New protecting groups as anchors for liquid phase peptide synthesis

Abstract

Object of the present invention are compounds of formula (1) as protecting groups to perform liquid phase peptide synthesis.

Description

DescriptionNew protecting groups as anchors for liquid phase peptide synthesis.Technical Field

[0001] The present invention refers to protecting groups for liquid phase peptide synthesis.Background Art

[0002] Peptide chemistry has experienced a significant grown in recent decades mainly due to the employment of peptides as therapeutics. Since the introduction of insulin almost a century ago, in fact, a large number of peptide drugs have reached the market for a wide range of diseases, including diabetes, cardiovascular diseases, gastrointestinal diseases, cancer, infectious diseases, vaccine development, HIV infection and chronic pain.

[0003] Therapeutic peptides are composed of a series of well-ordered amino acids, usually with molecular weights of 500-5000 Da. The high demand for this class of pharmaceutical agents has prompted great advances in peptide synthesis development.

[0004] The first approach used to prepare peptides is known as classical solution peptide synthesis (CSPS). This strategy, however, is tedious, highly timeconsuming and requires a great human effort. In practice, this technique is almost inapplicable for the preparation of medium-sized and long peptides.

[0005] A second approach, referred to as solid-phase peptide synthesis (SPPS), greatly simplified the workup and removed the need to purify intermediates (J. Am. Chem. Soc., 1963, 85, 2149-2154). This method shows poorer performance with respect to the classical solution peptide synthesis and requires the use of a large excess of solvents and reagents to compensate the poorer solubility and reactivity of the compound attached to the insoluble resin. Despite these disadvantages, however, SPPS significantly shorten the time required for the peptide preparation and can easily be scaled up. Some limits arise when SPPS is used to produce long peptides (>20 amino acids), as this strategy often results in the accumulation ofundesired byproducts and erroneous sequences that need to be removed through pricey chromatographic methods.

[0006] For these reasons, a third synthetic approach was developed, where the best attributes of SPPS and CSPS were combined. In this third approach, called liquid-phase peptide synthesis (LPPS), all the reactions are carried out in solution like in CSPS, allowing to reduce the excess of reagents and the huge amount of solvent required by SPPS. In LPPS the growing peptide chain is supported on a soluble tag or anchor, which confers the chain special properties that facilitate solution chemistry, while, at the same time, greatly simplifying the separation of peptide from excess reagents and byproducts, which is the advantage of SPPS chemistry.In practice, LPPS consists of the elongation of the peptide chain in solution thanks to a peculiar protecting group attached to one of its terminus that, after coupling reaction, is also the driver for the purification of each intermediate before the following amino acid addition.These tags / anchors attached to the peptide chain terminus, in fact, act as the polystyrene-based solid supports employed in standard SPPS, thus simplify the workup after each step of the synthesis. At the same time, however, maintain the peptide in solution and therefore realize the conditions of CSPS. At the end of the chain elongation, the tag / anchor is removed as any other protecting group used in peptide synthesis.

[0007] LPPS potential for scalability as well as its reduced use of reagents and solvents makes this technique more sustainable and environmentally friendly than any other chemical method developed up to date.

[0008] In order to be suitable as tags / anchors for LPPS, these peculiar protecting groups need to show simultaneously the property of easily dissolving in aqueous or organic solvents and a feature capable of significantly distinct the peptide from the other reagents and byproducts found in the mixture, so to simplify their removal by precipitation, filtration, or extraction at the end of each step.

[0009] It follows that the selection of adequate protecting groups suitable to be employed as tags / anchors is the primary criterion for a successful and effective LPPS strategy.

[0010] The first protecting groups used as LPPS tag were soluble polyethylene glycol polymers (PEGs). The application of these compounds for the preparation of peptides is reported for example in J. Am. Chem. Soc. 1974, 96, 7333-7336 or in J. Org. Chem. 1980, 45, 5364-5370.

[0011] Later, the so-called Membrane-enhanced peptide synthesis (MEPS) was developed as a variant of PEG tag-based technology, where the reagents are removed by organic solvent nanofiltration (Org. Process Res. Dev. 2010, 14, 1313-1325 and Macromolecules, 2017, 50, 1626-1634).

[0012] Another kind of protecting group developed for LPPS includes Perfluoroalkyl (fluorous) tags, which are simultaneously fluorophilic, lipophobic, and hydrophobic. This technology is described, for example, in Tetrahedron Letters, 2002, 43, 7809-7812, in Tetrahedron Letters 2003, 44, 9013-9016 and in J. Org. Chem. 2006, 71, 3299-3302.

[0013] Long aliphatic alkoxy substituents (PolyCarbon) were also employed as tags for LPPS, as it was found that these molecules were capable to confer differentiated properties to the growing peptide chains, allowing the isolation of the desired product via precipitation / extraction under proper conditions (Tetrahedron 2009, 65, 8014-8020).

[0014] Ionic Liquids have also been used as soluble supports for peptide synthesis (J. Org. Chem. 2005, 70, 3251-3255, Org. Letters 2007, 9, 2681-2684 and Tetrahedron 2010, 66, 8325-8334) as well as Polynorbornene supports, which are characterized by high loading capacity and enhanced solubility in organic solvents (Org. Letters 2013, 15, 5870-5873 and J. Org. Chem. 2014, 79, 11549- 11557).

[0015] All the above cited protecting groups, however, when employed as tags / anchors for LPPS, show some drawbacks such as limited loading, difficult monitoring of the peptide chain growing by standard analytical methods, disadvantageous process mass intensity index (PMI), which makes these tags / anchors not suitable for industrial application, and limited flexibility in solubility and / or crystalline properties provided to the growing peptides.

[0016] Thus, there is a need for protecting groups to be used as tags / anchors in LPPS at industrial scale characterized by high quality standards, low production costs and low environmental impact.

[0017] The compound of formula (8):

[0018] is disclosed in the prior art, for example, in the patent document WO2019019483 as well as in other documents. However, said compound was always disclosed only for the preparation of thermosetting resins or flameretardant resins with low polarity.Summary of the invention

[0019] The problem addressed by the present invention is therefore that of providing protecting groups to be used as tags / anchors in LPPS which simultaneously solve the following problems:• suitable for industrial application;• easy monitoring of the peptide chain growing by standard analytical methods, such as HPLC and / or mass determination;• provide improved peptide solubility, thus: o increasing process productivity; o allowing the reactions to be performed in solution as in CSPS; o allowing extractive work up after each reaction step;• provide crystallization properties, allowing isolation of the peptide after each step and / or at the end of the peptide chain synthesis.

[0020] As additional or alternative problem, it would be desirable to make available protecting groups to be used as tags / anchors in LPPS at industrial scale, wherein the solubility of the peptide protected by said tag / anchor can be modulated by selecting a specific compound within a class of compounds.

[0021] These problems are solved by employing as protecting groups for LPPS a compound of formula (1):(D, wherein n1, n2 and n3 are independently 0 or 1 and wherein X is OH, NH2 or halogen.

[0022] Specifically, the present invention is directed to the use of compound of formula (1) in LPPS.

[0023] As a further aspect, the present invention provides a process for preparing amino acids or amino acid derivatives protected with compound of formula (1), and a process for preparing a peptide which employes compound of formula (1) as tag / anchor for LPPS.

[0024] Further features and advantages of the present invention will result from the description hereafter reported and from the annexed claims whose definitions are integral part of the present description.Brief description of the drawings

[0025] Figure 1 shows the solubility trend of compound of formula (1) as a function of the molecular structure.

[0026] Figure 2 shows how compound of formula (1) can be attached to the carboxylic functional group of an amino acid or amino acid derivative.

[0027] Figure 3 shows how liquid-phase peptide synthesis using compound of formula (1) as C-terminus protecting group is performed.

[0028] Figure 4 shows how after cleavage of the C-terminus protecting group the by-product can be recovered and regenerated to obtain compound of formula (1).

[0029] Figure 5 shows1H NMR spectrum of compound of formula (7).

[0030] Figure 6 shows1H NMR spectrum of compound of formula (6).

[0031] Figure 7 shows1H NMR spectrum of compound of formula (8).

[0032] Figure 8 shows1H NMR spectrum of compound of formula (9).

[0033] Figure 9 shows1H NMR spectrum of compound of formula (34).

[0034] Figure 10 shows1H NMR spectrum of compound of formula (10).

[0035] Figure 11 shows1H NMR spectrum of compound of formula (13).

[0036] Figure 12 shows1H NMR spectrum of compound of formula (24).

[0037] Figure 13 shows1H NMR spectrum of compound of formula (25).

[0038] Figure 14 shows1H NMR spectrum of compound of formula (26).

[0039] Figure 15 shows1H NMR spectrum of compound of formula (43).

[0040] Figure 16 shows1H NMR spectrum of compound of formula (41).

[0041] Figure 17 shows the route of synthesis followed for the preparation of the pentapeptide of formula (32).Detailed description of the invention

[0042] The present invention is related to the use in LPPS of protecting groups of formula (1):wherein n1, n2 and n3 are independently 0 or 1 and wherein X is OH, NH2or halogen.

[0043] It was surprisingly found, in fact, that compound of formula (1) function as an ideal and improved tag / anchor for LPPS.

[0044] The reason is that compound of formula (1) surprisingly combines solubility properties, which are crucial to keep the peptide in solution during the reactions and the following work up, with the tendency to crystallinity, which is the key to allow the precipitation of the peptide, and therefore an easy purification after each step.

[0045] The properties of compound of formula (1) mentioned above can additionally be modulated by acting on the number of oxygen atoms to increase the solubility features (see Figure 1).

[0046] At the same time, by providing a rigid structure by means of the condensed- ring moiety, which brings a tendency to crystallinity, and thus the possibility of isolating the product by crystallization.

[0047] The structure depicted below, which is a 1,1'-biphenyl moiety substituted at the 2 and 2' position and which specifically characterizes compound of formula (1), is crucial in determining the peculiar properties mentioned above for compound of formula (1).

[0048] Indeed, it was surprisingly found that compound of formula (1) acts as an improved tag / anchor for LPPS, since the solubility of the peptide protected by said tag / anchor is improved.

[0049] Additionally, the solubility of the peptide protected by said tag / anchor can be modulated by selecting a specific compound within a class of compounds. In particular, it has been observed that the solubility of the peptide protected by said tag / anchor increases as the number of oxygen atoms found in the compound of formula (1) increases (see Figure 1).

[0050] Moreover, it has experimentally unexpectedly found that the amino acid or peptide protected with the compound of formula (1) can be easily purified, since said compound / s are easily crystallized, for example, by means of precipitation from an organic solution where said compound / s are dissolved by dosing of the solution over an antisolvent (refer to Example 12).

[0051] Furthermore, compound of formula (1) is suitable for industrial application and allows an easy monitoring of the peptide chain growing by standard analytical methods, such as mass determination, being a small molecule with a defined molecular weight and not a polymer, which is characterized by a non-homogeneous molecular weight.

[0052] Compound of formula (1) thus solves all the above-mentioned problems.

[0053] Object of the present invention is therefore the use of a compound of formula (1):(D, wherein n1, n2 and n3 are independently 0 or 1 and wherein X is OH, NH2 or halogen, in liquid phase peptide synthesis.

