 mg,

100%yield).

ESMS; m/z 785(M+1), 395 (M+2) 1H-NMR (MeOH-d4): δ 2.73 (t, J= 6.6 Hz, 2H), 3.10 (t, J= 6.6 Hz, 2H), 3.23-3.30 (m,

4H), 3.62-3.67 (m, 4H), 3.88 (m, 2H), 3.96 (m, 2H), 4.22 (m, 4H), 4.47 (s, 2H), 6.87 (dd, J

= 7.4, 1.7 Hz, IH), 7.02 (d, J= 7.9 Hz, IH), 7.08-7.24 (m, 4H), 7.76-7.80 (m, 2H), 8.38 (d,

J= 4.7 Hz, IH), 8.70 (br t, IH).

IR v _Max KBR/cm^" 3376 (br NH, NH₂), 1681 (C=O) Theoretical Mass: 748.33734. Measured Mass: 748.33682

Examples 4 to 17: Synthesis of SMC carrying 2 guanidϊne moieties

Benzyl 2-(2-bromo-5-iodophenoxy)ethylcarb amate

To a solution of 2-bromo-5-iodoanisole (12.5 g, 40 mmol) in DCM (200 mL) was added a 1.0 M DCM solution OfBBr₃ (40 mL) dropwise at 0 ⁰C. The reaction mixture was then allowed to warm to room temperature. After stirring overnight, the reaction mixture was cooled to 0 °C and MeOH (5 mL) was added dropwise. After stirring for 10 minutes at room temperature, the reaction mixture was washed with IN HCl (2 x 50 mL), water (50 mL) and brine (50 mL). The organic layer was dried over MgSO₄. filtered and concentrated under vacuum. The residue was then dissolved in DMF (100 mL) and Cs₂CO₃ (26 g, 80 mmol) and 2-(benzyloxycarbonylamino)ethyl methanesulfonate (11 g, 40 mmol) were added. After stirring at 80°C under nitrogen for 4 h, the reaction mixture was dissolved in EtOAc (100 mL). The organic layer was washed with brine (3 x 50 mL), dried over MgSO₄ and filtered. The solvent was removed under vacuum and the crude compound was purified by flash chromatography using cyclohexane/EtOAc (85/15) as eluent to afford the title compound (11.5 g, 60% yield) as a white solid. ESMS; m/z 476, 478 (M+l), 493, 495 (M+18) 1H-NMR (CDCl₃): 53.64 (q, J= 5.2 Hz, 2H), 4.05 (t, J= 4.7 Hz, 2H), 5.12 (s, 2H), 5.33 (bs, IH), 7.15-7.24 (m, 3H), 7.35 (bs, 5H).

¹³C-NMR (CDCl₃): 540.4, 67.0, 68.7, 92.4, 112.4, 122.7, 128.2, 128.2, 128.6, 131.5, 134.6, 136.4, 155.4, 156.4.

Theoretical Mass: (M + Na) 497.91777. Measured Mass: (M + Na) 497.91638 Microanalysis: %C 40.41 (40.36), %H 3.24 (3.18), %N 2.86 (2.94) M.p. 70⁰C Benzyl 2-(2-bromo-5-cyanophenoxy)ethylcarbamate

A mixture of benzyl 2-(2-bromo-5-iodophenoxy)ethylcarbarnate (1.9 g, 4 mmol), zinc cyanide (0.23 g, 2 mmol), Pd₂dba₃ (73 mg, 0.08 mmol), dppf (88 mg, 0.16 mmol), zinc acetate (0.14 g, 0.8 mmol) and zinc (52 mg, 0.8 mmol) in dimethylacetamide (20 mL) was heated at 140 °C for 10 minutes under microwave irradiation. The reaction mixture was diluted in EtOAc (25 mL) and water (100 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (7/3) as eluent to afford the title compound (1.13 g, 75% yield) as a white solid.

ESMS; m/z 375, 377 (M+l), 392, 394 (M+18)

¹H-NMR (CDCl₃): 53.69 (q, J= 5.2 Hz, 2H), 4.11 (t, J= 4.7 Hz, 2H), 5.12 (s, 2H), 5.26 (bs, IH), 7.08 (s, IH), 7.15 (dd, J= 8.1 Hz₅ J= 1.7 Hz, IH), 7.35 (bs, 5H), 7.64 (d, J= 8.1

Hz₅ IH).

¹³C-NMR (CDCl₃): 540.3, 67.0, 68.7, 112.3, 115.8, 118.0, 118.4, 125.9, 128.2, 128.3,

128.6, 134.4, 136.3, 155.3, 156.4.

Microanalysis: %C 54.48 (54.42), %H 4.09 (4.03), %N 7.35 (7.47) Theoretical Mass: (M + Na) 397.01637. Measured Mass: (M + Na) 397.01504

IR v _Max KBR/cπT3322 (br NH, NH₂), 2231 (CN), 1694 (C=ON)

M.p. 86-87°C

Benzyl 2-(4-cyano-2'-hydroxybiphenyl-2-yloxy)ethylcarbamate

A degassed mixture of benzyl 2-(2-bromo-5-cyanophenoxy)ethylcarbamate (0.7 g, 1.8 mmol), 2-hydroxyphenylboronic acid (0.3 g, 2.2 mmol), PdCl₂dppf. CH₂Cl₂ (73 mg, 0.09 mmol), potassium phosphate (0.83 g, 3.6 mmol), toluene (4 mL) and water (0.4 mL) was heated at 100⁰C for 18 h. The reaction mixture was diluted in EtOAc (25 mL) and water (25 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were dried over MgSO₄, filtered and concentrated under vacuum. The residue was purified by flash chromatography using cyclohexane/EtOAc (7/3) as eluent to afford the title compound (0.34 g, 49% yield) as transparent oil. ESMS; m/z 389 (M+l), 406 (M+18)

¹H-NMR (CDCl₃): 63.47 (m, 2H), 4.05 (m, 2H), 5.06 (s, 2H), 5.30 (bs, IH), 6.92-7.04 (m, 2H), 7.15 (m, 2H), 7.28-7.39 (m, 8H).

¹³C-NMR (CDCl₃): 540.3, 66.9, 68.4, 112.5, 115.8, 116.9, 118.5, 121.1, 124.3, 125.8, 128.1, 128.3, 128.6, 130.1, 131.0, 132.8, 133.2, 136.3, 153.2, 155.5, 156.5. Theoretical Mass: (M + Na) 411.13207. Measured Mass: (M + Na) 411.13099

Dibenzyl 2,2'-(4-cyanobiphenyl-2,2'-diyl)bis(oxy)bis(ethane-2,l-diyl)dicarbamate

Benzyl 2-(4-cyano-2'-hydroxybiphenyl-2-yloxy)ethylcarbamate (0.21 g, 0.54 mmol) was dissolved in DMF (7 mL) and Cs₂CO₃ (0.46 g, 1.4 mmol) and 2-

(benzyloxycarbonylamino)ethyl methanesulfonate (0.21 g, 0.77 mmol) were added. After stirring at 8O⁰C under nitrogen for 2 h, the reaction mixture was dissolved in EtOAc (25 mL). The organic layer was washed with brine (3 x 20 mL) and dried over MgSO₄. The solvent was removed under vacuum and the crude compound was purified by flash chromatography using cyclohexane/EtOAc (65/35) as eluent to afford (0.16g, 54% yield) as colourless oil. ESMS; m/z 566 (M+l), 583 (M+18) ¹H-NMR (CDCl₃): .53.33 (m, 4H), 3.90 (m, 4H), 4.80 (bs, IH), 4.97 (bs, 5H), 6.88 (d, J= 8.1 Hz, IH), 7.04 (m, 2H), 7.14 (dd, J= 7.5 Hz, J= 1.6 Hz, IH), 7.30 (m, 13H). 1³C-NMR (CDCl₃): 640.3, 66.9, 68.2, 68.3, 112.4, 113.3, 116.2, 119.9, 121.8, 125.6, 128.3, 128.6, 128.7, 129.9, 130.8, 132.1, 134.5, 136.4, 155.5, 156.0, 156.1. Theoretical Mass: (M + H) 566.22910. Measured Mass: (M + H) 566.22799 IR v _Max KBR/cm-3347 (br, NH), 2228 (CN), 1715 (C=O)

2, 2'-Bis{2-[iV, iV-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-4-cyanobiphenyl

Dibenzyl 2,2'-(4-cyanobiphenyl-2,2'-diyl)bis(oxy)bis(ethane-2, 1 -diyl)dicarbamate (0.12 g, 0.21 mmol) was dissolved in DCM (10 niL) and HBr (30% in acetic acid, 1.2 mL) was added dropwise. After stirring at room temperature for 1.5 h, the solvent was removed under vacuum and the crude diamine was carefully dried under vacuum for several hours. The resulting solid was suspended in DCM (5 mL) and Et₃N (0.29 mL, 2.1 mmol) and N, N-di-boc-N'-trifluoromethanesulfonyl-guanidine (0.17 g, 0.42 mmol) were added. The mixture was stirred over night at room temperature, diluted with DCM (20 mL) then washed with 2M NaHSO₄ 2M (25 mL) followed by sat. aq. NaHCO₃ (25 mL) and brine (25 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to afford the title compound (0.09 g, 54% yield) as a white solid.

¹H-NMR (CDCl₃): _.51.49 (m, 36H), 3.71 (q, J= 5 Hz, 4H), 4.05 (t, J= 4.6 Hz, 4H), 6.96 (d, J= 8.3 Hz, IH), 7.03 (t, J= 7.1 Hz, IH), 7.17 (d, J= 1.2 Hz, IH), 7.30-7.34 (m, 3H), 7.46 (d, J= 7.8 Hz, IH), 8.46 (m, 2H), 11.46 (bs, 2H). 1³C-NMR (CDCl₃): .628.1, 28.3, 39.9, 40.0, 66.7, 67.3, 79.4, 83.1, 83.3, 111.8, 112.0, 115.5, 118.7, 120.8, 124.9, 125.3, 129.5, 131.6, 132.9, 132.9, 153.0, 155.2, 155.8, 156.2, 163.4. Theoretical Mass: (M + H) 782.40885. Measured Mass: (M + H) 782.40963 Microanalysis (C₃₉H₅₅N₇O₁₀^H₂O): %C 56.90 (57.27), %H 7.12 (7.09), %N 11.58 (11.99) IR v _Max KBR/cm^" 3333 (br NH), 2228 (CN), 1721 (C=O), 1640 (C=N), 1618 (C=N) M.p. 82°C

N-{2, 2'-Bis{2-[iV, iV'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-biphenyl-4- ylmethyl}-3-[2-pyridyl)dithio]propionamide (Example 4)

2, 2'-Bis{2-[N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-4-cyanobiphenyl (0.06 g, 0.077 mmol) was dissolved in THF (2 mL) and NH₄OH (0.2 mL) was added followed by Raney Nickel (0.05 g). The mixture was stirred vigorously under H₂ atmosphere for 16 h. The catalyst was filtered off and washed several times with MeOH. Diethylenetriamine (leq) was added and the mixture stirred for 30 minutes then concentrated under vacuum. The residue was dissolved in EtOAc (10 mL) and washed with sat. aq. NaHCO₃ (10 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. To a solution of the crude amine and J-Pr₂EtN (26 μl, 0.15 mmol) in DCM (1.5 mL) was added N-succinimidyl-3-(2-pyridyldithio)propionate (34 mg, 0.11 mmol) and the mixture was stirred in the dark for 24 h. The solvent was removed under vacuum and the crude oil was purified by preparative TLC (ethyl acetate/hexane 1/1) to afford the title compound (20 mg, 26% yield).

¹H-NMR (CDCl₃): .81.48 (m, 36H), 2.68 (t, J= 5.0 Hz, 2H), 3.03 (t, J= 5 Hz, 2H), 3.67 (m, 4H), 3.99 (m, 4H), 4.49 (d, J= 5.2 Hz, 2H), 6.90-7.05 (m, 4H), 7.21-7.33 (m, 3H), 7.57 (m, 2H), 8.20 (d, J= 4.7 Hz, IH), 8.47 (m, 2), 11.46 (bs, 2H).

¹³C-NMR (CDCl₃): 828.1, 28.3, 29.7, 35.2, 35.8, 40.2, 53.4, 66.9, 79.3, 82.9, 83.1, 112.3, 112.7, 120.4, 121.0, 126.9, 128.5, 132.0, 132.3, 136.9, 138.4, 149.6, 152.9, 155.6, 155.8, 156.2, 159.4, 163.5, 170.6. Theoretical Mass: (M + Na) 1005.41899. Measured Mass: (M + Na) 1005.41622 N-[2, 2'-Bis(2-guanidino-ethyloxy)-biphenyl-4-ylmethyl]-3-[2- pyridyl)dithio]propionamide, formiate salt (Example 5)

N- {2, 2'-Bis {2~[N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy} -biphenyl-4-ylmethyl} - 3-[2-pyridyl)dithio]propionamide (15 mg, 0.015 mmol) was dissolved in formic acid (1 mL) and stirred at room temperature overnight. The mixture was then heated at 50⁰C for

Ih. Water was added and the product was lyophilised to give as colourless oil (13.6 mg,

100%).

¹H-NMR (MeOH-d4): 52.76 (t, J= 6.6 Hz, 2H), 3.11 (t, 2H, J= 6.3 Hz, 2H), 3.42 (m, 4H), 4.04 (m, 4H), 4.421 (s, 2H), 6.99-7.24 (m, 6H), 7.35 (t, J= 6.9 Hz, IH), 7.78-7.83 (m, 2H),

8.15 (m, IH), 8.39 (d, J= 4.6 Hz, IH), 8.57 (bs, 2H).

Theoretical Mass: (M + H) 583.22734. Measured Mass: (M + H) 583.22651

Benzyl 2-(4-cy ano-3 '-hydroxybiphenyl-2-yIoxy)ethylcarbamate

A degassed mixture of benzyl 2-(2-bromo-5-cyanophenoxy)ethylcarbamate (0.7 g, 1.8 mmol), 3-hydroxyphenylboronic acid (0.3 g, 2.2 mmol), PdCl₂dppf. CH₂Cl₂ (73 mg, 0.09 mmol), potassium phosphate (0.83 g, 3.6 mmol), toluene (4 mL) and water (0.4 mL) was heated at 100⁰C for 18 h. The reaction mixture was diluted in EtOAc (25 mL) and water (25 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (7/3) as eluent to afford the title compound (0.57 g, 81% yield) as transparent oil.

ESMS; m/z 389 (M+l), 406 (M+18)

¹H-NMR (CDCl₃): 53.63 (m, 2H), 4.01 (m, 2H), 5.10 (bs, 3H), 5.19 (bs, IH), 6.89 (m, 2H), 6.99 (d, J= 7.6 Hz, IH), 7.09-7.40 (m, 8H), 7.48 (d, J= 7.8 Hz, IH).

¹³C-NMR (CDCl₃): 640.4, 67.3, 67.9, 111.8, 115.6, 116.4, 117.0, 118.7, 121.0, 125.5, 128.2, 128.4, 128.6, 129.6, 131.3, 135.2, 136.0, 137.4, 155.5, 155.9, 156.8. Theoretical Mass: (M + H) 389.15012. Measured Mass: (M + H) 389.15110

Dibenzyl 2,2'-(4-cyanobiphenyl-2,3'-diyl)bis(oxy)bis(ethane-2,l-diyI)dicarbamate

To benzyl 2-(4-cyano-3'-hydroxybiphenyl-2-yloxy)ethylcarbamate (0.49 g, 1.26 mmol) dissolved in DMF (12 mL) was added Cs₂CO₃ (0.82 g, 2.5 mmol) and 2- (benzyloxycarbonylamino)ethyl methanesulfonate (0.38 g, 1.4 mmol). After stirring at 80 ⁰C under nitrogen for 3 h, further Cs₂CO₃ (0.41 g, 1.25 mmol) and 2-

(benzyloxycarbonylamino)ethyl methanesulfonate (0.2 g, 0.7 mmol) were added. After stirring for an additional 2 h, the reaction mixture was dissolved in EtOAc (25 mL). The organic layer was washed with brine (3 x 20 mL) and dried over MgSO₄. The solvent was removed under vacuum and the crude compound was purified by flash chromatography using cyclohexane/EtOAc (65/35) as eluent to afford the title compound (0.44g, 62% yield) as a colourless oil.

ESMS; m/z 566 (M+l), 583 (M+18)

¹H-NMR (CDCl₃): 53.51 (bs, 4H), 4.03 (bs, 4H), 5.07 (m, 5H), 5.32 (bs, IH), 6.87 (d, J = 7.6 Hz, IH), 7.04 (m, 2H), 7.21 (s, IH), 7.32-7.38 (m, 13H). 1³C-NMR (CDCl₃): 540.4, 40.6, 67.0, 68.0, 112.1, 114.0, 115.9, 118.6, 122.0, 125.5, 128.2, 128.3, 128.6, 129.5, 131.5, 135.8, 136.3, 136.4, 138.0, 155.5, 156.3, 156.5, 158.4. Theoretical Mass: (M + H) 566.22910. Measured Mass: (M + H) 566.22854 2, 3'-Bis{2-[N, iV'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-4-cyanobiphenyl

Dibenzyl 2,2'-(4-cyanobiphenyl-2,3'-diyl)bis(oxy)bis(ethane-2,l-diyl)dicarbamate (0.362 g, 0.64 mmol) was dissolved in DCM (10 mL) and HBr (30% in acetic acid, 3.5 mL) was added dropwise. After stirring at room temperature for 1.5 h, the solvent was removed under vacuum and the crude diamine was carefully dried under vacuum for several hours.

The resulting solid was suspended in DCM (5 mL) and Et₃N (0.88 mL, 6.4 mmol) and N,

N-di-boc-N'-trifluoromethanesulfonyl-guanidine (0.47 g, 1.2 mmol) were added. The mixture was stirred overnight at room temperature, diluted with DCM (20 mL) then washed with 2M NaHSO₄ 2M (25 mL), sat. aq. NaHCO₃ (25 mL) and brine (25 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to afford the title compound (0.33 g, 66% yield) as a white solid. 1H-NMR (CDCl₃): .61.48 (m, 36H), 3.79 (q, J= 5.2 Hz, 2H), 3.84 (q, J= 5 Hz, 2H), 4.09

(m, 4H), 6.94 (dd, J= 8.1 Hz, J= 1.7 Hz, IH), 6.99 (d, J= 1.4 Hz, IH), 7.18-7.22 (m, 2H),

7.34 (m, 2H), 7.40 (d, J= 7.8 Hz, IH), 8.63 (t, J= 5.1Hz, IH), 8.75 (bt, IH), 11.45 (bs,

2H).

¹³C-NMR (CDCl₃): 528.0, 28.3, 39.8, 40.2, 66.5, 67.4, 79.4, 79.5, 83.2, 83.2, 112.0, 114.2, 115.5, 115.8, 118.7, 122.7, 125.3, 129.2, 131.6, 135.8, 137.8, 153.0, 155.5, 156.3, 156.4,

158.6, 163.4, 163.5.

Theoretical Mass: (M + H) 782.40885. Measured Mass: (M + H) 782.40733

M.p. 89-91°C

IR v _Max KBR/crn 3333 (br NH), 2229 (CN), 1722 (C=O), 1641 (C=N), 1618 (C=N) N-{2, 3'-Bis{2-[iV, iV'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-biphenyl-4- yImethyl}-3-[2-pyridyl)dithio]propionamide (Example 6)

2, 3'-Bis{2-[iV, A/'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-4-cyanobiphenyl (0.5Og, 0.7mmol) was dissolved in THF (14 mL) and NH₄OH (2.4 mL) was added followed by Raney Nickel (0.50 g). The mixture was stirred vigorously under H₂ atmosphere for 16 h. The catalyst was filtered off and washed several times with MeOH. Diethylenetriamine (leq) was added and the mixture stirred for 30 minutes then concentrated under vacuum. The residue was dissolved in EtOAc (10 mL) and washed with sat. aq. NaHCO₃ (10 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. To a solution of the crude (0.040 g, 0.050 mmol) and Z-Pr₂EtN (10 μL, 0.10 mmol) in DCM (0.7 mL) was added N-succinimidyl-3-(2-pyridyldithio)propionate (15 mg, 0.053 mmol) and the mixture was stirred in the dark for 24 h. The solvent was removed under vacuum and the crude oil was purified by preparative TLC (ethyl acetate/hexane 1/1) to afford the title compound (8.5 mg, 16% yield).

¹H-NMR (CDCl₃): .51.49 (m, 36H), 2.67 (t, J= 5 Hz, 2H), 3.13 (t, J= 5 Hz, 2H), 3.73 (m, 2H), 3.84 (m, 2H), 4.05 (m, 4H), 4.50 (d, J= 4.1 Hz, 2H), 6.98-6.94 (m, 4H), 7.21-7.730 (m, 3H), 7.55 (m, 2H), 8.19 (d, J= 3.5 Hz, IH), 8.62 (t, J= 4 Hz, IH), 8.76 (m, IH), 11.46 (bs, 2H). 1³C-NMR (CDCl₃): 527.0, 28.0, 28.2, 28.5, 35.3, 35.8, 39.9, 40.2, 43.6, 66.3, 66.7, 79.3, 79.4, 83.1, 112.0, 113.1, 115.6, 120.6, 120.7, 121.1, 122.9, 128.8, 129.9, 131.1, 137.0, 139.0, 139.3, 149.51, 152.9, 155.47, 156.2, 156.3, 158.4, 159.1, 163.4, 163.5, 170.7, 171.9. Theoretical Mass: (M + H) 983.43705. Measured Mass: (M + H) 983.43839 IR v _Max KBR/cm^'3332 (NH), 1722 (C=O), 1641 (C=O). N- [2, 3 '-Bis(2-guanidino-ethyloxy)-biphenyl-4-ylmethyI]-3- [2- pyridyl)dithio]propionamide, formate salt (Example 7)

N- {2, 3'-Bis {2-[N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy} -biphenyl-4-ylmethyl} - 3-[2-pyridyl)dithio]propionamide (11 mg, 0.011 mmol) was dissolved in formic acid (1 mL) and stirred at room temperature overnight. The mixture was then heated at 50°C during Ih. Water was then added and the product was lyophilised to give the title compound as colourless oil (10 mg, 100%).

¹H-NMR (MeOH-d4): 52.71 (t, J= 6.6 Hz, 2H), 3.11 (t, 2H, J= 6.9 Hz, 2H), 3.50 (bt, 2H), 3.60 (bt, 2H), 4.12 (m, 4H), 4.41 (s, 2H), 6.92 (d, J= 7.5 Hz, IH), 6.99-7.12 (m, 4H), 7.18-

7.34 (m, 3H), 7.73-7.83 (m, 2H), 8.37 (d, J= 3.9 Hz, IH), 8.44 (bs, 2H).

Theoretical Mass: (M + H) 583.22734. Measured Mass: (M + H) 583.22816

Benzyl 2-(4-bromo-2'-hydroxybiphenyl-3-yloxy)ethylcarbamate

A degassed mixture of benzyl 2-(2-bromo-5-iodophenoxy)ethylcarbamate (1.43 g, 3 mmol), 2-hydroxyphenylboronic acid (0.37 g, 2.7 mmol), PdCl₂dppf. CH₂Cl₂ (0.12 g, 0.15 mmol), potassium phosphate (1.27 g, 6 mmol), toluene (6 mL) and water (0.6 mL) was heated at 100°C for 18 h. The reaction mixture was diluted in EtOAc (25 mL) and water (25 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (75/25) as eluent to afford the title compound (0.90 g, 75% yield) as a transparent oil.

ESMS; m/z 442, 444 (M+l), 459, 461 (M+18)

¹H-NMR (CDCl₃): 63.64 (m, 2H), 4.10 (m, 2H), 5.11 (s, 2H), 5.43 (bs, IH), 6.02 (s, IH), 6.97-7 '.03 (m, 4H), 7.25 (m, 2H), 7.35 (bs, 5H), 7.59 (d, J= 8 Hz, IH).