[0054] According to a preferred aspect, the invention is related to the use of a compound of formula (1) in liquid phase peptide synthesis, wherein n1, n2 and n3 are 0, or wherein n1 and n3 are 0 and n2 is 1, or wherein n1 and n2 are 0 and n3 is 1, or wherein n1 and n2 are 1 and n3 is 0, or wherein n2 and n3 are 1 and n1 is 0, or wherein n1, n2 and n3 are 1.

[0055] According to a more preferred aspect, the invention is related to the use of a compound of formula (1) wherein n1, n2 and n3 are independently 0 or 1 and wherein X is Br or NH2, in liquid phase peptide synthesis.

[0056] According to an even more preferred aspect, the invention is related to the use of a compound of formula (1) wherein n1, n2 and n3 are independently 0 or 1 and wherein X is Br, in liquid phase peptide synthesis.

[0057] According to a preferred aspect of the above said use, it is preferred a compound of formula (1), wherein n1 and n3 are 0 and n2 is 1, or wherein n1 and n2 are 0 and n3 is 1, or wherein n1 and n2 are 1 and n3 is 0, or wherein n2 and n3 are 1 and n1 is 0, or wherein n1, n2 and n3 are 1, since those compound provides the correspondent protecting peptide having improved solubility.

[0058] Indeed, it has to be considered, that improving of solubility means improving the industrially productivity since more matter can be produced in a defined reactor volume.

[0059] The improved solubility is intended as an improved solubility in organic solvents, such as DCM, EtOAc, acetonitrile, anisole or toluene.

[0060] According to a more preferred aspect of the above said use, it is preferred a compound of formula (1), wherein n1 and n2 are 1 and n3 is 0, or wherein n2 and n3 are 1 and n1 is 0, or wherein n1, n2 and n3 are 1, since thosecompounds provides the correspondent protecting peptide having an again more improved solubility.

[0061] According to an again more preferred aspect of the above said use, is preferred a compound of formula (1), wherein n1, n2 and n3 are 1, since those compounds provides the correspondent protecting peptide having the highest solubility.

[0062] Compound of formula (1) wherein n1, n2 and n3 are 0 corresponds to compound of formula (2):(2).

[0063] Compound of formula (1) wherein n1 and n3 are 0 and n2 is 1 corresponds to compound of formula(3).

[0064] Compound of formula (1) wherein n2 and n3 are 1 and n1 is 0 corresponds to compound of formula(4).

[0065] Compound of formula (1) wherein n1, n2 and n3 are 1 corresponds to compound of formula (5):

[0066] Compound of formula (1) wherein n1 and n2 are 0 and n3 is 1 corresponds to compound of formula (37):(37).

[0067] Compound of formula (1) wherein n1 and n2 are 1 and n3 is 0 corresponds to compound of formula (38):

[0068] According to another preferred aspect, the invention is related to a compound for use in liquid phase peptide synthesis selected among the following compounds:wherein X is OH, NH2or halogen.

[0069] According to a more preferred aspect, the present invention is related to the use of a compound in liquid phase peptide synthesis selected amongcompound of formula (2), or compound of formula (3), or compound of formula (4), or compound of formula (5), wherein X is Br or NH2.

[0070] According to an even more preferred aspect, the present invention is related to the use of a compound in liquid phase peptide synthesis selected among compound of formula (2), or compound of formula (3), or compound of formula (4), or compound of formula (5), wherein X is Br.

[0071] Compound of formula (1), or compound of formula (2), or compound of formula (3), or compound of formula (4), or compound of formula (5), wherein X is NH2 is preferred as tag for LPPS as, upon cleavage, the resulting peptide is obtained with a primary amide functional group at its C-terminus. Said primary amide functional group is often found in peptides used for pharmaceutical purposes, such as Exenatide, Liraglutide, Eptifibatide and Lixisenatide fragments.

[0072] Compound of formula (1), or compound of formula (2), or compound of formula (3), or compound of formula (4), or compound of formula (5), wherein X is halogen, and particularly wherein X is Br, is another preferred tag for LPPS. As depicted in Figure 2, in fact, when X is Br, the tag / anchor can be attached to an amino acid or amino acid derivative at its C-terminus by simply using a base, such as potassium carbonate, and no coupling / activating agent is needed.

[0073] Furthermore, as exemplified in Figure 4, upon cleavage of the anchor / tag from the peptide / amino acid, a methyl derivative is obtained (Anchor-CH3 in Figure 4), which can be converted into the corresponding brominated derivative Anchor-Br (that is to say a compound of formula (1) ready to be employed as tag for a new peptide synthesis) in a single synthetic step (refer to Example 13). Thereafter, the corresponding compound of formula (1) wherein X is NH2or OH can be obtained starting from the brominated derivative applying chemical transformation well known to the skilled in the art.

[0074] According to a particularly preferred aspect, the invention is related to the use of a compound in liquid phase peptide synthesis selected among the following compounds:

[0075] As already said, the compound of formula (8) is mentioned in prior art, for example in WO2019019483. In the prior art, however, compound of formula (8) is involved in the preparation of thermosetting resins or flame-retardant resins with low polarity, and according to the knowledge of the Applicant, said compound was never employed as protecting group for amino acids or in LPPS, nor in similar technical fields.

[0076] Another object of the present invention is therefore also a compound of formula (1):wherein n1, n2 and n3 are independently 0 or 1 and wherein X is OH, NH2or halogen, and wherein, when n2 is 1 and n1 and n3 are 0, X is not OH.

[0077] According to a preferred aspect, the invention is related to a compound of formula (1) wherein n1, n2 and n3 are 0, or wherein n1 and n3 are 0 and n2 is 1, or wherein n1 and n2 are 0 and n3 is 1, or wherein n1 and n2 are 1 and n3 is 0, or wherein n2 and n3 are 1 and n1 is 0, or wherein n1, n2 and n3 are1, except for compound of formula (8).wherein X is OH, NH2or halogen, with the exception of compound of formula (8):(8).

[0079] According to a more preferred aspect, the present invention is related to a compound selected among compound of formula (2), or compound of formula (3), or compound of formula (4), or compound of formula (5), wherein X is Br or NH2.

[0080] According to an even more preferred aspect, the present invention is related to a compound selected among compound of formula (2), or compound offormula (3), or compound of formula (4), or compound of formula (5), wherein X is Br or NH2.

[0081] According to a particularly preferred aspect, the invention is related to a compound selected among the following compounds:(12) (13) (36)

[0082] Compound of formula (1) or any one of its preferred aspects can be employed as protecting group of an amino acid or amino acid derivative, specifically for protecting its carboxylic functional group, as reported, for example, in the scheme below:Fmoc-Tyr(tBu)-OH Fmoc-Tyr(tBu)-Anchor

[0083] Object of the present invention is therefore also an amino acid or amino acid derivative protected with at least a compound of formula (1) or any one of its preferred aspects.

[0084] Object of the present invention is an amino acid or amino acid derivative protected with at least a compound of formula (1) or any one of its preferred aspects at its C-terminus or at a functional group found in the side chain.

[0085] According to a preferred aspect, the present invention is related to an amino acid or amino acid derivative having compound of formula (1) or any one of its preferred aspects as protecting group for the carboxylic functional group.

[0086] Object of the present invention is therefore also an amino acid or amino acid derivative protected with at least a compound of formula (1) or any one of its preferred aspects as protecting group.

[0087] The present invention also provides a process for the preparation of an amino acid or an amino acid derivative protected with at least a compound of formula (1) or any one of its preferred aspects.

[0088] Object of the present invention is also a method for protecting an amino acid or an amino acid derivative as depicted in Figure 2.

[0089] Specifically, another object is a method for protecting an amino acid or an amino acid derivative with a compound of formula (1) or any one of its preferred aspects, comprising the following steps: a) reacting the amino acid or amino acid derivative having a free carboxylic functional group, a protected N-terminus amino functional group and a side chain optionally protected at its functional groups, with a compound of formula (1) or any one of its preferred aspects, in the presence of: i) a base, when X is a halogen; or ii) a coupling agent and optionally an additive, when X is OH; or iii) a base, a coupling agent and / or an activating agent, when X is NH2; b) isolate the amino acid or amino acid derivative protected with compound of formula (1) obtained in step a).

[0090] According to a preferred aspect, the amino acid or amino acid derivative is protected with a Fmoc group at the N-terminus amino functional group.

[0091] According to another preferred aspect, the amino acid or amino acid derivative is protected with a Boc group at the N-terminus amino functional group.

[0092] According to a preferred aspect, in step a) case i) the base is potassium carbonate or sodium carbonate.

[0093] According to a more preferred aspect, in step a) case i) the base is potassium carbonate.

[0094] According to a particularly preferred aspect, step a) case i) is performed in a mixture of acetonitrile and DMF as solvent and using potassium carbonate as base.

[0095] According to another preferred aspect, in step a) case ii) the coupling agent is a carbodiimide selected from the group EDC, DCC, DIC, DSBC, DTBC or TBEC.

[0096] According to a more preferred aspect, in step a) case ii) the coupling agent is a carbodiimide selected from the group EDC, DCC, DIC, DSBC, DTBC or TBEC and the optional additive is DMAP.

[0097] According to an even more preferred aspect, in step a) case ii) the coupling agent is EDC.

[0098] According to a particularly preferred aspect, in step a) case ii) the additive is DMAP.

[0099] According to a particularly preferred aspect, step a) case ii) is performed in DCM as solvent using EDC as carbodiimide and DMAP as additive.

[0100] According to a preferred aspect, in step a) case iii) the base is DIPEA and the coupling agent and / or the activating agent is selected from the group EDC, DCC, HOBt, HOAt, HATU, HBTU, TBTU.

[0101] According to a more preferred aspect, step a) case iii) is performed using TBTU and DIPEA as base.

[0102] According to an even more preferred aspect, step a) case iii) is performed using TBTU and DIPEA as base in DCM as solvent.

[0103] By protecting the first amino acid or amino acid derivative with a compound of formula (1) when carrying out LPPS, said compound can act as an anchor in the following steps to produce the peptide, as exemplified in Figure 3.

[0104] Starting from the first amino acid (AA-i), having the N-terminus amino group functionalized with a protecting group such as Fmoc, for example, and wherein a compound of formula (1) or any one of its preferred aspects have been attached as an anchor / tag to the carboxylic functional group (step a1) in Figure 3), first step consists on a deprotection reaction to free the amine functional group of AA-, (step b1) in Figure 3). A purification step (step c1) in Figure 3) is then performed in order to remove excess reagents, by-products and impurities before proceeding with the coupling step. This purification can be achieved by extractive work up in a biphasic mixture comprising water and an organic solvent and / or by precipitation of the intermediate obtained in step b1) from the mixture. The second ammino acid (AA2), protected at its N-terminus amino function with a protecting group such as Fmoc, is then added to the mixture, and the coupling reaction under the typical peptide synthesis conditions, well known to the skilled in the art (step d1) in Figure 3) is carried out. These conditions comprise, for example, at least a coupling agent, such as TBTU, and at least a base, such as DIPEA.A purification step (step e1) in Figure 3) can be performed at this stage in order to remove excess reagents, by-products and impurities before proceeding with the following amino acid addition. This step e1) is optional, as in case the excess reagents and by-products are trapped by suitable scavengers, the synthesis could proceed without any further purification. Again, the purification could consist of an extractive work up and / or the precipitation of the obtained dipeptide from the organic solution, in order to discard impurities in the mother liquors. The product obtained is then ready for the following ammino acid addition, repeating the deprotection- purification-coupling cycle (step f1) in Figure 3).After the last amino acid has been added to the peptide chain, the anchor and eventually any other protecting group are cleaved, and the desired peptide can thus be obtained (step g1) in Figure 3).