¹³C-NMR (CDCl₃): 540.5, 67.0, 68.4, 111.6, 114.7, 116.2, 120.9, 123.1, 127.2, 128.2, 128.2, 128.6, 129.4, 130.2, 133.6, 136.3, 138.4, 152.6, 155.0, 156.6 Theoretical Mass: (M + Na) 464.04733. Measured Mass: (M + Na) 464.04803

Dibenzyl 2,2'-(4-bromobiphenyl-3,2'-diyl)bis(oxy)bis(ethane-2,l-diyl)dicarbamate

Benzyl 2-(4-bromo-2'-hydroxybiphenyl-3-yloxy)ethylcarbamate (0.8 g, 1.8 mmol) was dissolved in DMF (10 mL) and Cs₂CO₃ (1.17 g, 3.6 mmol) and 2- (benzyloxycarbonylamino)ethyl methanesulfonate (0.55 g, 2 mmol) were added. After stirring at 80 °C under nitrogen for 3.5 h, the reaction mixture was dissolved in EtOAc (25 mL). The organic layer was washed with brine (3 x 20 mL) and dried over MgSO₄. The solvent was removed under vacuum and the crude compound was purified by flash chromatography using cyclohexane/EtOAc (7/3) as eluent to afford the title compound (0.6 g, 54% yield) as a colourless oil. ESMS; m/z 619, 621 (M+l), 636, 638 (M+18)

¹H-NMR (CDCl₃): .63.50 (q, J= 5.1 Hz, 2H), 3.58 (m, 2H), 4.04 (t, J= 4.8 Hz, 2H), 4.08 (m, 2H), 5.08 (m, 5H), 5.39 (bs, IH), 6.97 (m, 2H), 7.06 (m, 2H), 7.25-7.33 (m, 12H), 7.53 (d, J= 8.1 Hz, IH). 1³C-NMR (CDCl₃): .640.6, 66.9, 67.7, 68.5, 111.1, 113.2, 115.2, 121.7, 123.6, 128.1, 128.5, 128.6, 129.3, 130.0, 130.7, 132.8, 136.4, 136.5, 139.2, 154.5, 155.2, 156.4, 156.5. Theoretical Mass: (M + Na) 641.12631. Measured Mass: (M + Na) 641.12487 Dibenzyl 2,2'-(4-cyanobiphenyl-3,2'-diyl)bis(oxy)bis(ethane-2,l-diyl)dicarbamate

A mixture of dibenzyl 2,2'-(4-bromobiphenyl-3,2'-diyl)bis(oxy)bis(ethane-2,l- diyl)dicarbamate (0.35 g, 0.56 mmol), zinc cyanide (0.1 g, 0.85 mmol), Pd₂dba₃ (26 mg, 0.03 mmol), dppf (39 mg, 0.07 mmol), zinc acetate (51 mg, 0.28 mmol) and zinc (18 mg, 0.28 mmol) in dimethylacetamide (4 mL) was heated at 160 ⁰C for 10 minutes under microwave irradiation. The reaction mixture was diluted in EtOAc (25 mL) and water (100 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over MgSO₄, filtered and concentrated under vacuum. The residue was purified by flash chromatography using cyclohexane/EtOAc (65/35) as eluent to afford the title compound (0.24 g, 76% yield) as a colourless oil. ESMS; m/z 566 (M+l), 583 (M+18) 1H-NMR (CDCl₃): .53.50 (q, J= 5.2 Hz, 2H), 3.58 (m, 2H), 4.06 (t, J= 4.9 Hz, 2H), 4.14 (m, 2H), 5.07 (m, 5H), 5.30 (bs, IH), 6.97 (d, J= 8.2 Hz, IH), 7.06-7.14 (m, 3H), 7.28-7.38 (m, 12H), 7.53 (d, J= 7.9 Hz, IH).

¹³C-NMR (CDCl₃): 540.3, 40.4, 66.9, 67.5, 68.1, 100.4, 113.0, 113.8, 116.4, 121.7, 122.5, 128.1, 128.2, 128.3, 128.5, 128.6, 129.2, 130.1, 130.7, 133.1, 136.3, 145.2, 155.2, 156.3, 156.4, 159.9. Theoretical Mass: (M + H) 566.22910. Measured Mass: (M + H) 566.22854 IR v w_ax KBR/cm^"3336 (br NH), 2224 (CN), 1712 (C=O)

3, 2'-Bis{2-[./V, 7V'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-4-cyanobiphenyl

Dibenzyl 2,2'-(4-cyanobiphenyl-3,2'-diyl)bis(oxy)bis(ethane-2,l-diyl)dicarbamate (0.21 g, 0.371 mmol) was dissolved in DCM (10 mL) and HBr (30% in acetic acid, 2 niL) was added dropwise. After stirring at room temperature for 1.5 h, the solvent was removed under vacuum and the crude diamine was carefully dried under vacuum for several hours. The resulting solid was suspended in DCM (5 mL) and Et₃N (0.51 mL, 3.7 mmol) and N, N-di-boc-N'-trifluoromethanesulfonyl-guanidine (0.29 g, 0.74 mmol) were added. The mixture was stirred overnight at room temperature, diluted with DCM (20 mL) then washed with 2M NaHSO₄ 2M (25 mL) follow by sat. aq. NaHCO₃ (25 mL) and brine (25 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to afford the title compound (0.17 g, 59% yield) as a white solid.

¹H-NMR (CDCl₃): .51.48 (m, 36H), 3.78 (q, J= 4.9Hz, 2H), 3.90 (q, J= 5.1Hz, 2H), 4.08(t, J= 4.8Hz, 2H), 4.23 (t, J= 5Hz, 2H), 6.98 (d, J= 8.2Hz, IH), 7.05 (m, 2H), 7.26-7.37 (m, 4H), 8.60 (t, J= 5.1Hz, IH), 8.82 (t, J= 5Hz, IH), 11.45 (bs, 2H). 1³C-NMR (CDCl₃): 528.1, 28.3, 39.9, 66.8, 67.5, 79.3, 79.4, 83.3, 83.3, 100.6, 112.3, 113.5, 116.3, 121.5, 123.2, 129.1, 130.0, 130.7, 133.2, 144.9, 153.0, 153.1, 155.1, 156.3, 156.5, 159.9, 163.4.

Theoretical Mass: (M + H) 782.40885. Measured Mass: (M + H) 782.40887 IR v _Max KBR/cin 3333 (br NH), 2226 (CN), 1723 (C=O), 1643 (C=N), 1615 (C=N) M.p. 88-90°C

iV-{3, 2'-Bis{2-[iV, N-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-biphenyl-4- ylmethyl}-3-[2-pyridyl)dithio]propionamide (Example 8)

To a solution of palladium on carbon 10% (25 mg), Raney nickel (25 mg) and NH₄OH (20 μL) in dioxane (40 mL) and water (10 mL) was added 3, 2'-bis{2-[N, N'-bis(tert- butoxycarbonyl)guanidino]-ethyloxy}-5-cyanobiphenyl (0.070 g, 0.09 mmol). The mixture was then hydrogenated (6 bar) overnight. The catalyst was filtered off and washed several times with MeOH. Diethylenetriamine (leq) was added and the mixture was stirred for 30 minutes then concentrated under vacuum. The residue was dissolved in EtOAc (10 mL) and washed with sat. aq. NaHCO₃ (10 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. To a solution of the crude and /-Pr₂EtN (31 μL, 0.18 mmol) in DCM (2 mL) was added N-succinimidyl-3-(2-pyridyldithio)propionate (39 mg, 0.126 mmol) and the mixture was stirred in the dark for 24 h. The solvent was removed under vacuum and the crude oil was purified by preparative TLC (ethyl acetate/hexane 1/1) to afford the title compound (14 mg, 16% yield). 1H-NMR (CDCl₃): δl.48 (m, 36H), 2.64 (t, 2H), 3.09 (t, J= 6.9 Hz, 2H), 3.76 (q, J= 5.1 Hz , 2H), 3.84 (q, J= 4.5 Hz, 2H), 4.05 (t, J= 5.1 Hz, 2H), 4.13 (t, J= 4.8 Hz, 2H), 4.51 (d, J= 6.0 Hz, 2H), 6.70 (t, J- 5.1 Hz, IH), 6.72-7.06 (m, 4H), 7.19 (d, J= 6.3 Hz, IH), 7.27- 7.38 (m, 4H), 7.53 (t, J = 6.3 Hz, IH), 7.62 (d, J= 8.1 Hz, IH), 8.33 (d, J= 3.9 Hz, IH), 8.63 (bt, IH), 8.78 (bt, IH), 11.45 (bs, IH), 11.52 (bs, IH). 1³C-NMR (CDCl₃): 528.0, 28.3, 34.6, 35.8, 39.0, 40.1, 40.2, 66.5, 67.0, 79.3, 79.4, 83.2, 83.4, 112.5, 112.8, 119.9, 120.7, 121.5, 122.8, 125.2, 128.8, 129.9, 130.6, 130.9, 136.2, 149.6, 153.0, 153.3, 155.3, 156.0, 156.3, 163.5, 170.3. Theoretical Mass: (M + H) 983.43705. Measured Mass: (M + H) 983.43757

iV-[3, 2'-Bis(2-guanidino-ethyloxy)-biphenyI-4-ylmethyl]-3-[2- pyridyl)dithio]propionamide, formate salt (Example 9)

N- {3, 2'-Bis{2-[N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-biphenyl-4-ylmethyl}- 3-[2-pyridyl)dithio]propionamide (14 mg, 0.014 mmol) was dissolved in formic acid (1 mL) and stirred at room temperature overnight. The mixture was then heated at 50°C during Ih. Water was then added and the product was lyophilised to give the title compound as a colourless oil (11.4 mg, 93%). ¹H-NMR (MeOH-(I₄): 52.72 (t, J= 6.1 Hz, 2H), 2.97 (t, 2H, J= 6.0 Hz, 2H), 3.49 (bs, 2H), 3.64 (bs, 2H), 4.08 (bs, 2H), 4.17 (bs, 2H), 4.45 (s, 2H), 7.05-7.09 (m, 3H), 7.20 (t, J= 5.7 Hz, IH), 7.28-7.35 (m, 3H), 7.70-7.92 (m, 2H), 8.37 (d, J= 4.5 Hz, IH), 8.40 (bs, 2H). Theoretical Mass: (M + H) 583.22734. Measured Mass: (M + H) 583.22816

Benzyl 2-(4-bromo-3 '-hydroxybiphenyI-3-yIoxy)ethylcarbamate

A degassed mixture of benzyl 2-(2-bromo-5-iodophenoxy)ethylcarbamate (1.43 g, 3 mmol), 2-hydroxyphenylboronic acid (0.37 g, 2.7 mmol), PdCl₂dppf. CH₂Cl₂ (0.12 g, 0.15 mmol), potassium phosphate (1.27 g, 6 mmol), toluene (6 mL) and water (0.6 mL) was heated at 100°C for 18 h. The reaction mixture was diluted in EtOAc (25 mL) and water (25 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (75/25) as eluent to afford the title compound (0.54 g, 45% yield) as a transparent oil.

ESMS; m/z 442, 444 (M+l), 459, 461 (M+18)

¹H-NMR (CDCl₃): 63.65 (q, J= 5 Hz, 2H), 4.08 (t, J= 4.8 Hz, 2H), 5.14 (s, 2H), 5.43 (bs, IH), 6.77 (s, IH), 6.88 (dd, J= 8.1 Hz, J= 1.7Hz, IH)₅ 6.98-7.06 (m, 4H), 7.24-7.36 (m, 6H), 7.52 (d, J= 8 Hz, IH).

¹³C-NMR (CDCl₃): .540.6, 67.2, 68.3, 111.5, 112.4, 114.1, 114.9, 119.1, 121.2, 128.2, 128.3, 128.6, 130.1, 133.4, 136.1, 141.6, 141.8, 154.9, 156.6, 156.9. Theoretical Mass: (M + Na) 464.04733. Measured Mass: (M + Na) 464.04628 Dibenzyl 2,2'-(4-bromobiphenyl-3,3'-diyI)bis(oxy)bis(ethane-2,l-diyl)dicarbamate

Benzyl 2-(4-bromo-3'-hydroxybiphenyl-3-yloxy)ethylcarbamate (0.46 g, 1.8 mmol) was dissolved in DMF (10 mL) and Cs₂CO₃ (1.17 g, 3.6 mmol) and 2-

(benzyloxycarbonylamino)ethyl methanesulfonate (0.55 g, 2 mmol) were added. After stirring at 80 °C under nitrogen for 3.5 h, the reaction mixture was dissolved in EtOAc (25 mL). The organic layer was washed with brine (3 x 20 mL) and dried over MgSO₄. The solvent was removed under vacuum and the crude compound was purified by flash chromatography using cyclohexane/EtOAc (7/3) as eluent to afford the title compound (0.33 g, 52 % yield) as a colourless oil. ESMS; m/z 619, 621 (M+l), 636, 638 (M+18)

¹H-NMR (CDCl₃): 53.65 (m, 4H), 4.10 (t, J= 4.5 Hz, 2H), 4.16 (t, J= 4.4 Hz, 2H), 5.12 (s, 4H), 5.32 (bs, IH), 5.40 (bs, IH), 6.88 (d, J= 6.8 Hz, IH), 7.05 (m, 3H), 7.14 (d, J= 7.7 Hz, IH), 7.35 (bs, 1 IH), 7.57 (d, J= 8.6 Hz, IH).

¹³C-NMR (CDCl₃): 640.6, 66.9, 67.0, 68.5, 111.7, 112.5, 113.5, 119.9, 121.2, 128.2, 128.6, 130.1, 133.5, 136.4, 141.7, 141.8, 155.1, 156.5, 158.9.

Theoretical Mass: (M + Na) 641.12631. Measured Mass: (M + Na) 641.12715 Microanalysis: %C 62.08 (62.04), %H 5.13 (5.04), %N 4.39 (4.52) IR v _Max KBR/cm^" 3339 (br, NH), 2226 (CN), 1723 (C=O), 1641 (C=N), 1620 (C=N)

Dibenzyl 2,2'-(4-cyanobiphenyl-3,3'-diyl)bis(oxy)bis(ethane-2,l-diyl)dicarbamate

A mixture of dibenzyl 2,2'-(4-bromobiphenyl-3,3'-diyl)bis(oxy)bis(ethane-2,l- diyl)dicarbamate (0.26 g, 0.42 mmol), zinc cyanide (74 mg, 0.63 mmol), Pd₂dba₃ (19 mg, 0.02 mmol), dppf (29 mg, 0.05 mmol), zinc acetate (39 mg, 0.21 mmol) and zinc (14 mg, 0.21 mmol) in dimethylacetamide (3 mL) was heated at 160⁰C for 10 minutes under microwave irradiation. The reaction mixture was diluted in EtOAc (25 mL) and water (100 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (65/35) as eluent to afford the title compound (0.2 g, 84% yield) as a colourless oil. ESMS; m/z 566 (M+l), 583 (M+18)

¹H-NMR (CDCl₃): 53.61-3.71 (m, 4H), 4.10 (t, J= 4.6 Hz, 2H), 4.22 (t, J= 4.7 Hz, 2H), 5.11 (s, 4H), 5.27 (bs, IH), 5.37 (bs, IH), 6.93 (d, J= 8.1 Hz, IH), 7.07-7.22 (m, 4H), 7.29- 7.40 (m, 1 IH), 7.59 (d, J= 8 Hz, IH).

¹³C-NMR (CDCl₃): 540.3, 40.4, 66.9, 67.1, 68.2, 100.9, 111.1, 113.7, 114.5, 120.2, 128.1, 128.2, 128.2, 128.5, 128.6, 130.3, 134.0, 136.3, 140.9, 147.4, 156.5, 159.0, 160.4. Theoretical Mass: (M + H) 566.22910. Measured Mass: (M + H) 566.22964 IR v _Max KBR/cm^"3339 (br, NH), 2225 (CN), 1713 (C=O)

3, 3'-Bis{2-[iV, iV'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-4-cyanobiphenyl

Dibenzyl 2,2'-(4-cyanobiphenyl-3,3^l-diyl)bis(oxy)bis(ethane-2,l-diyl)dicarbamate (0.15 g, 0.26 mmol) was dissolved in DCM (10 mL) and HBr (30% in acetic acid, 1.5 mL) was added dropwise. After stirring at room temperature for 1.5 h, the solvent was removed under vacuum and the crude diamine was carefully dried under vacuum for several hours. The resulting solid was suspended in DCM (5 mL) and Et₃N (0.36 mL, 2.6 mmol) and N, N-di-boc-N'-trifluoromethanesulfonyl-guanidine (0.21 g, 0.53 mmol) were added. The mixture was stirred overnight at room temperature, diluted with DCM (20 mL) then washed with 2M NaHSO₄2M (25 mL) followed by sat.aq. NaHCO₃ (25 mL) and brine (25 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to afford the title compound (0.18 g, 86% yield) as a white solid.

¹H-NMR (CDCl₃): 61.50 (m, 36H), 3.86 (q, J= 5.1 Hz, 2H), 3.93 (q, J= 5.2 Hz, 2H), 4.15 (t, J= 5 Hz, 2H), 4.29 (t, J= 5.1 Hz, 2H), 6.99 (dd, J= 8.1 Hz, J=2 Hz, IH), 7.10-7.16 (m, 3H), 7.21 (d, J= 8 Hz, IH), 7.38 (t, J= 8 Hz₅ IH), 7.60 (d, J= 8 Hz, IH), 8.62 (bt, IH), 8.82 (bt, IH), 11.45 (bs, 2H). 1³C-NMR (CDCl₃): 527.8, 28.1, 28.3, 39.9, 40.2, 66.6, 67.6, 79.4, 83.2, 83.3, 101.2, 111.3, 114.1, 114.7, 116.0, 120.2, 120.3, 130.2, 134.1, 141.0, 147.4, 153.0, 153.1, 156.4, 156.5, 159.1, 160.4, 163.5.

Theoretical Mass: (M + H) 782.40885. Measured Mass: (M + H) 782.40656 IR v _Max KBR/cnϊ 3332 (br, NH), 2226 (CN), 1723 (C=O), 1641 (C=N), 1620 (C=N) M.p. 82-84°C

N-{3, 3'-Bis{2-[W, iV-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-biphenyl-4- ylmethyl}-3-[2-pyridyl)dithio]propionamide (Example 10)

To a solution of palladium on carbon 10% (25 mg), Raney nickel (25 mg) and NH₄OH (20 μL) in dioxane (40 mL) and water (10 mL) was added 3, 3'-Bis{2-[N, N'-bis(tert- butoxycarbonyl)guanidino]-ethyloxy}-5-cyanobiphenyl (0.070 g, 0.09 mmol). The mixture was then hydrogenated (6 bar) overnight. The catalyst was then filtered off and washed several times with MeOH. Diethylenetriamine (leq) was added and the mixture was stirred for 30 minutes then concentrated under vacuum. The residue was dissolved in EtOAc (10 mL) and washed with sat. aq. NaHCO₃ 10 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. To a solution of the crude and J--Pr₂EtN (31 μL, 0.18 mmol) in DCM (2 mL) was added N-succinimidyl-3-(2-pyridyldithio)propionate (39 mg, 0.126 mmol) and the mixture was stirred in the dark for 24 h. The solvent was removed under vacuum and the crude oil was purified by preparative TLC (ethyl acetate/hexane 1/1) to afford the title compound (8 mg, 11% yield).

¹H-NMR (CDCl₃): .81.50 (m, 36H), 2.64 (t, 2H), 3.08 (t, J= 6.9 Hz, 2H), 3.89 (m , 4H), 4.17 (m, 4H), 4.50 (d, J= 5.7 Hz, 2H), 6.81 (bt, IH), 6.92 (dd, J= 7.8 Hz, J= 2.4 Hz, IH), 7.02-7.15 (m, 5H), 7.32 (t, J= 4.5 Hz, IH), 7.42 (d, J= 8.1 Hz, IH), 7.53-7.63 (m, 2H), 8.34 (d, J= 3.6 Hz, IH), 8.78 (m, 2H), 8.78 (bt, IH), 11.49 (bs, IH), 11.54 (bs, IH).

¹³C-NMR (CDCl₃): 828.0, 28.3, 34.4, 35.3, 38.5, 39.9, 40.1, 66.3, 66.7, 79.3, 79.5, 83.1, 83.4, 111.5, 112.9, 120.1, 120.4, 125.7, 129.2, 130.1, 136.0, 140.9, 141.3, 148.0, 153.7, 156.9, 159.6, 163.5, 170.5. Theoretical Mass: (M + H) 983.43705. Measured Mass: (M + H) 983.43928

iV-[3, 3'-Bis(2-guanidino-ethyloxy)-biphenyl-4-ylmethyl]-3-[2- pyridyl)dithio]propionamide, formate salt (Example 11)

N- {3, 3'-Bis {2-[N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy} -biphenyl-4-ylmethyl} - 3-[2-pyridyl)dithio]propionamide (8 mg, 0.0081 mmol) was dissolved in formic acid (1 mL) and stirred at room temperature overnight. The mixture was then heated at 50°C for

Ih. Water was added and the product was freeze-dried to give the title compound as colourless oil (7.1 mg, 100%).

¹H-NMR (MeOH-Cl₄): 82.69 (t, J= 6.8 Hz, 2H), 3.09 (t, 2H, J= 6.5 Hz, 2H), 3.60-3.68 (m, 4H), 4.18(t, J= 4.9 Hz, 2H), 4.24 (t, J= 4.8 Hz, 2H), 4.46 (s, 2H), 6.95 (d, J= 7.5 Hz, IH), 7.18-7.24 (m, 4H), 7.31-7.39 (m, 2H), 7.71-7.78 (m, 2H), 8.36 (d, J= 4.6 Hz, IH), 8.43 (bs,

2H).

Theoretical Mass: (M + H) 583.22734. Measured Mass: (M + H) 583.22796

iV-(2-(4-cyanophenoxy)ethyl)-2-phenyIacetamide

3-Bromo-4-hydroxybenzonitrile (7.25 g, 36.6 mmol) was dissolved in DMF (20 mL) and

Na₂CO₃ (3.7 g, 73.2 mmol) and 2-(benzyloxycarbonylamino)ethyl methanesulfonate (12 g, 42 mmol) were added. After stirring at 80 °C under nitrogen for 15 h, the reaction mixture was dissolved in EtOAc (25 mL). The organic layer was washed with brine (3 x 20 mL), dried over MgSO₄ and filtered. The solvent was removed under vacuum and the crude compound was purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to afford the title compound (9.4 g, 68% yield) as a white solid. 1H-NMR (CDCl₃): 53.70 (q, J= 5.3 Hz, 2H), 4.13 (m, 2H), 5.11 (s, 2H), 5.23 (bs, IH), 6.90

(d, J= 8.5 Hz, IH), 7.35 (m, 5H), 7.56 (d, J= 8.5 Hz, IH), 7.81 (d, J= 1.9 Hz, IH).

¹³C-NMR (CDCl₃): 540.2, 67.0, 68.5, 105.7, 112.7, 113.0, 117.6, 128.2, 128.3, 128.6,

133.2, 136.3, 136.8, 156.4, 158.4.

Theoretical Mass: (M + Na) 397.01637. Measured Mass: (M + Na) 397.01539. Microanalysis: %C 54.10 (54.42), %H 4.23 (4.03), %N 7.19 (7.47)

IR v _Max KBR/cm^"3351(br NH, NH₂), 2227 (CN), 11704 (C=ON)

M.p. 65-68°C

Benzyl 2-(5-cyano-2'-hydroxybiphenyl-2-yIoxy)ethylcarbamate

A degassed mixture of N-(2-(4-cyanophenoxy)ethyl)-2-phenylacetamide (1.9 g, 5 mmol), 2-hydroxyphenyl boronic acid (0.83 g, 6 mmol), Pd(OAc)₂ (10 mg, 0.05 mmol) , 2- dicyclohexylphosphino-2',6'-dimethoxybiphenyl (40 mg, 0.1 mmol), potassium phosphate (2.1 g, 10 mmol), toluene (10 mL) and water (1 mL) was heated at 100°C for 1 h. The reaction mixture was diluted in EtOAc (50 mL) and water (50 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (7/3) as eluent to afford the title compound (1.07 g, 55% yield) as a colourless oil. 1H-NMR (CDCl₃): _.63.48 (m, 2H), 4.13 (m, 2H), 5.06 (bs, 2H), 5.32 (bs, IH), 6.34 (bs, IH), 6.91-7.02 (m, 3H), 7.13 (d, J= 7Hz, IH), 7.27-7.33 (m, 6H), 7.58 (m, 2H). 1³C-NMR (CDCl₃): 640.2, 66.9, 68.0, 105.2, 112.8, 116.7, 118.8, 121.1, 123.7, 128.1, 128.3, 128.6, 128.9, 130.0, 131.2, 133.6, 135.8, 136.3, 153.1, 156.5, 158.7. Theoretical Mass: (M + H) 389.15012. Measured Mass: (M + H) 389.15078

Dibenzyl 2,2'-(5-cyanobiphenyl-2,2'-diylbis(oxy))bis(ethane-2,l-diyl)tetracarbamate

Benzyl 2-(5-cyano-2'-hydroxybiphenyl-2-yloxy)ethylcarbamate (1.06 g, 2.7 mmol) was dissolved in DMF (7 mL) and Cs₂CO₃ (1.8 g, 5.4 mmol) and 2-

(benzyloxycarbonylamino)ethyl methanesulfonate (0.75 g, 2.7 mmol) were added. After stirring at 80 ⁰C under nitrogen overnight, further Cs₂CO₃ (0.9 g, 2.7 mmol) and 2- (benzyloxycarbonylamino)ethyl methanesulfonate (0.4 g, 1.5 mmol) were added. After stirring for a further 4 h, the reaction mixture was dissolved in EtOAc (25 mL). The organic layer was washed with brine (3 x 20 mL) and dried over MgSO₄. The solvent was removed under vacuum and the crude compound was purified by flash chromatography using cyclohexane/EtOAc (1/1) as eluent to afford the title compound (1.23 g, 80% yield) as a colourless oil. ¹H-NMR (CDCl₃): δ3,32(m, 2H), 3.80 (t, J= 8.9 Hz, 2H), 4.00 (m, 2H), 4.39 (t, J= 8.7 Hz, 2H), 5.04 (s, 2H), 5.22 (s, 2H), 5.30 (bs, IH), 5.54 (bs, IH), 6.86 (m, 2H), 7.02 (t, J= 7.3 Hz, IH), 7.13 (dd, J= 8.9 Hz, J= 1.7 Hz, IH), 7.34 (m, HH), 7.46 (d, J= 2 Hz, IH), 7.53 (dd, J= 8.5 Hz, 1.8 Hz, IH). 1³C-NMR (CDCl₃): 640.1, 40.3, 66.8, 68.0, 68.3, 104.7, 112.8, 113.5, 118.9, 121.9, 126.4,

128.1, 128.3, 128.5, 128.6, 129.7, 129.8, 131.0, 133.5, 134.9, 135.4, 136.4, 155.6, 156.2,

159.2, 163.6.