[0105] The present invention is therefore also related to a process for performing solution-phase peptide synthesis comprising a compound of formula (1) or any one of its preferred aspects as C-terminus protecting group.

[0106] Object of the invention is thus a method for performing solution-phase peptide synthesis comprising a compound of formula (1) or any one of its preferred aspects as C-terminus protecting group, comprising the following steps: a1) protecting the carboxylic functional group of an amino acid or amino acid derivative with a compound of formula (1) or any one of its preferred aspects, according to the method mentioned above; b1) deprotecting the amino functional group of the protected amino acid or amino acid derivative obtained in step a1); c1) purifying the deprotected amino acid or amino acid derivative obtained in step b1); d1) reacting the deprotected amino acid or amino acid derivative obtained in step c1) with another amino acid or amino acid derivative protected at the amine function, to obtain a peptide or a peptide derivative; e1) optionally purifying the peptide or peptide derivative obtained in step d1); f1) repeating step b1), c1) d1) and e1) to add other amino acids or amino acid derivatives to the peptide chain, until the complete peptide chain is obtained; g1) isolating the protected peptide obtained in step f1); hi) removing the C-terminus protecting group and any other protecting groups from the chain to obtain the peptide.

[0107] According to a preferred aspect, the amino acids or amino acid derivatives employed in the above-mentioned method are protected at the N-terminus amino functional group with a Fmoc protecting group.

[0108] According to a preferred aspect, therefore, the amino function of the amino acids or amino acid derivatives that are added to the peptide chain is protected with a Fmoc group.

[0109] According to another preferred aspect, the amino acids or amino acid derivatives employed in the above-mentioned method are protected at the N-terminus amino functional group with a Boc protecting group.

[0110] According to a preferred aspect, therefore, the amino acid or amino acid derivative in step a1) and in step d1) of the above-mentioned method are protected at the amino functional group with a Fmoc or with a Boc protecting group.

[0111] According to another preferred aspect, the deprotection in step b1) is carried out with piperidine.

[0112] According to a more preferred aspect, the deprotection in step b1) is carried out in a solvent selected from the group consisting of ethyl acetate, isopropyl acetate, acetonitrile, toluene, anisole, DCM or CPME.

[0113] According to a particularly preferred aspect, the deprotection in step b1) is performed with piperidine in DCM as solvent.

[0114] According to a preferred aspect, the amino acid or amino acid derivative in step a1) and in step d1) are protected at the amino functional group with Fmoc protecting group and the deprotection in step b1) is performed with piperidine.

[0115] According to a particularly preferred aspect, the amino acid or amino acid derivative in step a1) and in step d1) are protected at the amino functional group with Fmoc protecting group and the deprotection in step b1) is performed with piperidine in DCM as solvent.

[0116] According to a preferred aspect, in step c1) the purification is performed by extractions of the mixture obtained in step b1) with aqueous acidic solutions.

[0117] According to a more preferred aspect, in step c1) the purification is performed by extractions of the mixture obtained in step b1) with aqueous ammonium chloride solutions.

[0118] According to a particularly preferred aspect, in step c1) the purification is performed by extractions of the mixture obtained in step b1) with 25% w / w aqueous ammonium chloride solutions.

[0119] According to a preferred aspect, the reaction in step d1) is carried out in a solvent selected from the group consisting of ethyl acetate, isopropyl acetate, acetonitrile, toluene, anisole, DCM or CPME.

[0120] According to another preferred aspect, the reaction in step d1) is carried out using TBTU as coupling agent and DIPEA as base.

[0121] According to an even more preferred aspect, the reaction in step d1) is carried out using TBTU as coupling agent and DIPEA as base in DCM as solvent.

[0122] According to a preferred aspect, in step e1) the purification consists of the peptide isolation as a solid.

[0123] According to a more preferred aspect, in step e1) the purification consists of peptide crystallization.

[0124] According to a preferred aspect, in step e1) and / or step g1) the purification and / or isolation is performed by dosing a solution of the peptide in a solvent selected from the group ethyl acetate, isopropyl acetate, acetonitrile, DCM or CPME into a solvent selected from the group n-hexane, n-heptane, diethyl ether, di-isopropyl ether, MTBE or MIBE, to obtain peptide precipitation.

[0125] According to a more preferred aspect, in step e1) and / or step g1) the purification and / or isolation is performed by dosing a solution of the peptide in a solvent selected from the group ethyl acetate, isopropyl acetate, acetonitrile, DCM or CPME into a solvent selected from the group n-hexane, n-heptane, MTBE or MIBE to obtain peptide precipitation, keeping the temperature below T=10°C.

[0126] According to an even more preferred aspect, in step e1) and / or step g1) the purification and / or isolation is performed by dosing a solution of the peptide in a solvent selected from the group ethyl acetate, isopropyl acetate, acetonitrile, DCM or CPME into a solvent selected from the group n-hexane, n-heptane, MTBE or MIBE to obtain peptide precipitation, keeping the temperature at T = 0°C.

[0127] According to a particularly preferred aspect, in step e1) and / or step g1) the purification and / or isolation is performed by dosing a solution of the peptide in DCM into a solvent selected from the group n-hexane, n-heptane, MTBE or MIBE to obtain peptide precipitation.

[0128] According to an even more preferred aspect, in step e1) and / or step g1) the purification and / or isolation is performed by dosing a solution of the peptide in a solvent selected from the group ethyl acetate, isopropyl acetate, acetonitrile, DCM or CPME into n-heptane to obtain peptide precipitation.

[0129] According to an even more preferred aspect, in step e1) and / or step g1) the purification and / or isolation is performed by dosing a solution of the peptide in DCM into n-heptane, keeping the mixture at T=0°C to obtain peptide precipitation.

[0130] Another important achievement allowed by the use of compound of formula (1) in LPPS is the fact that compound of formula (1) can be recovered and recycled after the final anchor cleavage step by simple organic chemistry reactions, as exemplified in Figure 4.

[0131] Cleavage of compound of formula (1) or any one of its preferred aspects as protecting group can be realized, as exemplified in Figure 4 and described in Example 13, by catalytic hydrogenation. The result of the cleavage is a methyl-derivative (Anchor-CH3in Figure 4), which is a precursor of compound of formula (1) or any one of its preferred aspects.

[0132] Starting from said methylated -derivative, the brominated compound of formula (1) (Anchor-Hal), that is compound of formula (1) wherein X = Br, can easily be obtained by applying well known organic chemistry techniques, as described, for example, in Example 13.

[0133] Brominate derivatives of compound of formula (1) can in turn be converted into hydroxyl (Anchor-OH in Figure 4) or amine (Anchor-NH2 in Figure 4) derivatives, following again chemical transformations well known to the skilled in the art.

[0134] According to a preferred aspect, therefore, the present invention is also related to a process for performing solution-phase peptide synthesis comprising a compound of formula (1) or any one of its preferred aspects as C-terminus protecting group, comprising the steps from a1) to hi) as described above, further comprising step J1) of regeneration of the C- terminus protecting group obtained after deprotection in step hi) into a compound of formula (1) or any one of its preferred aspects to be employed in step a1).

[0135] According to another preferred aspect, the present invention is also related to a peptide or a peptide derivative comprising at least a compound of formula (1) or any one of its preferred aspects as protecting group.

[0136] According to another aspect, the present invention is related to a kit for peptide synthesis comprising at least a compound of formula (1) or any one of its preferred aspects and at least a peptide synthesis coupling agent.

[0137] According to a preferred aspect, the present invention is related to a kit for peptide synthesis comprising at least a compound of formula (1) or any one of its preferred aspects, a carbodiimide and DMAP.

[0138] According to another preferred aspect, the present invention is related to a kit for peptide synthesis comprising at least a compound of formula (1) or any one of its preferred aspects and potassium carbonate.

[0139] According to another preferred aspect, the present invention is related to a kit for peptide synthesis comprising at least a compound of formula (1) or any one of its preferred aspects and TBTU.

[0140] The present invention also provides tags / anchors to be used in LPPS, characterized by a significant flexibility in solubility and / or crystalline properties provided to the growing peptide.

[0141] Specifically, the present invention is related to tags / anchors to be used in LPPS, wherein the solubility and / or crystalline properties of said tags / anchors can be modulated by selecting a specific compound within a class of compounds.

[0142] The present invention is therefore also related to a screening kit for peptide synthesis comprising at least two or a plurality of compounds of formula (1) or any one of its preferred aspects.

[0143] The screening kit mentioned above allows, for example, to select the most suitable compound among those of general formula (1), which maximizes the solubility of the peptide to be prepared in a given solvent, so to reduce the amount of solvent needed to maintain the peptide in solution, and thus improving process productivity.

[0144] Said screening kit also allows to select the most suitable compound among those of general formula (1), which makes it easier to isolate the peptide by crystallization, thus simplifying and optimizing the product purification.

[0145] Moreover, the screening kit provides a number of modular compounds that can be tested for a specific peptide, allowing to select the best solution for a given synthetic strategy (i.e. isolation by crystallization of the growingpeptide only at the end of the synthesis or after each amino acid added to the growing chain).

[0146] Another object is also the use of compound of formula (1) or any one of its preferred aspects for the preparation of the pentapeptide Thymopentin, having formula H-Arg-Lys-Asp-Val-Tyr-OH.

[0147] Another object is therefore the use of compound of formula (1) or any one of its preferred aspects for the preparation of the pentapeptide Thymopentin, having formula H-Arg-Lys-Asp-Val-Tyr-OH, in liquid phase peptide synthesis.

[0148] According to a preferred aspect, the present invention is related to the use of compound of formula (8) for the preparation of the pentapeptide Thymopentin, having formula H-Arg-Lys-Asp-Val-Tyr-OH.

[0149] Thus, according to a preferred aspect, is preferred the use wherein compound of formula (1) has the formula of compound (8) for the preparation of the pentapeptide Thymopentin, having formula H-Arg-Lys- Asp-Val-Tyr-OH.

[0150] According to a particularly preferred aspect, the present invention is related to the use of compound of formula (8) for the preparation of the pentapeptide Thymopentin, having formula H-Arg-Lys-Asp-Val-Tyr-OH, in liquid phase peptide synthesis.

[0151] All the features and preferred aspects of the process of the present invention given above can be combined in each possible combination to carry out the claimed process.Experimental Section

[0152] Glossary:

[0153] Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Other substituents also found within the molecule are called "side chain".

[0154] In biochemistry, non-coded or non-proteinogenic amino acids are distinct from the 22 proteinogenic amino acids, which are naturally encoded in the genome of organisms for the assembly of proteins. However, over 140 non- proteinogenic amino acids occur naturally in proteins and many more mayoccur in nature or be synthesized in laboratory. By ammino acid derivative it is therefore intended in the present document a natural or chemically synthesized amino acid which is not comprised in the proteinogenic amino acid group.

[0155] Protecting group or protective group (PG) is a reversibly formed derivative of an existing functional group in a molecule; it is introduced into a molecule by chemical modification of a functional group to obtain chemo-selectivity in a subsequent chemical reaction.

[0156] Within a peptide, the amine group of an amino acid is bonded to the carboxylic group of another amino acid, making it a chain. That leaves a free carboxylic group at one end of the peptide, called the C-terminus, and a free amine group on the other end, called the N-terminus.

[0157] As intended herein, by amino acid or amino acid derivative protected at the N-terminus amino function it is intended an amino acid or amino acid derivative wherein a protecting group has been introduced at the amino group at one end of the amino acid.

[0158] As intended herein, by amino acid or amino acid derivative protected at the side chain it is intended an amino acid or amino acid derivative wherein at least a protecting group has been introduced at one functional group found in the side chain.

[0159] In this document, the peptide notation commonly employed in the state of the art is used. The sequence of a peptide is therefore notated as a string of letters, according to the order of the amino acids from the N-terminus to the C-terminus of the peptide. Thus, for example, H-Ala-Tyr-OH represent a dipeptide consisting of the amino acid Alanine linked at its carboxylic function to the amino function of the amino acid Tyrosine.

[0160] The three-letter code notation used in the state of the art to identify the proteinogenic amino acids was adopted also in this document to represent each amino acid in the sequence. Thus, for example, H-Ala-OH represent the amino acid Alanine, and Fmoc-Tyr(tBu)-OH represent the amino acid Tyrosine which is protected at its amino function with the Fmoc group and at the phenol functional group of its side chain with a terbutyl group.

[0161] As intended in this document, a protecting group is for example: Fmoc = Fluorenylmethyloxycarbonyl; Boc = tert-Butoxycarbonyl; tBu = terbutyl; Ac = Acetyl; Bz = Benzoyl; Piv = Pivaloyl; terbutyl; dimethylacetal; TMS = trimethylsilyl; TBDMS = tert-Butyldimethylsilyl; Bn = Benzyl; Trt = Trityl; Mtt = 4-Methyltrityl; DMT = Dimethoxytrityl; PMB = p- Methoxybenzyl ether; MEM = Methoxyethoxymethyl ether; Cbz = Carbobenzyloxy; Ts = Tosyl; Alloc = N-Allyloxycarbonyl; Pbf = 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl.

[0162] For tag or anchor in this document it is intended a molecule which is grafted to a first amino acid or amino acid derivative and remains attached to that amino acid all along the synthesis of the peptide, and which is finally removed only after completion of the elongation of the peptide chain.

[0163] SPPS = solid-phase peptide synthesis is a process in which a peptide anchored at its C-terminus to an insoluble polymer is assembled.

[0164] LPPS = liquid phase peptide synthesis is a process for peptide synthesis where the peptide elongation is carried out in solution and the growing peptide chain is supported on a soluble tag, which confers characteristic properties.

[0165] Peptide coupling reagents or activating reagents are compounds used to facilitate the formation of peptide bonds between amino acids, enabling the synthesis of peptides.

[0166] As intended in this document, coupling reagents or activating reagents are for example EDC = 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; DCC = dicyclo hexylcarbodiimide; DIC = diisopropyl carbodiimide; DSBC= N,N'- disec butylcarbodiimide; DTBC = N,N' -ditertbutyl carbodiimide TBEC = 1- tert-Butyl-3-ethylcarbodiimide; HOBt = 1-hydroxy-benzotriazole; HOAt = 1- hydroxy-7-aza-benzotriazole; HBTU = hexafluorophosphatebenzo triazole tetramethyl uranium; HATU = hexafluoro phosphate aza benzotriazole tetramethyl uranium; HBTU = 2-(1H-Benzotriazole-1-yl)-1, 1,3,3- tetramethyluronium hexafluoro phosphate; TBTU = 2-(1H-Benzotriazole-1- yl)-1,1,3,3-tetramethyl aminiumtetrafluoroborate, BOP = Benzotriazole-1-yl- oxy-tris-(dimethyl amino)-phosphonium hexafluorophosphate; COMU = 1- [(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholino)] uranium hexafluorophosphate; PyClock = 6- Chloro-benzotriazole-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate; PyAOP = (7-Azabenzo triazol-1- yloxy)trispyrrolidinophosphonium hexafluorophosphate; TSTU = N,N,N,N- Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate.

[0167] The following abbreviation were also used in the present document: TFA = trifluoroacetic acid; TEA = triethylamine; DIPEA = N,N-diisopropyl ethylamine; DMAP = 4-dimethylaminopyridine; DCM = dichloromethane; 1,2-DME = 1,2-dimethoxyethane; DMF = dimethylformamide; EtOAc = ethyl acetate; IPAC= isopropyl acetate; MeOH = methanol; EtOH = ethanol; MTBE = Methyl tert-butyl ether; MIBE = Methyl iso-butyl ether; CPME = Cyclopentyl methyl ether; THF = tetrahydrofuran; ACN = acetonitrile; Pd / C 10% = Palladium 10% w / w on carbon powder.

[0168] As intended in this document, work-up refers to the series of manipulations required to isolate and purify the product(s) of a chemical reaction. The term is used colloquially to refer to these manipulations, which may include deactivating any unreacted reagents by quenching a reaction, changing the protonation state of the products or impurities by adding an acid or base, separating the reaction mixture into organic and aqueous layers by liquidliquid extraction or removal of solvents by evaporation. The work-up steps required for a given chemical reaction may require one or more of the above-mentioned manipulations. Written experimental procedures will describe work-up steps but will usually not formally refer to them as a workup.

[0169] All the raw materials reported in the experimental section are commercially available, for example by Merck, Apollo Scientific, Iris Biotech GmbH or abcr Gmbh.

[0170] Room temperature (RT) means a temperature that is comprised in a range from 20 to 25°C, it is defined as comfortable temperature range indoors.

[0171] As intended herein, drying the wet material under vacuum means to heat the material to a given temperature for a given time while applying a reduced pressure. This operation is performed to remove residual solvents from the product.

[0172] As intended herein, to concentrate a mixture under vacuum means to heat the material to a given temperature for a given time while applying a reduced pressure. This operation is performed to remove solvents from the mixture.

[0173] The term "volume" (V) means volume of solvent per unit of product, thus, for example, 1 volume is 1 Liter per 1 Kilo, or 1 mL per 1 gram, or 1 microliter per 1 milligram. Thus, 10 volumes mean for example 10 Liters per 1 Kilogram of substance.

[0174] As intended in this document, purification refers to the combination of processes used to isolate and refine components of a mixture. Purification can be obtained for example by selective extraction in an organic solvent under proper conditions, by separation based on equilibria, or by precipitation, crystallization, or re-slurry.

[0175] By precipitation or crystallization, it is intended the process of transforming a dissolved substance into an insoluble solid which remains suspended in the mixture. For promoting the precipitation or crystallization of a compound various methods are available, for example concentrating the solution until compound saturation is realized, cooling down said solution to lower compound solubility, adding a solvent in which the compound is poorly soluble or insoluble or seeding said solution with a small amount of the compound (seed). The solid obtained is then usually isolated by filtration or centrifugation.

[0176] By re-slurry it is intended that a compound is suspended in a solvent or in mixture of solvents. The solid obtained is then usually isolated by filtration or centrifugation.

[0177] Molar equivalent or equivalent (eq) means that the molar amount of a substance reacts with a molar amount of another substance in a given chemical reaction

[0178] The expression "w / w" stands for weight / weight, representing the weight of a solute in relation to the total weight of the solution.

[0179] Molar concentration or molarity is a measure of the concentration of a chemical species of a solute in a solution, in terms of moles of substance per unit volume of solution. In chemistry, the most commonly used unit for molarity is the number of moles per Liter, having the unit symbol mol / L. Asolution with a concentration of 1 mol / L is said to be 1 molar, commonly designated as 1 M.

[0180] Example 1: Preparation of compound of formula (7).

[0181] STEP 1 - preparation of compound of formula (15).In a round bottom flask equipped with a half-moon stirrer, butyllithium (32.3 g of a 2.7 M solution in heptane) was slowly added at T= -78°C to a precooled solution of 4-Bromotoluene (20 g) in anhydrous THE (80 mL). After stirring at this temperature for 30 minutes, zinc chloride (245.6 mL of a 0.5 M solution in THE) was added, and the mixture was kept stirring at T = -78°C for further 2 hours. Phosphorus trichloride (16.9 g) was finally added at T = - 78°C and the mixture was allowed to reach room temperature. After 1 hour stirring, the mixture was distilled to residue under reduced pressure and then diluted with THE (80 mL).

[0182] STEP 2- preparation of compound of formula (16).In a round bottom flask equipped with a half-moon stirrer, 2,2'- dibromobiphenyl (25.54 g) was dissolved in anhydrous THE (120 mL). The solution was cooled to T = -78°C and then butyllithium (47.4 mL of a 2.7 M solution in heptane) was slowly added, keeping T = -78°C. After 30 minutes stirring at this temperature, the solution obtained in Step 1 was slowly added to the mixture at T = -78°C. The mixture was allowed to react at T = -78°Cfor 1 hour and then warmed to room temperature. After reaction completion, the reaction was quenched with water (200 mL). The resulting biphasic mixture was distilled at reduced pressure to remove most of THF, and then diluted with DCM. The phases were separated, and the aqueous phase was extracted two times with DCM (2 x 200 mL). The combined organic layers were finally washed with water (200 mL), and the resulting organic phase was distilled at reduced pressure to residue.

[0183] STEP 3- preparation of compound of formula (17).In a round bottom flask equipped with a half-moon stirrer, the residue obtained in Step 2 (30 g) was diluted with DCM (126 mL). After cooling the resulting solution to T = 0°C, hydrogen peroxide (37.2 g) was added. The mixture was allowed to reach room temperature and kept reacting for 1 hour. After quenching by addition of a sodium bisulfite solution in water (34.1 g of sodium bisulfite in 45 mL of water), the phases were separated, washing the organic layer with an aqueous solution of sodium bisulfite (11.4 g of sodium bisulfite in 69 mL of water) and again with water (90 mL). The combined aqueous phases were extracted with DCM (90 mL). After phase separation, the organic layer was distilled under vacuum to residue. Dilution of the residue n-heptane allowed to obtain a solid, which was isolated by filtration.

[0184] STEP 4- preparation of compound of formula (7).In a round bottom flask equipped with a half-moon stirrer and masked from the light, compound of formula (17) obtained in Step 3 (5 g), benzoyl peroxide (0.33 g), N-Bromo succinimide (4.6 g) and 50 mL of degassed acetonitrile were charged. The mixture was heated to reflux and stirred at this temperature for 1.5 h. After reaction completion, the mixture was cooled down to room temperature and diluted with a 5% w / w aqueous solution of sodium hydrogen carbonate (250 mL). The phases were separated, and the aqueous phase was extracted with MTBE (250 mL). The combined organic layers were then washed with a 10% w / w aqueous solution of sodium sulfite (250 mL) and finally with brine (250 mL). The resulting solution was concentrated to 3 residual volumes and diluted with acetonitrile (450 mL); this operation was repeated until complete removal of MTBE and until reaching a water content lower than KF = 0.5%. Thereafter, the mixture wasdiluted with acetonitrile (350 mL) and cooled down to T = 0°C. DIPEA (1.56 g) and diethyl phosphite (1.67 g) were added, and the mixture was then warmed to room temperature. After addition of a citric acid solution (5.0 g of citric acid dissolved in 50 mL of water) and MTBE (50 mL), the phases were separated. The organic layer was washed 3 times with a sodium carbonate solution (1.25 g of sodium carbonate dissolved in 25 mL of water). The combined aqueous layers were extracted with MTBE (50 mL). The combined organic layers were further washed with water (25 mL). After phase separation, the organic layer was anhydrified by azeotropic distillation with IPAC until reaching a water content lower than KF = 0.5%. The solution was finally concentrated under vacuum to 2 residual volumes, clarified by filtration and then diluted with MTBE (4 volumes). The mixture was kept stirring for 3 hours at room temperature, then the slurry was filtered washing the wet cake with MTBE (1.5 volumes). The solid was dried under vacuum at 45°C for 12 hours. 15.9 g of compound of formula (7) were obtained.1H NMR (400 MHz, CDCI3): 7.85-7.82 (dd, 2H), 7.74-7.70 ppm (t, 2H), 7.64-7.59 ppm (m, 4H), 7.41-7.39 ppm (m, 4H), 4.45 ppm (s, 2H).31P NMR (162 MHz, CDCI3): 33.11 ppm.