Theoretical Mass: (M + Na) 588.21104. Measured Mass: (M + Na) 588.21104 IR v _Max KBR/cm^" 3342 (br, NH), 2224 (CN), 1721 (C=O)

2, 2'-Bis{2-[N, Λ^'-bis(tert-butoxycarbonyI)guanidino]-ethyloxy}-5-cyanobiphenyl

Dibenzyl 2,2'-(5-cyanobiphenyl-2,2'-diylbis(oxy))bis(ethane-2,l-diyl)tetracarbamate (0.9 g, 1.6 mmol) was dissolved in DCM (50 mL) and HBr (30% in acetic acid, 8 mL) was added dropwise. After stirring at room temperature for 1.5 h, water (25 mL) was added to the mixture, the layers were separated and the aqueous layer was washed with DCM (2 x 25 mL). The water was then removed under vacuum and the crude diamine was carefully dried under vacuum for several hours. The residue was suspended in DCM (7 mL) and Et₃N (1.2 mL, 8 mmol) and N, N-di-boc- N'-trifluoromethanesulfonyl-guanidine (1.3 g, 3.2 mmol) were added. The mixture was stirred overnight at room temperature, diluted with DCM (20 mL) then washed with 2M NaHSO₄ 2M (25 mL) follow by sat. aq. NaHCO₃ (25 mL) and brine (25 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to afford the title compound (0.28 g, 22% yield) as a white solid. ESMS; m/z 782(M+1), 391(M+2) ¹H-NMR (CDCl₃): 51.48 (m, 36H), 3.69 (m, 4H), 4.05 (t, J= 5.2 Hz, 2H), 4.13 (t, J= 5.2 Hz, 2H), 6.95-7.06 (m, 3H), 7.22 (dd, 7=7.5, 1.7 Hz, IH), 7.32 (td, J= 7.2, 1.7 Hz, IH), 7.55 (d, J= 2 Hz, IH), 7.59 (dd, J= 8.5, 2.1 Hz, IH), 8.40 (m, 2H), 11.42 (bs, 2H). 1³C-NMR (CDCl₃): 628.1, 28.3, 39.8, 40.1, 66.7, 66.9, 79.3, 79.4, 83.1, 104.1, 112.2, 112.4, 119.2, 121.0, 125.6, 129.1, 129.4, 131.3, 133.2, 135.2, 152.9, 155.6, 156.3, 159.2, 163.4. Theoretical Mass: (M + Na) 804.39079. Measured Mass: (M + Na) 804.39290 IR v _Max KBR/crn 3333 (br, NH), 2226 (CN), 1722 (C=O), 1642 (C=N), 1619 (C=N) M.p. 82°C

2, 2'-Bis{2-[iV, iV-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-5- (aminomethyl)biphenyl (Example 12)

2, 2'-Bis {2-[N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy} -5-cyanobiphenyl (0.1O g, 0.12 mmol) was dissolved in THF (7 mL) and NH₄OH (1.4 mL) was added followed by Raney Nickel (0.1 g). The mixture was stirred vigorously under H₂ atmosphere for 16 h. The catalyst was filtered off and washed several times with MeOH. Diethylenetriamine (leq) was added and the mixture stirred for 30 minutes then concentrated under vacuum. The residue was dissolved in EtOAc (10 mL) and washed with sat. aq. NaHCO₃ (10 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using DCM/MeOH (97/3) as eluent to afford the title compound (45 mg, 48% yield). ESMS; m/z 786(M+1), 394(M+2) 1H-NMR (CDCl₃): .51.47 (m, 36H), 3.63 (m, 4H), 3.81 (s, 2H), 4.02 (m, 4H), 6.90-6.99 (m, 3H), 7.22 (m, 4H), , 8.40 (m, 2H), 11.42 (bs, 2H).

¹³C-NMR (CDCl₃): 528.1, 28.3, 40.2, 40.3, 67.1, 79.1, 79.2, 82.8, 82.9, 112.9, 113.0, 121.0, 127.0, 127.8, 128.0, 128.5, 130.6, 131.8, 135.7, 152.8, 154.7, 155.7, 156.2, 163.4. IR v _Max KBR/cm^~3338 (NH, NH₂), 1723 (C=O), 1641(C=N), 1614 (C=N)

N-{2, 2'-Bis{2-[iV, N-bis(tert-butoxycarbonyl)guanidino]-ethyIoxy}-biphenyI-5- yImethyl}-3-[2-pyridyl)dithio]propionamide (Example 13)

To a solution of 2, 2'-Bis{2-[N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-5- (aminomethyl)biphenyl (0.042 g, 0.053 mmol) and Z-Pr₂EtN (10 μL, 0.10 mmol) in DCM (0.7 mL) was added N-succinimidyl-3-(2-pyridyldithio)propionate (15 mg, 0.053 mmol) and the mixture was stirred in the dark for 24 h. The solvent was removed under vacuum and the crude oil was purified by preparative TLC (ethyl acetate/hexane 1/1) to afford the title compound (8.5 mg, 16% yield).

¹H-NMR (CDCl₃): δl.48 (m, 36H), 2.61 (t, J= 6.8 Hz, 2H), 3.09 (t, J= 6.7 Hz, 2H), 3.65 (m, 4H), 4.02 (m, 4H), 4.42 (d, J= 5.4 Hz, 2H), 6.89-7.03 (m, 6H), 7.23-7.30 (m, 3H), 7.58 (m, 2H), 8.23 (m, 2H), 8.23 (dd, J= 3.8, 1.5Hz, IH), 8.36 (bt, IH), 8.47 (bt, IH), 11.66 (bs, 2H).

Theoretical Mass: (M + H) 983.43705. Measured Mass: (M + H) 983.43763 IR v _Max KBR/cm^" 3332 (NH), 1722 (C=O), 1641 (C=O).

N-[2, 3'-Bis(2-guanidino-ethyloxy)-biphenyI-5-ylmethyl]-3-[2- pyridyl)dithio]propionamide, trifluoroacetate salt (Example 14)

N- {2, 3'-Bis {2-[N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy} -biphenyl-5- ylmethyl}-3-[2-pyridyl)dithio]propionamide (8.5 mg, 0.0086 mmol) was dissolved in a mixture of TFA/ H₂O/triisopropylsilane 95/2.5/2.5 (1 mL) and stirred at room temperature for 3 h. The solvent was removed under vacuum to give the title compound as a colourless oil (8 mg, 100%).

¹H-NMR (MeOH-d4): 52.71 (t, J= 6.7Hz, 2H), 3.07 (t, 2H, J= 6.7Hz, 2H), 3.41 (m, 4H), 4.05 (m, 4H), 4.35 (s, 2H), 7.07 (m, 4H), 7.18 (m, 4H), 7.27 (m, 2H), 7.35 (m, 2H), 7.75 (m, 2H), 8.37 (m, IH). Theoretical Mass: (M + H) 583.22734. Measured Mass: (M + H) 583.22566 IR v _Max KBR/cm^"3336 (NH), 1642 (C=O) Benzyl 2-(5-cyano-3 '-hydroxybiphenyI-2-yloxy)ethylcarbamate

A degassed mixture of iV-(2-(4-cyanophenoxy)ethyl)-2-phenylacetamide (3.7 g, 9.8 mmol), 3-hydroxyphenyl boronic acid (1.6 g, 11.6 mmol), Pd(OAc)₂ (22 mg, 0.1 mmol) 2- dicyclohexylphosphino-2',6'-dimethoxybiphenyl (82 mg, 0.2 mmol) potassium phosphate

(4.2 g, 20 mmol), toluene (20 mL) and water (2 mL) was heated at 100⁰C for 1 h. The reaction mixture was diluted in EtOAc (50 mL) and water (50 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were dried over MgSO₄, filtered and concentrated under vacuum.

The residue was then purified by flash chromatography using cyclohexane/EtOAc (7/3) as eluent to afford the title compound (2.2 g, 59% yield) as a colourless solid.

ESMS; m/z 389 (M+ 1)

¹H-NMR (CDCl₃): 53.40 (m, 2H), 3.60 (m, 2H), 5.08 (s, 2H), 5.23 (m, 2H), 6.89 (m, 4H), 6.95 (2, IH), 7.18 (m, 56H), 7.53 (m, IH).

Microanalysis: C₂₃H₂₀N₂O₄- H₂O: %C 67.81 (67.97), %H 5.41 (5.46), %N 6.85 (6.89).

IR v _Max KBR/cm^'3347(br NH, NH₂), 2225(CN), 1714 (C=ON)

Mp >200°C

Λ^L{2-[5'-Cyano-2'-(2-phenylacetylamino-ethoxy)-biphenyl-3-yloxy]-ethyl}-2-phenyl- acetamide

Benzyl 2-(5-cyano-3'-hydroxybiphenyl-2-yloxy)ethylcarbamate (0.62 g, 1.6 mmol) was dissolved in DMF (8 mL) and Cs₂CO₃ (1.04 g, 3.2 mmol) and 2- (benzyloxycarbonylamino)ethyl methanesulfonate (0.44 g, 1.6 mmol) were added. After stirring at 80 ⁰C under nitrogen for 3.5 h, the reaction mixture was dissolved in EtOAc (25 mL). The organic layer was washed with brine (3 x 20 mL) and dried over MgSO₄. The solvent was removed under vacuum and the crude compound was purified by flash chromatography using cyclohexane/EtOAc (1/1) as eluent to afford the title compound (0.86 g, 95% yield).

¹H-NMR (CDCl₃): 63.70 (q, J= 5.3 Hz, 2H), 4.13 (m, 2H), 5.11 (s, 2H), 5.23 (bs, IH), 6.90 (d, J= 8.5 Hz, IH), 7.35 (m, 5H), 7.56 (d, J= 8.5 Hz, IH), 7.81 (d, J= 1.9 Hz, IH). 1³C-NMR (CDCl₃): D40.2, 67.0, 68.5, 105.7, 112.7, 113.0, 117.6, 128.2, 128.3, 128.6, 133.2, 136.3, 136.8, 156.4, 158.4. IR v w_ax KBR/crn 3335(br NH, NH₂), 2225(CN), 1714 (C=ON)

N-{2-[5'-Cyano-2'-(2-phenylacetylamino-ethoxy)-biphenyl-3-yloxy]-ethyl}-2-phenyl- acetamide (1.7 g, 3 mmol) was dissolved in DCM (25 mL) and HBr (30% in acetic acid, 4.0 mL) was added dropwise. After stirring at room temperature for 1.5 h, water (25 mL) was added to the mixture, the layers were separated and the aqueous layer was washed with DCM (2 x 25 mL). The water was then removed under vacuum and the crude diamine was carefully dried under vacuum for several hours. The resulting solid was suspended in DCM (20 mL) and /-Pr₂EtN (3mL, 39 mmol) and N, N-di-boc-N'-trifluoromethanesulfonyl-guanidine (3.7 g, 9 mmol) were added. The mixture was stirred overnight at room temperature, diluted with DCM (40 mL) then washed with 2M NaHSO₄ 2M (25 mL) followed by sat. aq. NaHCO₃ (25 mL) and brine (25 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (8/2) as eluent to afford the title compound (1.102 g, 47% yield) as a white solid. ESMS; m/z 782(M+1), 391(M+2) ¹H-NMR (CDCl₃): .51.48 (m, 36H), 3.80 (m, 2H), 3.83 (m, 2H), 4.12 (m, 4H), 6.81-6.98 (m, 2H), 7.09 (d, J- 9.2 Hz, IH), 7.16 (d, J= 7.8 Hz, IH), 7.34 (t, J= 7.9 Hz, IH), 7.60 (m, 2H), 8.60 (t, J= 5.3 Hz, IH), 8.76 (t, J= 4.9 Hz, IH), 11.54 (bs, 2H). 1³C-NMR (CDCl₃): 528.0, 28.3, 39.6, 40.2, 66.5, 67.0, 79.3, 79.4, 83.2, 83.3, 104.6, 112.7, 113.9, 115.9, 119.0, 122.7, 129.2, 131.9, 133.2, 134.5, 137.5, 153.0, 156.4, 158.6, 158.7, 163.4, 163.5.

Microanalysis: C₃₉H₅₅N₇O₁₀: %C 60.30 (59.91), %H 7.20 (7.09), %N 12.45 (13.54). IR v _Max KBR/crn 3333 (br, NH), 2226 (CN), 1722 (C=O), 1642 (C=N), 1619 (C=N) Mp 120-121⁰C

2, 3'-BiS(Z-[N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-5- (aminomethyl)biphenyl (Example 15)

2, 3 '-Bis {2- [N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-5-cyanobiphenyl (0.54 g, 0.7 mmol) was dissolved in THF (14 mL) and NH₄OH (2.4 mL) was added followed by

Raney Nikel (0.5 g). The mixture was stirred vigorously under H₂ atmosphere for 16 h. The catalyst was filtered off and washed several times with MeOH. Diethylenetriamine (leq) was added and the mixture stirred for 30 minutes then concentrated under vacuum. The residue was dissolved in EtOAc (10 mL) and washed with sat. aq. NaHCO₃ (10 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using DCM/MeOH (97/3) as eluent to afford the title compound (0.213 g, 39% yield).

ESMS; m/z 787 (M+l), 394 (M+2)

¹H-NMR (CDCl₃): 51.47 (m, 36H), 3.59 (m, 2H), 3.78 (m, 4H), 4.03 (t, J= 4.9 Hz, 2H), 4.10 (t, J= 4.9 Hz, 2H), 6.86 (dd, J= 8, 2Hz, IH), 6.93 (d, J= 8.3 Hz, IH), 7.20-7.30 (m,

4H), 8.63 (t, J= 5.2 Hz, IH), 8.74 (t, J= 5.1 Hz, IH), 11.54 (bs, 2H). ¹³C-NMR (CDCl₃): 528.1, 28.3, 40.1, 40.3, 45.9, 66.3, 67.1, 79.3, 83.0, 83.1, 112.9, 113.2,

115.6, 123.0, 127.3, 128.9, 129.9, 130.9, 136.2, 139.7, 152.9, 153.0, 154.2, 156.3, 156.3,

158.4, 163.5, 163.5.

Microanalysis: C₃₉H₅₉N₇O₁₀- H₂O: %C 58.60 (58.27), %H 7.4 (7.65), %N 12.20 (11.91)

IR v _Max KBR/crn 3338 (br NH, NH₂), 1722 (C=ON), 1641 (C=O)

Mp 124-125⁰C

iV-{2, 3'-Bis{2-[iV, N'-bis(tert-butoxycarbonyl)guanidino]-ethyIoxy}-biphenyI-5- ylmethyl}-3-[2-pyridyI)dithio]propionamide (Example 16)

To a solution of 2, 3'-Bis{2-[N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-5- (aminomethyl)biphenyl (0.10 g, 0.12 mmol) and Z-Pr₂EtN (40 μL, leq) in DCM (1.5 mL) was added N-succinimidyl-3-(2-pyridyldithio)propionate (44 mg, 0.12 mmol) and the mixture was stirred in the dark for 24 h. The solvent was removed under vacuum and the crude oil was purified by preparative TLC (ethyl acetate/hexane 1/1) to afford the title compound (25 mg, 21% yield). ESMS; m/z 983(M+1), 492(M+2)

¹H-NMR (CDCl₃): 61.49 (m, 36H), 2.62 (t, J= 6.5 Hz, 2H), 2.72 (t, J= 6.5 Hz, 2H), 3.76 (m, 2H), 3.83 (m, 2H), 4.08 (m, 4H), 4.45 (d, J= 5.4 Hz, 2H), 6.95 (m, 4H), 7.21 (m, 4H), 7.55 (m, 2H), 8.10 (d, J= 4.8 Hz, IH), 8.46 (t, J= 4.7 Hz, IH), 8.74 (bs, IH), 11.47 (bs, 2H).

¹³C-NMR (MeOH-d4): 528.0, 28.3, 34.5, 35.3, 39.5, 39.8, 43.3, 66.3, 67.0, 79.3, 81.3, 83.1, 110.5, 111.2, 113.3, 120.5, 120.9, 122.8, 128.1, 128.9, 130.9, 131.0, 1369, 149.5, 151.2, 153.0, 156.3, 158.8, 159.1, 163.5, 170.5. IR v _Max KBR/cm3332 (NH), 1722 (C=O), 1641 (C=O) iV-[2, 3'-Bis(2-guanidino-ethyloxy)-biphenyl-5-ylmethyl]-3-[2- pyridyl)dithio]propionamide, trifluoroacetate salt (Example 17)

N- {2, 3'-Bis{2-[N, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-biphenyl-5-ylmethyl}- 3-[2-pyridyl)dithio]propionamide (25 mg, 0.025 mmol) was dissolved in a mixture of

TFA/H₂O/triisopropylsilane 95/2.5/2.5 (1 mL) and stirred at room temperature for 3 h. The solvent was removed under vacuum to give the title compound as colourless oil (20 mg,

80%).

ESMS; m/z 582(M+1), 292(M+2) 1H-NMR (MeOH-d4): 52.65 (t, J= 6.7 Hz, 2H), 3.07 (t, 2H, J= 6.7 Hz, 2H), 3.50 (m, 2H),

3.60 (m, 2H), 4.10 (m, 4H), 4.35 (s, 2H), 6.91 (d, J= 7.4 Hz, IH), 7.10 (m, 7H), 7.74 (m,

2H), 8.35 (d, J= 4.7 Hz, IH).

¹³C-NMR (MeOH-d4): 526.9, 29.7, 40.4, 67.2, 67.7, 104.6, 112.8, 115.4, 116.8, 119.0,

121.0, 128.1, 128.3, 128.6, 129.6, 131.4, 133.1, 134.9, 135.9, 137.1, 156.0, 156.8, 158.7. IR v _Max KBR/cm^"3376 (NH), 1681(C=ON)

Examples 18 to 20: Synthesis of SMOC carrying 3 guanidine moieties

Tribenzyl 2,2',2"-(5-cyanobiphenyl-2,2',3'-triyltris(oxy))tris(ethane-2,l- diyl)tetracarbamate

ZHN

A degassed mixture of N-(2-(4-cyanophenoxy)ethyl)-2-phenylacetamide (1.0 g, 2.67 mmol) 2,2'-(3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-l,2-phenylene)bis(oxy)bis (ethane- 2,l-diyl)dicarbamate (2.07 g, 3.47 mmol), PdCl₂dppf.CH₂Cl₂ (100 mg, 4 mol%), potassium phosphate (1.13 g, 5.34 mmol), toluene (2 mL) and water (0.133 mL) was heated at 100°C for 18 h. The reaction mixture was diluted in EtOAc (25 mL) and water (25 mL) was added. The layers were separated and the aqueous layer was extracted with EtOAc (3 x 25 mL). The combined organic layers were dried over MgSO4, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (75/35) as eluent to afford the title compound (1.9 g, 93% yield). 1H-NMR (CDCl₃): 53.15 (m, 2H), 3.38 (m, 2H), 3.46 (m, 2H), 3.60 (m, 2H), 3.68 (m, 2H), 4.12 (m, 4H), 5.00 (bs, 2H), 5.03 (bs, 2H) 5.07 (bs, 2H), 5.25 (bs, IH), 5.32 (bs, IH), 5.97 (bs, IH), 6.61-6.93 (m, 6H), 7.07-7.61 (m, 15H). IR v _Max KBR/cm 3335(br, NH), 2225(CN), 1714 (C=O)

2, 2', 3'-Tris{2-[iV, N'-bis^ert-butoxycarbony^guanidinol-ethyloxyJ-S-cyanobiphenyl

Tribenzyl 2,2',2"-(5-cyanobiphenyl-2,2',3^I-triyltris(oxy))tris(ethane-2, 1 -diyl) tetracarbamate

(1.6 g, 2.11 mmol) was dissolved in DCM (10 mL) and HBr (30% in acetic acid, IQmL) was added dropwise. After stirring at room temperature for 2 h, water (25 mL) was added to the mixture, the layers were separated and the aqueous layer was washed with DCM (2 x

25 mL). The water was then removed under vacuum and the crude diamine was carefully dried under vacuum for several hours.

ESMS; m/z 357 (M+l)

¹H-NMR (MeOH-Cl₄): 53.05 ((t, J= 5.1 Hz, 2H), 3.30 (m, 2H), 3.52 (t, J= 4.7 Hz, 2H), 3.98 (t, J= 5.3 Hz, 2H), 3.68 (m, 2H), 4.12 (m, 4H), 5.00 (bs, 2H), 5.03 (bs, 2H) 5.07 (bs, 2H), 5.25 (bs, IH), 5.32 (bs, IH), 5.97 c, 6.99 (dd, J= 6.9, 2.3Hz, IH), 7.17-7.26 (m, 2H), (d, J= 8.6 Hz, IH), 7.65 (s, IH), 7.80 (dd, J= 8.6, 2.1 Hz, IH).

The resulting solid was suspended in DCM (10 mL) and Z-Pr₂EtN (1.6 mL) and N, N-di- boc-N'-trifluoromethanesulfonyl-guanidine (2.47 g, 3eq) were added. The mixture was stirred overnight at room temperature, diluted with DCM (40 mL) then washed with 2M NaHSO₄ (25 mL) followed by sat. aq. NaHCO₃ (25 mL) and brine (25 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using cyclohexane/EtOAc (80/20) as eluent to afford the title compound (1.3 g, 57% yield) as a white solid. ESMS; m/z 1084(M+l), 542(M+2)

¹H-NMR (CDCl₃): δl.43-1.53 (m, 54H), 3.43 (m, 2H), 3.73 (m, 2H), 3.84 (m, 2H), 3.91 (m, 2H), 4.07-4.18 (m, 5H), 6.88 (m, IH), 6.97 (m, IH), 7.06 (m, 2H), 7.56 (bs, IH), 8.41 (bt, IH), 8.44 (bt, IH), 8.80 (bt,lH), 11.42 (bs, IH), 11.47 (bs, IH), 11.10 (bs, IH). 1³C-NMR (CDCl₃): .627.8, 28.0, 28.1, 28.3, 38.9, 39.7, 40.6, 66.5, 71.0, 73.5, 79.3, 82.8, 83.1, 85.9, 104.9, 112.7, 114.7, 124.5, 124.1, 127.7, 130.8, 135.9 , 145.8, 149.0, 151.4, 153.0, 156.2, 156.3, 159.5, 163.4.

IR v _Max KBR/cm^" 3326 (br NH, NH₂), 2226 (CN), 1724 (C=O), 1619 (C=O) Mp 74-75⁰C

2, 2', 3'-Tris{2-[iV, iV'-bis(tert-butoxycarbonyI)guanidino]-ethyIoxy}-5- (aminomethyl)biphenyl (Example 18)

BocHN

2,2',3'-Tris {2-[N,N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy} -5-cyanobiphenyl (0.100 g, 0.092 mmol) was dissolved in THF (1.2 mL) and NH₄OH (0.30 mL) was added followed by Raney Nickel (0.010 g). The mixture was stirred vigorously under H₂ atmosphere for 16 h. The catalyst was filtered off and washed several times with MeOH. Diethylenetriamine (leq) was added and the mixture stirred for 30 minutes then concentrated under vacuum. The residue was dissolved in EtOAc (10 mL) and washed with sat. aq. NaHCO₃ (10 mL). The organic layer was dried over MgSO₄, filtered and concentrated under vacuum. The residue was then purified by flash chromatography using MeOH/DCM (5/95) as eluent to afford the title compound (70 mg, 69% yield).