[0185] Example 2: Preparation of compound of formula (6).In a vial equipped with a magnetic stirrer compound of formula (7) (1.65 g) and potassium acetate (0.50 g) were dissolved in DMF (2 mL). After 2 hours stirring at room temperature, water (20 mL) was added to the mixture. After phase separation, the aqueous phase was extracted with ethyl acetate (1 x 30 mL and then 2 x 20 mL). The combined organic layers were washed with a 15% w / w aqueous solution of sodium sulfate (20 mL). After anhydrification on sodium sulfate, the organic solution was concentrated under vacuum to residue. The resulting oil was diluted with water (5 mL) and THF (6.6 mL) and potassium hydroxide (0.35 g) was added. The mixture was allowed to reactuntil complete conversion of the starting material (additional 0.23 g of potassium hydroxide were added to push the conversion after 22 hours, total reaction time 23 hours). After distillation under reduced pressure to remove THF, the mixture was diluted with water (35 mL), a 10% w / w aqueous solution of ammonium chloride (12 mL) and DCM (30 mL). The phases were separated, and the aqueous phase was extracted twice with DCM (2 x 30 mL). The combined organic layers were anhydrified on sodium sulfate, and then distilled to residue under reduced pressure. After chromatographic purification on silica gel (EtOAdOO % to EtOAc / MeOH 95:5, Rf:0.25), compound (6) was obtained as a yellow powder (1.32 g).1H NMR (400 MHz, acetone-d6): 8.10-8.00 ppm (dd, 2H), 7.73-7.67 ppm (m, 4H), 7.60-7.54 ppm (m, 2H), 7.52-7.43 ppm (m, 2H), 4.7 ppm (d, 2H).31P NMR (162 MHz, acetone d-6): 30.5 ppm.

[0186] Example 3: Preparation of compound of formula (8).(21 ) (22) (8)In a 2L reactor equipped with a half-moon stirrer, compound of formula (21) (100 g), [1,1'-Bis (diphenylphosphino)ferrocene]dichloro palladium (II) (Pd(dppf)CI2, 6.8 g), DMF (1660 mL), 1,2-DME (186 ml) and DIPEA (105 ml) were charged. The mixture was vigorously stirred at T = 20°C under nitrogen atmosphere for 10 minutes. Then, 4-bromo benzylalcohol (86.6 g) was added. The mixture was heated to T = 115°C and stirred under nitrogen atmosphere for 10 hours. After cooling down to T = 20°C, the mixture was treated with activated charcoal (10 g) and filtered on a celite pad. The resulting solution was distilled under reduced pressure to residual 3.5 volumes (350 mL). This solution was slowly dosed under vigorous stirring at to a biphasic mixture comprising MTBE (700 mL) and an aqueous 1 M solution of hydrochloric acid (700 mL). The resulting slurry was filtered washing the cake with water (300 mL) and MTBE (300 mL). The solid was re-slurried in MTBE (300 mL) and then isolated by filtration. 125 g of compound of formula (8) were obtained. ESI-MS [M + H]+=323; calculated [M + H]+= 323.0831 Da.1H NMR (400 MHz, CDCI3) 5 8.15 - 7.95 (m, 2H), 7.81 (dd, J = 13.1, 8.2 Hz, 2H), 7.71 (ddt, J = 8.3, 7.3, 1.3 Hz, 1H), 7.61 (ddd, J = 13.9, 7.7, 1.4 Hz, 1H), 7.53 - 7.35 (m, 4H), 7.35 - 7.22 (m, 2H), 4.78 (d, J = 5.5 Hz, 2H), 2.22 (t, J = 5.9 Hz, 1H).13C NMR (101 MHz, DMSO) 5 148.61 (d, J = 8.1 Hz, 1C), 148.46 (d, J = 2.9 Hz, 1C), 134.87 (d, J = 5.5 Hz, 1C), 133.43 (d, J = 2.6 Hz, 1C), 131.52 (d, J = 11.5 Hz, 2C) , 130.89 (s, 1C), 130.37 (d, J = 12.1 Hz, 1C), 128.79 (d, J = 13.6 Hz, 1C), 127.21 (d, J = 143.5 Hz, 1C), 126.53 (d, J = 14.3 Hz, 2C), 125.71 (s, 1C), 124.95 (d, J = 127.4 Hz, 1C), 124.89 (s, 1C), 124.28 (d, J = 7.9 Hz, 1C), 121.73 (d, J = 11.0 Hz, 1C), 120.23 (d, J = 5.5 Hz, 1C), 64.67 (s, 1C).31P NMR (162 MHz, DMSO) 5 23.96.

[0187] Example 4: Preparation of compound of formula(8)

[0188] To a dry and degassed round bottom flask triphenylphosphine (13 g) was dissolved in DCM (200 mL). The mixture was cooled to 0°C and NBS (8,8 g) was added in portions. A solution of compound of formula (8) (10.0 g) in DCM (500 mL) was prepared and then dosed over the previously prepared mixture. The resulting mixture was then heated to 40°C and stirred at this temperature for 1 hour. After cooling down to room temperature, water was added (500 mL) and the phases were separated. The organic layer was dried over sodium sulphate and then concentrated to residue under reduced pressure yielding 7.0 g of compound of formula (9). ESI-MS [M + H]+= 385 (79Br isotope); 387 (81Br isotope); [M + Na]+= 407 (79Br isotope); 410 (81Br isotope); calculated [M + H]+= 383.9914 Da(79Br isotope).1H NMR (400MHz, DMSO-d6, ppm), 5: 8.25 (dd, J = 8.2 Hz, 4.9 Hz, 1H, aromatic ring CH), 8.25 (dd, J = 8.0 Hz, 1.6 Hz, 1H, aromatic ring CH), 7.85-7.78 (m, 1H, aromatic ring CH), 7.73 (d, J = 8.2 Hz, 1H, aromatic ring CH), 7.70 (d, J = 8.2 Hz, 1H, aromatic ring CH), 7.65-7.59 (m, 2H, aromatic ring CH), 7.58-7.52 (m, 2H, aromatic ring CH), 7,48 (t, J = 7.6 Hz, 1H, aromatic ring CH), 7.39-7.30 (m, 2H, aromatic ringCH), 4.74 (s, 2H, CH2Br).13C NMR (100MHz, DMSO-d6, ppm), 5: 148.5 (d, J = 9.0 Hz, 1C, aromatic ring), 143.4 (d, J = 3.0 Hz, 1C, aromatic ring), 134.9 (d, J = 6.0 Hz, 1C, aromatic ring), 133.6, (d, J = 2.0 Hz, 1C, aromatic ring), 132.0 (d, J = 11.0 Hz, 1C, aromatic ring), 131.0 (s, 1C, aromatic ring), 130.5 (d, J = 12.0 Hz, 1C, aromatic ring), 129.8 (s, 1C, aromatic ring), 129.7 (d, J = 14.0 Hz, 1C, aromatic ring), 128.9 (d, J = 14.0 Hz, 1C, aromatic ring), 128.4 (s, 1C, aromatic ring), 125.7 (s, 1C, aromatic ring), 125.1, 125.0 (s, 1C, aromatic ring), 124.3 (d, J = 9.0 Hz, 1C, aromatic ring), 123.8 (s, 1C, aromatic ring), 121.6 (d, J = 11.0 Hz, 1C, aromatic ring), 120.2 (d, J = 6.0 Hz, 1C, aromatic ring), 32.9 (d, J = 0.9 Hz, 1C, CH2).31P NMR (162 MHz, DMSO-d6, ppm), 5: 23.3.

[0189] Example 5: Preparation of compound of formula (34).

[0190] STEP 1- preparation of compound of formula (40).In a round bottom flask equipped with a half-moon stirrer, were charged compound of formula (9) (2.0 g) and DMF (10 mL). To the resulting solution potassium phthalimide (compound of formula (39), 1.48 g) was added, and the mixture was heated to T=100°C for 14 hours. After reaction completion the mixture was cooled down to room temperature and diluted with DCM (50 mL) and water (50 mL). The phases were separated, and the organic layer was concentrated to residue under vacuum. Compound of formula (40) thus obtained (2.10 g) was employed directly in the following step.

[0191] STEP 2- preparation of compound of formula (34).The distillation residue obtained in Step 1 was diluted with hydrazine hydrate (0.90 g), and the mixture was allowed to react until complete conversion. After dilution with water (50 mL) and DCM (50 mL), the phases were separated. The aqueous phase was extracted with DCM (50 ml_), then the combined organic layers were anhydrified over sodium sulfate. Compound of formula (34) was isolated by concentration of the filtrate solution to small volume under vacuum (0.4 g isolated yield). ESI-MS [M + H]+=322; calculated [M + H]+= 322.0991 Da.1H NMR (400 MHz, CDCI3): 5 8.08 - 7.99 (m, 2H), 7.78 (dd, J = 13.0, 7.9 Hz, 2H), 7.73 - 7.55 (m, 2H), 7.52 - 7.36 (m, 4H), 7.28 (dtd, J = 9.5, 7.9, 1.4 Hz, 2H), 3.94 (s, 2H), 2.35 (s, 2H). In the spectrum, the two singlets found at around 5 3 ppm do not belong to compound of formula (34) but corresponds to the residual solvent DMF, found in the sample analyzed.

[0192] Example 6: Preparation of compound of formula (10).STEP 1 STEP 2

[0193] STEP 1: preparation of compound of formula (44).In a round bottom flask equipped with a half-moon stirrer, compound of formula (21) (5.0 g) was dissolved in degassed toluene (50.0 mL). N- chlorosuccinimide (NCS, 3,40 g) was added, and the mixture was stirred at room temperature for 2.5 hours. After filtration to remove the insoluble, the resulting solution was concentrated to residue under reduced pressure yielding compound of formula (44) (6.2 g), which was employed in the following step without purification.

[0194] STEP 2: preparation of compound of formula (10).In a round bottom flask equipped with a half-moon stirrer, compound of formula (45) (500 mg) was dissolved in THE (5.0 mL). After addition of sodiumhydroxide (NaOH, 160 mg), the mixture was heated to 40°C for 30 minutes, observing the precipitation of a red solid. Compound of formula (44) obtained in Step 1 (1.0 g) was dissolved in DCM (5.0 mL), and the solution was dosed over the suspension previously prepared. After 5 hours stirring at room temperature, the mixture was filtered to remove the insoluble. The organic solution was then concentrated to residue by distillation at reduced pressure to obtain compound of formula (10) (1.2 g). ESI-MS [M + H]+= 339.3; calculated [M + H]+= 339.0781 Da.1H NMR (400 MHz, DMSO-d6, ppm), 5: 8.01 (m, 3H, aromatic ring CH), 7.78 (t, J = 8.0 Hz, 1H, aromatic ring CH), 7.55 (m, 1H, aromatic ring CH), 7.43 (t, J = 8.2 , 1H , aromatic ring CH), 7.33 (t, J = 8.2 Hz, 1H, aromatic ring CH), 7.28 (m, 3H, aromatic ring CH), 7.06 (dd, J = 8.0 Hz , J = 3.0 Hz, 2H, aromatic ring CH), 4.65 (s, 2H, CH2OH), 1.28 (s, 1H, OH).31P NMR (162 MHz, DMSO-d6, ppm), 5: 6.51.