¹H-NMR (CDCl₃): .51.47 (m, 54H), 3.38 (m, 2H), 3.70 (m, 2H), 3.79 (m, 4H), 3.89 (m, 2H), 3.70 (m, 2H), 4.01 (m, 2H), 6.70-6.95 (m, 4H), 7.05 (m, 2H), 8.34 (bt, IH), 8.42 (bt, IH), 8.80 (m, IH), 11.43 (m, 2H), 11.46 (bs, IH).

¹³C-NMR (CDCl₃): 528.1, 28.9, 30.34, 38.72, 40.1, 41.5, 41.4, 45.8, 50.6, 66.7, 67.2, 68.1, 71.0, 72.4, 78.8, 79.2, 82.6, 82.8, 83.0, 112.8, 123.8, 124.4, 127.1, 127.2, 128.5, 131.0, 131.4, 132.5, 136.0, 145.2, 151.6, 152.8, 152.9, 154.3, 156.0, 156.3, 156.3, 163.4. IR v M_ax KBR/cm- 3335 (br NH, NH₂), 1722 (C=O), 1641 (C=ON)

N-{2, 2',3'-Tris{2-[N, N'-bis(tert-butoxycarbonyI)guanidino]-ethyloxy}-biphenyl-5- ylmethyl}-3-[2-pyridyl)dithio]propionamide (Example 19)

To a solution of 2,2',3'-Tris{2-[iV, N'-bis(tert-butoxycarbonyl)guanidino]-ethyloxy}-5-

(aminomethyl)biphenyl (50 mg, 0.045 mmol) and Z-Pr₂EtN (10 μL, 0.10 mmol) in DCM

(0.7 mL) was added N-succinimidyl-3-(2-pyridyldithio)propionate (13 mg, 0.045 mmol) and the mixture was stirred in the dark for 24 h. The solvent was removed under vacuum and the crude oil was purified by preparative TLC (ethyl acetate/hexane 35/75) to afford the title compound (35 mg, 60 % yield).

ESMS; m/z 1285 (M+l), 643 (M+2), 429 (M+3).

¹H-NMR (CDCl₃): .51.43-1.53 (m, 54H), 2.64 (t, J= 7.1 Hz, 2H), 3.10 (t, J= 6.8 Hz, 2H), 3.40 (m, 2H), 3.71 (m, 2H), 3.83 (m, 2H), 3.91 (m, 2H), 4.03 (m, 2H), 4.15 (m, 2H), 4.38

(d, J= 5.6 Hz, 2H), 6.84-7.06 (m, 7H), 7.23 (m, IH), 7.61 (m, 2H), 8.31 (dd, J= 4.1, IHz, IH), 8.39 (bt, IH), 8.52 (bt, IH), 8.80 (bt,lH), 11.40 (bs, IH), 11.45 (bs, IH), 11.05 (bs, IH).

¹³C-NMR (CDCl₃): 521.0, 25.5, 28.0, 28.1, 28.3, 34.7, 35.6, 40.0, 41.3, 43.0, 60.3, 66.7, 67.1, 70.6, 79.1, 79.2, 82.9, 83.0, 112.1, 112.7, 119.9, 120.7, 123.8, 124.3, 127.0, 128.8, 130.5, 131.4, 132.7, 136.9, 145.4, 1495, 151.6, 152.8, 153.0, 154.8, 156.1, 156.2, 156.3, 159.7, 163.3, 163.5, 170.5.

N-[2, 2\ 3'-Tris(2-guanidino-ethyloxy)-biphenyl-5-ylmethyl]-3-[2- pyridyl)dithio]propionamide, trifluroacetate salt (Example 20)

N- {2, 3 '-Bis {2-[N, N'-bis^ert-butoxycarbony^guanidinoJ-ethyloxy} -biphenyl-5- ylmethyl}-3-[2-pyridyl)dithio]propionamide (6 mg) was dissolved in a mixture of TFA/ H₂O/triisopropylsilane 95/2.5/2.5 (1 mL) and stirred at room temperature for 3 h. The solvent was removed under vacuum to give the title compound as colourless oil (4 mg, 60%).

ESMS; m/z 343 (M+2)

¹H-NMR (MeOH-d₄): δ 2.68 (t, J= 6.8Hz, 2H), 3.07 (t, J= 6.7 Hz, 2H), 3.20 (m, 2H), 3.42 (m, 2H), 3.65 (m, 2H), 3.90 (m, 2H), 4.08 (m, 2H), 4.17 (m, 2H), 4.37 (bs, 2H), 6.86 (m, IH), 7.05-7.29 (m, 6H), 7.73 (m, 2H), 8.37 (m, IH). 1³C-NMR (CDCl₃): δ 35.4, 35.8, 42.3, 43.0, 43.6, 68.3, 68.8 67.1, 72.7, 79.1, 114.1, 114.5, 121.2, 122.4, 125.0, 125.6, 128.9, 129.1, 130.9, 132.7, 134.6, 139.1, 146.5, 150.4, 152.7, 156.1, 159.3, 161.0, 173.6.

Theoretical Mass: (M + H) 684.28572. Measured Mass: (M + H) 684.28417 IR v Max KBR/cm^"3412 (br NH, NH₂), 1677 (C=ON) Example 21: Observation of cellular uptake via live microscopy

Geminin and 15k were expressed and purified according to the method described in R. V. Stevens, G.S. Bisacchi, Journal of Organic Chemistry 1982, 47, 2396-2399, the entirety of which is incorporated herein by reference. Geminin was conjugated with Alexa Fluor 488 using a Molecular Probes Protein Labelling kit, according to the manufacturer's protocol (Invitrogen). Coupling reactions of proteins with different SMOCs have been described in Okuyama M. et al, Nat. Methods 2007, 4, 153-159, the entirety of which is incorporated herein by reference. The efficiency of coupling was assessed by spectrophotometric quantification of pyridyl at 343 run and monitoring release of sulfhydryl groups using Ellman's Reagent (Pierce, Rockford, IL, USA) according to Pierce protocol 22582.

Human U2OS and WI-38 human diploid fibroblasts (HDF), were cultured in DMEM supplemented with 10% FCS, 100 LVmL penicillin and 0.1 mg/mL streptomycin.

To determine the ability of compounds of the invention to carry biomolecules into cells, the nuclear protein Geminin, a repressor of origin licensing, was coupled to compounds of the invention via a disulfide bond. Geminin is a 23.5 kDa protein that blocks DNA replication. Three compounds of the invention, Examples 3, 17 and 20 were conjugated to Geminin labelled with AlexaFluor 488 via thiol exchange, giving compounds 11-g*, 33-g* and 35- g* respectively. The cellular uptake, followed via live microscopy in two cell lines (WI-38 human diploid fibroblast and human U2OS osteosarcoma cells) is shown in Figure 1.

When U2OS cell line was treated with the fluorescent tagged protein, a higher uptake was detected when the cells were incubated with 11-g* and a lower when treated with 23-g*. In the case of WI-38 HDF the difference in the uptake was not so obvious by this method, but a preferential perinuclear and nuclear localisation was observed when Geminin was linked to the compounds 35-g* and 11-g* containing 3 and 4 guanidine moieties respectively. These data suggested a correlation between the number of guanidine moieties and efficiency of the delivery and cargo localisation. For detection of the uptake of compounds of the invention coupled with Geminin- AlexaFluor488 into live cells, exponentially growing cells (WI-38 HDF and U2OS) were cultured on glass coverslips. Cells were washed in PBS, and incubated with fresh medium containing 10 μM solutions of compounds of the invention coupled with Geminin- AlexaFluor488. Coverslips were washed extensively in PBS, placed in a plate containing medium without Red Phenol (Gibco) and observed by live confocal fluorescence microscopy (MP-UV, Leica Microsystems GmbH, Wetzlar, Germany) using 40^χ and 63 x water immersion objectives, hi order to obtain similar fluorescence intensities, WI-38, and U2OS cells required incubation with the protein conjugate for 1 and 5 hours respectively.

Example 22: Analysis of cellular uptake by FACS analysis

To evaluate and compare the ability of different compounds of the invention to carry biomolecules into different human cells and provide a measure of the efficiency of cell penetration compounds of the invention coupled with Geminin-AlexaFluor488 molecules were treated with WI-38 and U2OS.

Cells were seeded in 6-well plates and incubated with 10 μM solutions of compounds of the invention coupled with Geminin-AlexaFluor488 for 3 h (WI-38) or 5 h (U2OS). Cells were then washed extensively with PBS, trypsinised, resuspended in medium, washed again in cold PBS and immediately analysed by Flow Cytometry (FACSCalibur). A total of 10,000 cells per sample were counted. The mean fluorescence intensity of treated cell populations was compared with untreated control cells and cells incubated with AF488 or AF488-15k only.

Figure 2 shows the mean value of fluorescence obtained from this experiment in both cell lines. Cells treated with labelled Geminin conjugated to compounds of the invention show a clear shift in the FACS profile towards higher FLlH values compared to the controls, revealing that proteins conjugated to compounds of the invention can enter efficiently into the cells (approximately 100% of U2OS cells show uptake, a range between 75 and 90% in the case of WI-38 HDF). Both cell lines showed similar trend which means that the relative efficiency of compounds of the invention was identical in normal and cancer cells. However, the fluorescence was more intense in WI-38 HDF meaning that the compounds of the invention were more efficient carriers in this cell line.

Several compounds of the invention, Examples 9, 11, 5, 7, 17, 14, 20 and 3 were coupled to Geminin labelled with AlexaFluor488, giving compounds 23-g*, 26-g*, 18-g*, 20-g*, 33- g*, 31-g*, 35-g* and 11-g*, respectively.

The first six bars of the chart shown in Figure 2 represent the uptake obtained using bis- guanidines (23-g*, 26-g*, 18-g*, 20-g*, 33-g* and 31-g*) as carrier, the seventh and eighth bar represent the data for the tris-guanidine compound 35-g* and the tetra-guanidine compound 11-g* respectively. It appears that when more guanidine moieties are present on the carrier, its efficiency is higher. Using the data obtained from the bis-guanidine, it was possible to make a correlation between the position of the guanidine moiety and the linker moiety and the ability of the compounds to carry a biomolecule. In the case of compounds 18-g*, 20-g*, 33-g* and 31-g* the guanidine moieties are in the same position but the linker moiety is in a different position. The efficiency was higher when the linker was para to the guanidine moiety. Moreover, when the guanidine moiety is ortho (compounds 23-g* and 26-g*) to the linker moiety, a reduction in the ability to carry the cargo into the cells was observed.

Example 22: Cell proliferation assay

To assess whether 15k Geminin retains its ability to inhibit DNA replication in cycling cells when coupled with compounds of the invention, 10 μm solutions of compounds of the invention coupled with 15k were added to WI-38 and U2OS and replenished every 24 h, for a total 72 h treatment. Seventy-one hours after the initial treatment, cells were pulse- labelled for 1 h with 10 μM BrdU, then fixed with 4% paraformaldehyde, and treated as described in Okayama et al, Nat Methods 2007, 4 153-159. Confocal fluorescence microscopy was performed on a Leica TCS DMRE confocal microscope. The results are shown in Figure 3. The functional activity of Geminin was retained.

Claims

1. A process for the production of a compound of formula I, or a pharmaceutically acceptable salt thereof,

wherein

X₁, X₂ and X₃ are each independently

NR₂ Nv' ^NR, NR₃R₄ where Y is an alkylene, alkenylene or alkynylene group, each of which may be optionally substituted with one or more substituents selected from alkyl, halo, CF₃, OH, alkoxy, NH₂, CN, NO₂ and COOH; W is absent or is O, S or NH;

R₁, R₂, R₃ and R₄ are each independently selected from H, alkyl, aryl and a protecting group

Pi;

R₇, R₈ and R₉ are each independently selected from H, alkyl, halo, CF₃, OH, alkoxy, NH₂, CN, NO₂ and COOH; - q and r are each independently 1, 2, 3 or 4; q¹ and r' are each independently O, 1, 2 or 3, where q + q' and r + r' each equal 4; p is 1, 2, 3, 4 or 5, and p' is O, 1, 2, 3 or 4, where p + p' is 5; n is 0, 1, 2, 3, 4, 5 or 6; and

L is (Z)_1nNR₅R₆ wherein Z is a hydrocarbyl group and m is 0 or 1 wherein R₅ and R₆ are each independently H, CO(CH₂)_jQi or C=S(NH)(CH₂)_kQ₂, where j and k are each independently 0, 1, 2, 3, 4 or 5, and Q₁ and Q₂ are each independently selected from COOH, a chromophore,

or R₅, R₆ and the nitrogen to which they are attached together form

which process comprises:

(a) coupling a compound of formula II to a compound of formula III to form a compound of formula IV

IV wherein: n, p, p', q, q', r and r' are defined as above; at least one X₁ ¹, X₂' and X₃' moiety is -W-Y-NR₁Ri₀ or -W-Y-NR₁-CC=NRz)-NR₃R₄ and the other X₁', X₂' and X₃' moieties are each independently selected from OH, SH, NH₂, -W-Y-NR₁R₁₀, and -W-Y-NR₁ -C(=NR₂)-NR₃R4 wherein R₁, W and Y are defined as above and R₁₀ is H or a protecting group P₂; one of J₁ and J₂ is a leaving group LG₁, and the other is a boronic acid, a boronic ester, a borane group or a trihalogenoborate salt group; L'" is L, as defined above, a leaving group LG₂, -COR₁₈, -(Z)_1nNHR₁₂, or a group which can be reduced to a moiety -(Z)_mNH₂, where Z and m are defined as above, R₁₂ is a protecting group P₃ and R]₈ is hydrogen or a C₁-C₄ alkyl group; and

(b) if any of X₁ ', X₂' and X₃ ¹ in the formula (IV) is other than -W-Y-NR₁ -C(=NR₂)- NR₃R₄, alkylating the X₁', X₂ ¹ and X₃' moieties so that they each represent a group of formula

where W, Y, R₁, R₂, R₃ and R₄ are defined as above, and, if necessary, converting the moiety L'" to a moiety L as defined above, to obtain a compound of formula I.