[0195] Example 7: Preparation of compound of formula (13).

[0196] STEP 1- preparation of compound of formula (19).In a round bottom flask equipped with an anchor stirrer, 2,2'-biphenol (20.0 g, 0.107 mol, 1.0 eq) was dissolved in 2.5 vol of dioxane (50 mL, 4.0 vol). Water (1.93 g, 0.107 mol, 1.0 eq) was added. The reaction mixture was heated to reflux. Phosphorus trichloride (14.7 g, 0.107 mol, 1.0 eq) was added dropwise over 4 hours keeping the same temperature. Basic scrubber (diluted aqueous KOH) was installed at the end of the apparatus to quench any HCI gasdeveloped during the reaction. After the end of phosphorus trichloride dosing, the solvent was evaporated under vacuum to yield compound of formula (19) as a pale brown oil.

[0197] STEP 2- preparation of compound of formula (20).In a round bottom flask equipped with anchor stirrer, 1,3,5-trichloro-1,3,5- triazinane-2, 4, 6-trione (9.3 g, 0.04 mol, 0.4 eq), was dissolved in 7 vol (175 mL) of acetonitrile. Compound of formula (19) obtained in Step 1 (23.0 g, 0.10 mol, 1 eq) was dissolved in 2 vol (46 mL) of acetonitrile and slowly added at T = 0 - 5 ° C. The reaction mixture was stirred at room temperature for 1 hour. During the activation the reaction mixture turned to a heavy suspension. Once the starting material was totally consumed, the reaction mixture was cooled to T = 0 - 5 ° C. A preformed solution of triethylamine (15.1 g, 21 mL, 0,15 mol, 1.5 eq) and para-cresol (10.7 g, 0,10 mol, 1.0 eq) was added at T = 0 - 5 ° C in 30 min. The reaction mixture was allowed to reach room temperature and was stirred for 2 hours. Once complete conversion was reached, the reaction mixture was cooled to T = 0 - 5 ° C and citric acid 10 %w / w (5 vol) aqueous solution was added, the reaction mixture was allowed to reach room temperature, and phases were separated. Aqueous phase was back extracted with 5 vol of MTBE. Enriched organic phases were washed with 5 vol of citric acid 10 % w / w aqueous solution to remove residual triethylamine, followed by additional wash with sodium hydrogen carbonate 6% w / w aqueous solution (5 vol) to pH = 8. A final wash with brine (4 vol) was carried out, followed by drying over sodium sulphate. The inorganic salts were filtered off and washed with 1 vol MTBE. The solvent was evaporated under high vacuum to yield compound of formula (20) as a deep orange oil.

[0198] STEP 3- preparation of compound of formula (13).In a round bottom flask equipped with anchor stirrer, compound of formula (20) obtained in Step 2 (27.0 g, 0.08 mol, 1.0 eq) was dissolved in 10 vol (270 mL) of deoxygenated acetonitrile. Azobisisobutirronitrile (AIBN, 1.31 g, 0.008 mol, 0.1 eq) and N-Bromo succinimide (NBS, 21.31 g, 0.12 mol, 1.5 eq) were added at room temperature and the reaction mixture was heated to reflux. Additional 0.8 eq of NBS were added 3 times to push the reaction to completion. Thereafter, the reaction mixture was cooled to roomtemperature and sodium sulphite 10 % w / w (10 vol) was added. MTBE (5 vol) was added to improve phase separation. Aqueous layer was back extracted with 5 vol MTBE. Enriched organic phases were washed with citric acid 10 % w / w (5 vol) for two times to pH 3. The solution was then washed twice with potassium carbonate 10 % w / w to remove succinimide and then with brine (5 vol). After phase separation, the organic phase was dried over sodium sulphate, salts were filtered off and washed with 1 vol of MTBE. After concentration to residue, the crude compound of formula (13) was isolated as an oil (21 g) with 97.13% A / A purity by HPLC. If needed, purification by chromatography or crystallization from MTBE can be carried out to improve product purity.1H NMR (400 MHz, CD3CN): 7.72-7.70 ppm (dd, 2H), 7.59- 7.55 ppm (m, 2H), 7.53-7.48 (m, 4H), 7.41-7.38 (m, 2H), 7.34-7.31 ppm (m, 2H).31P NMR (162 MHz, CD3CN): -4.3 ppm.

[0199] Example 8: Preparation of compound of formula (24).

[0200] In a round bottom flask equipped with a magnetic stirrer, compound of formula (23) (Fmoc-Tyr(tBu)-OH, 6.50 g) was dissolved in DMF (32.5 mL). A suspension of compound of formula (7) (5.98 g) in acetonitrile (32.5 mL) was then added. After charging potassium carbonate (2.93 g), the mixture was diluted with DMF (13 mL) and acetonitrile (13 mL) and allowed to react at room temperature overnight. Thereafter, ethyl acetate (163 mL) and a 10% w / w aqueous solution of citric acid (98 mL) were added. The phases were separated, and the organic layer was washed with a 20% w / w aqueous sodium chloride solution twice (2 x 98 mL). The combined aqueous phases were back extracted with ethyl acetate (30 mL). The combined organic layers were then concentrated to residue under reduced pressure yielding 12.6 g ofcompound of formula (24). HPLC purity: 92.84% A / A.1H NMR (400 MHz, DMSO-d6, ppm), 5: 8.13 (dd, J = 8.0, 2.8 Hz, 2H, aromatic ring CH), 7.91 (d, J = 8.1 Hz, 1H, NH), 7.86 (d, J = 7.6 Hz, 2H, aromatic ring CH), 7.70 (t, J = 7.9 Hz, 4H, aromatic ring CH), 7.63 (dd, J = 7.6, 3.1 Hz, 2H, aromatic ring CH), 7.52-7.43 (m, 4H, aromatic ring CH), 7.42-7.32 (m, 4H, aromatic ring CH), 7.26 (q, J = 7.0 Hz, 2H, aromatic ring CH), 7.11 (d, J = 8.4 Hz, 2H, aromatic ring CH), 6.80 (d, J = 8.4 Hz, 2H, aromatic ring CH), 5.11 (dd, J = 13.2 Hz, 2H, O-CH2TAG), 4.32-4.25 (m, 1H, CH Tyr), 4.24-4.16 (m, 2H, O-CH2Fmoc), 4.15- 4.08 (m, 1H, CH Fmoc), 3.05-2.95 (m, 1H, CAH Tyr), 2.91-2.82 (m, 1H, CAH Tyr), 1.18 (s, 9H, C(CH3)3).13C NMR (100 MHz, DMSO-d6, ppm), 5: 171.7, 155.9,153.6, 150.8, 143.7, 141.2, 141.0, 140.7, 140.3, 133.7, 132.7, 131.9, 131.7, 130.7,130.6, 129.8, 129.7, 129.6, 129.5, 129.4, 128.9, 127.8, 127.7, 127.6, 127.0, 126.0, 125.2, 123.4, 122.1, 122.0, 121.4, 120.1, 77.6, 65.7, 65.3, 55.6, 46.6, 35.8, 28.5.31P NMR (162 MHz, DMSO-d6, ppm), 5: 31.2.

[0201] Example 9: Preparation of compound of formula (25).In a round bottom flask equipped with a half-moon stirrer, compound of formula (8) (6.0 g) and DCM (60 mL) were charged. A suspension of compound of formula (23) (Fmoc-Tyr(tBu)-OH, 8.55 g) in DCM (60 mL) was added and the mixture was cooled down to T=0°C. Keeping this temperature, EDC HCI (3.75 g) was added, followed by DMAP (0.23 g). The mixture was warmed to room temperature and allowed to react for 2 hours. Thereafter, a 20% w / w aqueous solution of ammonium chloride (120 mL) and a 20% w / w aqueous solution of brine (120 mL) were added. The phases were separated, and the organic layer was dried over magnesium sulfate. The resulting solution was concentrated to residue under reduced pressureyielding 15.8g of compound of formula (25). HPLC purity: 92.45% A / A.1H NMR (400 MHz, DMSO-d6, ppm), 5: 8.32-8.28 (m, 1H, aromatic ring CH), 8.25 (d, J = 8.1 Hz, 1H, aromatic ring CH), 7.94 (d, J = 7.9 Hz, 1H, NH), 7.86- 7.77 (m, 3H, aromatic ring CH), 7.69-7.60 (m, 4H, aromatic ring CH), 7.56- 7.50 (m, 2H, aromatic ring CH), 7.49-7.45 (m, 1H, aromatic ring CH), 7.44- 7.39 (m, 2H, aromatic ring CH), 7.38-7.31 (m, 4H, aromatic ring CH), 7.30-7.23 (m, 2H, aromatic ring CH), 7.13 (d, J = 8.6 Hz, 2H, aromatic ring CH), 6.81 (d, J = 6.8 Hz, 2H, aromatic ring CH), 5.17 (d, J = 6.5 Hz, 2H, O-CH2TAG), 4.35- 4.28 (m, 1H, CH Tyr), 4.25-4.17 (m, 2H, O-CH2Fmoc), 4.16-4.10 (m, 1H, CH Fmoc), 3.06-2.97 (m, 1H, CAH Tyr), 2.94-2.84 (m, 1H, CAH Tyr), 1.19 (s, 9H, C(CH3)3).13c NMR (100 MHz, DMSO-d6, ppm), 5: 171.7, 155.9, 153.6, 148.6, 148.5, 143.7, 143.6, 141.5, 140.7, 133.6, 131.8, 131.7, 131.0, 130.5, 130.4, 129.7, 128.9, 128.8, 127.6, 127.6, 127.4, 127.0, 125.8, 125.2, 125.0, 124.4, 124.3, 123.9, 123.4, 121.7, 121.6, 120.3, 120.2, 120.1, 77.6, 65.7, 65.2, 55.6, 46.5, 35.8, 28.5.31P NMR (162 MHz, DMSO-d6, ppm), 5: 23.3.

[0202] Example 10: Preparation of compound of formula (26).In a round bottom flask equipped with a magnetic stirrer, compound of formula (23) (Fmoc-Tyr(tBu)-OH, 2.0 g) was dissolved in DMF (8 mL). Potassium carbonate (0.9 g) was added, and the mixture was kept stirring at room temperature for 30 minutes. Thereafter, a solution of compound of formula (13) (1.91 g) in acetonitrile (10 mL) was added dropwise into the mixture. After stirring at room temperature overnight, additional 0.2g of compound of formula (13) were added to push conversion. Thereafter, ethyl acetate (100 mL) and a 10% w / w aqueous solution of citric acid (20 mL) were added. The phases were separated, and the organic layer was washed witha 20% w / w aqueous sodium chloride solution twice (2 x 20 mL). The combined organic layers were then concentrated to residue under reduced pressure yielding 3.8 g of compound of formula (26). HPLC purity: 91.08% A / A.1H NMR (400 MHz, DMSO-d6, ppm), 5: 7.99 (d, J = 8.1 Hz, 1H, NH), 7.87 (d, J = 7.6 Hz, 2H, aromatic ring CH), 7.74 (dd, J = 7.7, 1.8 Hz, 2H, aromatic ring CH), 7.66 (d, J = 7.5 Hz, 2H, aromatic ring CH), 7.58 (d, J = 7.4 Hz, 2H, aromatic ring CH), 7.51 (d, J = 7.4 Hz, 2H, aromatic ring CH), 7.45 (d, J = 8.0 Hz, 2H, aromatic ring CH), 7.44-7.36 (m, 4H, aromatic ring CH), 7.34-7.26 (m, 4H, aromatic ring CH), 7.15 (d, J = 8.2 Hz, 2H, aromatic ring CH), 6.83 (d, J = 8.4 Hz, 2H, aromatic ring CH), 5.12 (d, J = 6.8 Hz, 2H, O-CH2Fmoc), 4.35- 4.28 (m, 1H, CH Tyr), 4.27-4.19 (m, 2H, O-CH2Fmoc), 4.18-4.12 (m, 1H, CH Fmoc), 3.03 (dd, J = 13.8, 5.5 Hz, 1H, CAH Tyr), 2.90 (dd, J = 13.8, 9.8 Hz, 1H, CAH Tyr), 1.20 (s, 9H, C(CH3)3).13C NMR (100 MHz, DMSO-d6, ppm), 5: 171.7, 155.9, 153.6, 149.3, 149.2, 146.9, 146.8, 143.7, 143.7, 140.7, 133.8, 131.8, 130.8, 130.4, 129.8, 129.7, 127.6, 127.4, 127.3, 127.0, 125.2, 123.4, 121.3, 121.2, 120.1, 120.0, 120.0, 77.6, 65.7, 65.2, 55.7, 46.6, 35.9, 28.5.31P NMR (162 MHz, DMSO- d6, ppm), 5: -4.44.

[0203] Example 11: Preparation of compound of formula (43).In a round bottom flask equipped with a half-moon stirrer, compound of formula (34) (200 mg), compound of formula (23) (286 mg) and acetonitrile (4 mL) were charged. DIPEA was then added (322 mg) followed by TBTU (220 mg). The mixture was stirred at room temperature for 30 minutes, then distilled to residue under reduced pressure. The residue was diluted with EtOAc (4 mL) and an aqueous solution of ammonium chloride (400 mg of ammonium chloride in 1,6 mL of water). After phase separation, the organic layer was washed two more times with ammonium chloride solution (2 x 400mg of ammonium chloride in 1,6 mL of water). The resulting organic layer was distilled to residue under reduced pressure yielding compound of formula (43) as a white solid (600 mg).1H NMR (400 MHz, DMSO-d6, ppm), 5: 8.59 (t, 1H) 8.30 (m, 1H), 8.25 (d, 1H) 7.86 (d, 2H), 7.81 (m, 1H), 7.67 (q, 3H), 7.62 (d, 2H), 7.55 (m, 2H), 7.48 (t, 1H), 7.42 - 7.38 (m, 2H), 7.36 - 7.28 (m, 6H), 7.17 (d, 2H), 6.80 (dd, 2H), 4.36 (t, 1H), 4.32 - 4.25 (m, 1H), 4.19 - 4.11 (m, 3H), 2.98 (d, 1H), 2.80 (d, 1H), 2.69 (s, 1H), 1.22 (t, 1H), 1.17 (s, 8H).31P NMR (162 MHz, DMSO-d6, ppm), 5: 23.7.

[0204] Example 12: Preparation of compound Thymopentin of formula (32): H-Arg- Lys-Asp-Val-Tyr-OH according to the scheme reported in Figure 13.

[0205] Anchoring Step - Preparation of compound of formula (25): Fmoc-Tyr(tBu)- Anchor.Refer to Example 9 of the present document.

[0206] Cycle 1 - Preparation of compound of formula (27): Fmoc-Val-Tyr(tBu)- Anchor.To a round bottom flask equipped with a half-moon stirrer, compound of formula (25) (Fmoc-Tyr(tBu)-Anchor, 4.0g), DCM (80 mL) and piperidine (17.84 g) were charged. The mixture was stirred at room temperature for 15 minutes. Thereafter, a solution of ammonium chloride (24 g) in water (96 mL) was added. The biphasic mixture was stirred for 10 minutes and then the phases were separated. The organic layer was washed three more times with a solution of ammonium chloride (24 g) in water (96 mL). Fmoc-Val-OH (2.13 g), TBTU (2.02 g) and DIPEA (1.62 g) were added to the organic solution and the mixture was allowed to react at room temperature until completion. To the resulting mixture was added a solution of ammonium chloride (16 g) in water (64 mL). The biphasic mixture was stirred for 10 minutes and then the phases were separated. The organic layer was washed three more times with a solution of ammonium chloride (16 g) in water (64 mL). The organic layer was then concentrated to 2 residual volumes under reduced pressure and the resulting solution was dosed over n-hexane (40 mL) pre-cooled at T = 0°C. The slurry was then filtered, and the wet solid was washed with n-hexane (20 mL). After drying under vacuum at T =25°C, the product obtained (4.0 g) was employed directly in the following step.

[0207] Cycle 2 - Preparation of compound of formula (28): Fmoc-Asp(tBu)-Val- Tyr(tBu)-Anchor.To a round bottom flask equipped with a half-moon stirrer, compound of formula (27) (Fmoc-Val-Tyr(tBu)-Anchor, 2.0g), DCM (40 mL) and piperidine (7.89 g) were charged. The mixture was stirred at room temperature for 15 minutes. Thereafter, a solution of ammonium chloride (12 g) in water (48 mL) was added. The biphasic mixture was stirred for 10 minutes and then the phases were separated. The organic layer was washed again with a solution of ammonium chloride (12 g) in water (48 mL). An additional wash of the organic layer with a solution of ammonium chloride (6 g) in water (48 mL) was performed, diluting the biphasic mixture with DCM (120 mL) before phase separation. After a last wash with a solution of ammonium chloride (12 g) in water (48 mL), the organic layer was concentrated under vacuum to 60 mL. Fmoc-Asp(tBu)-OH (1.62 g), TBTU (1.26 g) and DIPEA (2.64 g) were added to the organic solution, and the mixture was allowed to react at room temperature until complete conversion. To the resulting mixture, a solution of ammonium chloride (4 g) in water (16 mL) was added. After 10 minutes stirring, the phases were separated. The organic layer was washed three times with a solution of ammonium chloride (4 g) in water (16 mL). The organic layer was then concentrated to residue under reduced pressure and then diluted with DCM (20 mL). The resulting solution was dosed over n- heptane (100 mL) pre-cooled at T=0°C. The slurry was then filtered, and the wet solid was washed with n-heptane (20 mL). After drying under vacuum at T =25°C, the product obtained (2.15 g) was employed directly in the following step.

[0208] Cycle 3 - Preparation of compound of formula (29): Fmoc-Lys(Boc)-Asp(tBu)- Val-Tyr(tBu)-Anchor.To a round bottom flask equipped with a half-moon stirrer, compound of formula (28) (Fmoc-Asp(tBu)-Val-Tyr(tBu)-Anchor, 2.0 g), DCM (40 mL) and piperidine (6.59 g) were charged. The mixture was stirred at room temperature for 65 minutes. Thereafter, a solution of ammonium chloride (12 g) in water (48 mL) was added. The biphasic mixture was stirred for 10 minutes and then the phases were separated. The organic layer was washedthree more times with a solution of ammonium chloride (12 g) in water (48 mL). The organic layer was concentrated under vacuum to 20 mL. The mixture was diluted with 40 mL of DCM and distilled again to 20 mL. This operation was repeated four more times, and finally the solution was diluted with DCM (40 mL). Fmoc-Lys(Boc)-OH (1.09 g), TBTU (0.75 g) and DIPEA (1.20 g) were added to the organic solution and the mixture was allowed to react at room temperature until reaction completion. After dilution with DCM (20 mL), a solution of ammonium chloride (4 g) in water (16 mL) was added into the mixture. The biphasic mixture was stirred for 10 minutes and then the phases were separated. The organic layer was washed three more times with a solution of ammonium chloride (4 g) in water (16 mL). The organic layer was then concentrated to residue under reduced pressure, diluted with DCM (20 mL) and concentrated to residue again. The mixture was finally diluted with DCM (40 mL), and the resulting solution was dosed over n-heptane (200 mL) pre-cooled at T = 0°C. The slurry was then filtered, and the wet solid was washed with n-heptane (20 mL). After drying under vacuum at T=25°C, the product obtained (2.55 g) was employed directly in the following step.

[0209] Cycle 4 - Preparation of compound of formula (30): Fmoc-Arg(Pbf)-Lys(Boc)- Asp(tBu)-Val-Tyr(tBu)-Anchor.To a round bottom flask equipped with a half-moon stirrer, compound of formula (29) (Fmoc-Lys(Boc)Asp(tBu)-Val-Tyr(tBu)-Anchor, 2.4 g), DCM (36 mL) and piperidine (6.48 g) were charged. The mixture was stirred at room temperature for 50 minutes. Thereafter, a solution of ammonium chloride (14.4 g) in water (57.6 mL) was added. The biphasic mixture was stirred for 10 minutes and then the phases were separated. The organic layer was washed three more times with a solution of ammonium chloride (14.4 g) in water (57.6 mL). The organic layer was anhydrified by distilling the mixture to residue under reduced pressure and diluting with DCM before repeating the distillation. The distillation residue was finally diluted with DCM (36 mL) and Fmoc-Arg(Pbf)-OH (1.23 g), TBTU (0.61 g) and DIPEA (1.18 g) were added to the organic solution. The mixture was allowed to react at room temperature until reaction completion. A solution of ammonium chloride (4.8 g) in water (19.2 mL) was added into the mixture. The biphasic mixture was stirred for 10minutes and then the phases were separated. The organic layer was washed three more times with a solution of ammonium chloride (4.8 g) in water (19.2 mL). The organic layer was then concentrated to residue under reduced pressure. The residue was diluted with DCM (48 mL) and the resulting solution was dosed over n-heptane (240 mL) pre-cooled at T = 0°C. The slurry was then filtered, and the wet solid was washed with n-heptane (2 x 24 mL). After drying under vacuum at T=25°C, the product obtained (1.55 g) was employed directly in the following step.

[0210] Side chain PG cleavage: Preparation of compound of formula (31): H-Arg- Lys-Asp-Val-Tyr-Anchor.In a round bottom flask equipped with a half-moon stirrer, compound of formula (30) (Fmoc-Arg(Pbf)-Lys(Boc)-Asp(tBu)-Val-Tyr(tBu)-Anchor, 1.4 g) and DCM (98 mL) were charged, followed by piperidine (2.85 g). The mixture was stirred at room temperature for 1 hour, then diluted with DCM (14 mL) and an aqueous solution of ammonium chloride (8.4 g of ammonium chloride in 33.6 mL of water). The phases were separated, and the aqueous layer was extracted with EtOAc (50 mL).The combined organic layers were solvent switched to EtOAc by distillation of the mixture to residue under reduced pressure followed by dilution with EtOAc (overall 162 mL). An aqueous solution of ammonium chloride (8.4 g of ammonium chloride in 33.6 mL of water) was added to the mixture. The phases were separated, and the organic layer was washed two times with ammonium chloride solution (8.4 g of ammonium chloride in 33.6 mL of water). The organic solution was concentrated to residue under reduced pressure and then diluted with DCM (14 mL). The solution was slowly dosed into n-heptane pre-cooled at T =0°C. After 20 minutes stirring at T =0°C, the resulting slurry was filtered, washing the wet cake with n-heptane (2 x 28 mL). The solid was then charged in a round bottom flask equipped with a halfmoon stirrer and TEA (8.40 mL), DCM (4.20 mL) and water (1.40 mL) were added. After 6.5 hours stirring at room temperature, the solution was diluted with DCM (1 mL) and then dosed slowly over MTBE pre-cooled to T =0°C. The resulting slurry was stirred at T =0°C for 20 minutes and then filtered, washing the wet cake with MTBE (2 x 5 mL). After drying under vacuum at T=40°C toremove residual solvents, 300 mg of compound of formula (31) were obtained. ESI-MS [M + H]+= 984.6; calculated [M + H]+= 984.4379 Da. ESI-MS[M-H]- = 982.5; calculated [M-H]’ = 982.4236 Da.