2. A process according to claim 1, wherein at least one X₃' is -W-Y-NR₁Ri₀ or -W-Y- NR₁-C(=NR₂)-NR₃R₄.

3. A process according to claim 1, wherein when any of the Xi', X₂ ¹ and X₃' moieties in the formula (IV) are OH, SH or NH₂, alkylation so that all X₁', X₂' and X₃' moieties represent a group of formula -W-Y-NRi -C(=NR₂)-NR₃R₄ is effected by either: (i) alkylating any X₁', X₂' and X₃' moieties that are OH, SH or NH₂ so that they represent -W-Y-NR₁R_1O, deprotecting any protected amine groups on -W-Y-NRiRi₀ moieties present at the Xi¹, X₂' and X₃' positions and guanidinylating the deprotected moieties so that they each represent a group of formula -W-Y-NRi -C(=NR₂)- NR₃R₄; or (ii) alkylating any Xi', X₂' and X₃' moieties that are OH, SH or NH₂ so that they each represent a group of formula -W-Y-NR₁ -C(=NR₂)-NR₃R₄, deprotecting any protected amine groups on -W-Y-NRiRi₀ moieties present at the Xi', X₂' and X₃' positions and guanidinylating the deprotected moieties so that they each represent a group of formula -W-Y-NR_]-C(=NR₂)-NR₃R₄.

4. A process according to claim 3 wherein the alkylation of hydroxy, thiol and amino groups at the Xi', X₂' and X₃' positions so that they represent -W-Y-NRiRi₀ is effected with a compound of formula LG₃-Y-NR₁R₁₀ where R₁ and Y are as defined in claim 1, R₁₀ is H or a protecting group P₂ and LG₃ is a leaving group.

5. A process according to claim 3 wherein the alkylation of any X₁', X₂' and X₃' moieties that are OH, SH or NH₂ so that they each represent a group of formula -W-Y-NR₁- C(=NR₂)-NR₃R₄ is effected by a compound of formula LG₇-Y-NRj -C(=NR₂)-NR₃R4.

6. A process according to claim 1 wherein all of the X₁', X₂' and X₃' moieties are -W-Y-NR₁R₁₀ or -W-Y-NR₁ -C(=NR₂)-NR₃R4 and any necessary alkylation of Xi¹, X₂' and X₃' moieties so that they each represent -W-Y-NR₁ -C(=NR₂)-NR₃R₄ is effected by deprotecting any protected amine groups on -W-Y-NR₁Ri₀ moieties present at the X₁ ¹, X₂' and X₃' positions and guanidinylating the deprotected moieties so that they represent a group of formula -W-Y-NR₁ -C(=NR₂)-NR₃R4.

7. A process according to any one of claims 3, 4, 5 and 6, wherein said guanidinylation of deprotected -W-Y-NRiRi₀ moieties at the X₁', X₂' and X₃' positions is effected either by a compound of formula V, or a tautomer thereof, where R₂, R₃ and R₄ are

R₄R₃N LG₄

R₂HN

V as defined in claim 1 and LG₄ is a leaving group; or by a compound of formula V\ or a tautomer thereof,

R₄R₃N

R₂N

V

where R₂, R₃ and R₄ are as defined in claim 1 and LG₄' is a leaving group.

8. A process according to claim 7, wherein LG₄ is a triflyl group or LG₄' is a pyrazole group.

9. A process according to claim 1, wherein at least one X₁', X₂' and X₃ ¹ moiety is -W- Y-NR₁R_1O and the other X₁', X₂' and X₃ ¹ moieties are each independently selected from OH, SH, NH₂ and -W-Y-NR₁R₁₀ wherein R₁, W and Y are as defined in claim 1 and R₁₀ is H or a protecting group P₂, and wherein in step (b) when any X₁', X₂' and X₃' moieties are OH, SH or NH₂, alkylation is effected so that all X₁', X₂' and X₃' moieties represent -W-Y-NRiR₁₀, any protected amine groups on the X₁', X₂' and X₃ ¹ moieties are deprotected and the X₁', X₂' and X₃' moieties are guanidinylated so that they each represent a group of the formula -W-Y- NRrC(=NR₂)-NR₃R₄ and, if necessary, the moiety L"' is converted to a moiety L, to obtain a compound of formula (I).

10. A process according to any one of the preceding claims wherein L'" is a group which can be reduced to a moiety -(Z)_mNH₂, and the conversion of the moiety L'" to a moiety L in step (b) comprises (i) reducing L'" and (ii) optionally reacting the thus obtained compound with a compound of formula LG₅CO(CH₂)_JQJ or LG₆C=S (NH)(CH₂)kQ₂ where j, k, Q₁ and Q₂ are as defined in any one of the preceding claims and LG₅ and LG₆ are leaving groups.

11. A process according to claim 10 wherein L'" is CN.

12. A process according to any one of claims 1 to 9 wherein L'" is a leaving group LG₂, and the conversion of the moiety L'" to a moiety L in step (b) comprises (i) cyanating with a source of CN^' in the presence of a suitable catalyst, (ii) reducing the thus obtained CN moiety and (iii) optionally reacting the thus obtained compound with a compound of formula LG₅CO(CH₂)_JQ₁ or LG₆C=S (NH)(CH₂)_kQ₂ where j, k, Q₁, Q₂, LG₅ and LG₆ are as defined in any one of the preceding claims.

13. A process according to claim 11 or 12, wherein the CN moiety is reduced under a hydrogen atmosphere, in the presence of a catalyst.

14. A process according to any one of claims 1 to 13, wherein, in step (b), the alkylation of the X₁', X₂' and X₃' moieties so that they each represent a group of formula -W-Y-NR₁- C(=NR₂)-NR₃R₄ is effected before the conversion of the moiety L'" to a moiety L.

15. A process according to any one of claims 1 to 9 and 14, wherein L'" is -COR₁₈, and the conversion of the moiety L'" to a moiety L in step (b) comprises (i) reductively aminating the -COR₁₈ moiety and (ii) optionally reacting the thus obtained compound with a compound of formula LG₅CO(CH₂)JQ₁ or LG₆C=S(NH)(CH₂)_kQ₂.

16. A process according to claim 15 wherein reductive amination of the -COR₁₈ moiety is effected by (a) reaction with NH₂OR^ or NHR₅R₆ and (b) reduction of the carbon nitrogen double bond, wherein R^ is H or an alkyl group.

17. A process according to any one of the preceding claims wherein W is O and/or Y is CH₂CH₂.

18. A process according to any one of the preceding claims, wherein R₁ and R₃ are hydrogen, and R₂ and R₄ are each independently selected from H and a buryloxycarbonyl (Boc) protecting group.

19. A compound according to any one of the preceding claims, wherein p, q and r are each independently 1 or 2.

20. A process according to any one of the preceding claims, wherein in formula IV, R₁ is H and R₁₀ is a carbobenzyloxy (Cbz) protecting group.

21. A process according to any one of the preceding claims, wherein n is 0.

22. A process according to any one of the preceding claims, wherein Ji is a boronic acid, a boronic ester, a borane group or a trihalogenoborate salt group and J₂ is a leaving group LGi.

23. A process according to any one of the preceding claims wherein the boronic ester is -B(OR₁₃)(OR₁₄) Or

where R₁₃ and R₁₄ are each independently selected from C₁-C₆ alkyl groups and R₁₅ is a C₁-C₆ alkyl or phenyl group.

24. A process according to any one of claims 1 to 22 wherein the borane is -BR₁₆R₁₇ where R₁₆ and R₁₇ are each independently selected from C₁-C₆ alkyl groups.

25. A process according to any one of claims 1 to 22 wherein said compound of formula II is of formula Ha, lib, Hc or Hd and/or said compound III is of formula Ilia, IHb, IHc, IHd or HIe.

Ha πb Hc πd

IHa nib me

ma me

26. A compound of formula (I), as defined in any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein r is 1 or 2 and the phenyl ring which carries L has no substituents ortho to the L moiety.

27. A compound according to claim 26, wherein the phenyl ring which carries L has a hydrogen atom ortho to the moiety

28. A compound according to claim 26, wherein r is 1 and L is para to the X₃ moiety.

29. A compound of formula Via, VIb, VIc, VId or VIe or a pharmaceutically acceptable salt thereof,

VId Vie

wherein X₁, X₃ and L are as defined in any one of the preceding claims.

30. A compound according to claim 29 which is selected from the following:

31. A compound of formula VII, or a pharmaceutically acceptable salt thereof,

VII wherein:

X], X₃, R₇, Rg and L are as defined in any one of the preceding claims; r is 1, 2, 3 or 4 and r' is 0, 1, 2 or 3, wherein r + r' is 4; p is 1, 2, 3, 4 or 5 and p¹ is 0, 1, 2, 3 or 4, where p + p¹ is 5; and p + r is 3.

32. A compound according to claim 31 which is:

33. A process for preparing a conjugate, which process comprises: (i) preparing a compound of formula I, or a pharmaceutically acceptable salt thereof, in which L is other than a moiety -(Z)_m-phthalimide, wherein Z and m are as defined in claim 1, by a process according to any one of claims 1 to 25; and (ii) reacting said compound of formula I with a cargo moiety selected from a protein, a peptide, an oligonucleotide, a nucleotide, a diagnostic agent, a biologically active compound, an antibody and a drug.

34. A process according to claim 33, wherein in step (ii) a nucleophilic group on the cargo moiety displaces a leaving group in the moiety L in the compound of formula (I).

35. A process according to claim 33 or 34, wherein the cargo moiety is a drug.

36. A process according to claim 35, wherein the drug is a cytotoxic drug.

37. A process according to claim 35, wherein the drug is selected from DNA damaging agents, anti-metabolites, anti-tumour antibiotics, natural products and their analogues, dihydrofolate reductase inhibitors, pyrimidine analogues, purine analogues, cyclin- dependent kinase inhibitors, thymidylate synthase inhibitors, DNA intercalators, DNA cleavers, topoisomerase inhibitors, anthracyclines, vinca drugs, mitomycins, bleomycins, cytotoxic nucleosides, pteridine drugs, diynenes, podophyllotoxins, platinum containing drugs, differentiation inducers and taxanes.

38. A process according to claim 37, wherein the drug is selected from methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, tri-substituted purines such as olomoucine, roscovitine and bohemine, flavopiridol, staurosporin, cytosine arabinoside, melphalan, leurosine, actinomycin, daunorubicin, doxorubicin, mitomycin D, mitomycin A, carninomycin, aminopterin, tallysomycin, podophyllotoxin (and derivatives thereof), etoposide, cisplatinum, carboplatinum, vinblastine, vincristine, vindesin, paclitaxel, docetaxel, taxotere retinoic acid, butyric acid, acetyl spermidine, tamoxifen, irinotecan and campotothecin.

39. A conjugate obtainable by reacting a compound as defined in any one of claims 26 to 32 with a cargo moiety as defined in any one of claims 33 and 35 to 38, or a pharmaceutically acceptable salt of said conjugate.

40. Use of a compound or conjugate according to any one of claims 26 to 32 or 39 in the manufacture of a medicament for use delivering a drug to a patient transdermally.

41. A skin patch which comprises a compound or conjugate according to any one of claims 26 to 32 or 39 and a pharmaceutically acceptable carrier or diluent.