[0211] Anchor cleavage: Preparation of compound of formula (32): H-Arg-Lys-Asp- Val-Tyr-OH.In an autoclave equipped with a turbine stirrer, compound of formula (31) (H-Arg-Lys-Asp-Val-Tyr-Anchor, 300 mg), MeOH (25 mL), DCM (15 mL) and Pd / C 10% (60 mg) were charged. The mixture was hydrogenated at T=25°C and 2 bar hydrogen pressure until reaction completion, then the catalyst was removed by filtration on a celite pad washing the cake with MeOH (15 mL). The resulting solution was concentrated to residue under vacuum and then diluted with aqueous acetic acid (3 mL of a 10% w / w solution in water) and DCM (3 mL). After phase separation the aqueous layer was washed two times with DCM (2 x 3 mL). Compound of formula (32) was obtained by distillation of the resulting aqueous solution to residue (100 mg isolated solid). ESI-MS [M + H]+= 680.4; calculated [M + H]+= 680.3726 Da. ESI-MS [M-H]’ = 678.5; calculated [M-H]’ = 678.3580 Da.

[0212] Example 13: cleavage of the anchor and re-generation of compound of formula (9).

[0213] STEP 1: preparation of compound of formula (41).In a round bottom flask equipped with a half-moon stirrer, compound of formula (25) (Fmoc-Tyr(tBu)-Anchor, 5.5 g) was dissolved in DCM (110 mL). Piperidine (24.5 g) was added, and the mixture was allowed to react at room temperature for 1 hour. After reaction completion, an aqueous solution of ammonium chloride (33 g of ammonium chloride dissolved in 132 mL of water) was added, and the phases were separated. The organic layer was washed three more times with aqueous ammonium chloride (3 x 33 g of ammonium chloride dissolved in 132 mL of water). The organic layer was concentrated to residue under reduced pressure, then diluted with TFA (12.0 mL), DCM (6.0 mL) and water (2.0 mL). The mixture was stirred at room temperature for 90 minutes, and then concentrated to residue under vacuum. The residue was then stripped repeating the distillation to residue under vacuum with MTBE (4 x 50 mL). The residue was diluted with methanol (48 mL) and transferred into an autoclave, where Pd / C 10% w / w (240 mg) was added. The resulting mixture was hydrogenated at 1 bar hydrogen pressure for 17 hours, filtered on a celite pad and then concentrated to residue under vacuum. The residue was diluted with a 10% w / w aqueoussolution of acetic acid (24 mL) and DCM (24 mL). After phase separation, the aqueous layer was extracted with DCM (2 x 24 mL). The deprotected amino acid Tyrosine (compound of formula (42)) can be isolated from the aqueous layer, for example by precipitation. The combined organic layers were then distilled to residue under vacuum, yielding 1.5 g of compound of formula (41). ESI-MS [M + H]+= 307.3; [2M + H]+=613.3; calculated [M + H]+= 680.3726 Da.1H NMR (400 MHz, DMSO-d6, ppm), 5: 8.29 (dd, J = 8.2 Hz, 4.9 Hz, 1H, aromatic ring CH), 8.24 (dd, J = 8.0 Hz, 1.6 Hz, 1H, aromatic ring CH), 7.84- 7.76 (m, 1H, aromatic ring CH), 7.62 (d, J = 8.1 Hz, 1H, aromatic ring CH), 7.59 (d, J = 8.1 Hz, 1H, aromatic ring CH), 7.55 (d, J = 4.6 Hz, 1H, aromatic ring CH), 7.53-7.49 (m, 1H, aromatic ring CH), 7.47 (d, J = 7.6 Hz, 1H, aromatic ring CH), 7.40-7.34 (m, 3H, aromatic ring CH), 7.32 (dd, J = 8.2 Hz, 0.8 Hz, 1H, aromatic ring CH), 2.37 (s, 3H, CH3).13C NMR (100 MHz, DMSO-d6, ppm), 5: 148.6 (d, J = 9.0 Hz, 1C, aromatic ring), 143.7 (d, J = 3.0 Hz, 1C, aromatic ring), 134.8 (d, J = 6.0 Hz, 1C, aromatic ring), 133.4, (d, J = 3.0 Hz, 1C, aromatic ring), 131.7 (d, J = 12.0 Hz, 1C, aromatic ring), 130.9, 130.3 (d, J = 12.0 Hz, 1C, aromatic ring), 129.5 (d, J = 16.0 Hz, 1C, aromatic ring), 128.8, (d, J = 13.0 Hz, 1C, aromatic ring), 126.7, 125.7, 125.6, 125.3, 124.9, 124.4, 124.2 (d, J = 9.0 Hz, 1C, aromatic ring), 121.7 (d, J = 13.0 Hz, 1C, aromatic ring), 120.2 (d, J = 6.0 Hz, 1C, aromatic ring), 21.2 (d, J = 1.1 Hz, 1C, CH3).31P NMR (162 MHz, DMSO-d6, ppm), 5: 24.1.

[0214] STEP 2: preparation of compound of formula (9).In a round bottom flask equipped with a magnetic stirrer and masked from the light, 600 mg of compound of formula (41) were dissolved in degassed acetonitrile (6.0 mL), and benzoyl peroxide (50 mg) was then added. N- bromo succinimide (NBS, 520 mg) was then added portion wise over the mixture in five portions, each portion carried out 30 minutes after the previous one. The mixture was finally heated to T=80°C for 2 hours in order to push the reaction to completion. Thereafter, a 10% w / w aqueous solution of sodium thiosulfate (6.0 mL) was charged, and after 10 minutes stirring at room temperature MTBE (6.0 mL) was added. The phases were separated, and the aqueous layer was extracted with MTBE (2 x 6.0 mL). The combined organic layers were washed two times with a 10% w / w aqueous solution ofpotassium carbonate (2 x 6.0 mL) and finally with brine (6.0 mL). The resulting organic solution was distilled under vacuum to residue, yielding compound of formula (9) (800 mg). If needed, purification by chromatography or recrystallization can be carried out to improve product purity. ESI-MS [M + H]+= 385 (79Br isotope); 387 (81Br isotope); [M + Na]+= 407 (79Br isotope); 410 (81Br isotope); calculated [M + H]+= 383.9914 Da(79Br isotope).1H NMR (400 MHz, DMSO-d6, ppm), 5: 8.25 (dd, J = 8.2 Hz, 4.9 Hz, 1H, aromatic ring CH), 8.25 (dd, J = 8.0 Hz, 1.6 Hz, 1H, aromatic ring CH), 7.85-7.78 (m, 1H, aromatic ring CH), 7.73 (d, J = 8.2 Hz, 1H, aromatic ring CH), 7.70 (d, J = 8.2 Hz, 1H, aromatic ring CH), 7.65-7.59 (m, 2H, aromatic ring CH), 7.58-7.52 (m, 2H, aromatic ring CH), 7,48 (t, J = 7.6 Hz, 1H, aromatic ring CH), 7.39-7.30 (m, 2H, aromatic ring CH), 4.74 (s, 2H, CH2Br).13C NMR (100 MHz, DMSO-d6, ppm), 5: 148.5 (d, J = 9.0 Hz, 1C, aromatic ring), 143.4 (d, J = 3.0 Hz, 1C, aromatic ring), 134.9 (d, J = 6.0 Hz, 1C, aromatic ring), 133.6, (d, J = 2.0 Hz, 1C, aromatic ring), 132.0 (d, J = 11.0 Hz, 1C, aromatic ring), 131.0 (s, 1C, aromatic ring), 130.5 (d, J = 12.0 Hz, 1C, aromatic ring), 129.8 (s, 1C, aromatic ring), 129.7 (d, J = 14.0 Hz, 1C, aromatic ring), 128.9 (d, J = 14.0 Hz, 1C, aromatic ring), 128.4 (s, 1C, aromatic ring), 125.7 (s, 1C, aromatic ring), 125.1, 125.0 (s, 1C, aromatic ring), 124.3 (d, J = 9.0 Hz, 1C, aromatic ring), 123.8 (s, 1C, aromatic ring), 121.6 (d, J = 11.0 Hz, 1C, aromatic ring), 120.2 (d, J = 6.0 Hz, 1C, aromatic ring), 32.9 (d, J = 0.9 Hz, 1C, CH2).31P NMR (162 MHz, DMSO-d6, ppm), 5: 23.3.

Claims

ClaimsUse of a compound of formula (1):wherein n1, n2 and n3 are independently 0 or 1 and wherein X is OH, NH2 or halogen, in liquid phase peptide synthesis.2) Use of a compound of formula (1) according to claim 1, wherein n1, n2 and n3 are 0, or wherein n1 and n3 are 0 and n2 is 1, or wherein n1 and n2 are 0 and n3 is 1, or wherein n1 and n2 are 1 and n3 is 0, or wherein n2 and n3 are 1 and n1 is 0, or wherein n1, n2 and n3 are 1.3) Use of a compound of formula (1) according to claim 1 or claim 2, wherein X is Br or NH2.4) Use of a compound of formula (1) according to any one of the claims from 1 to 3, selected among the following compounds:(12) (13) (36).5) A method for protecting an amino acid or an amino acid derivative with a compound of formula (1) according to any one of the claims from 1 to 4, comprising the following steps: a) reacting the amino acid or amino acid derivative having a free carboxylic functional group, a protected N-terminus amino functional group and a side chain optionally protected at its functional groups, with a compound of formula (1) according to the claims from 1 to 4, in the presence of: i) a base, when X is a halogen; or ii) a coupling agent and optionally an additive, when X is OH; or iii) a base, a coupling agent and / or an activating agent, when X is NH2; b) isolate the amino acid or amino acid derivative protected with compound of formula (1) obtained in step a).6) A method according to claim 5, wherein in step a) case i) the base is potassium carbonate, in step a) case ii) the coupling agent is a carbodiimide selected from the group EDC, DCC, DIG, DSBC, DTBC or TBEC and the optional additive is DMAP and wherein in step a) case iii) the base is DIPEA and the coupling agent and / or the activating agent is selected from the group EDC, DCC, HOBt, HOAt, HATU, HBTU, TBTU.7) A method for performing solution-phase peptide synthesis comprising a compound of formula (1) according to any one of the claims from 1 to 4 as C- terminus protecting group, comprising the following steps: a1) protecting the carboxylic functional group of an amino acid or amino acid derivative with a compound of formula (1) according to anyone of the claims from 1 to 4, according to the method of claim 5 or claim 6; b1) deprotecting the amino functional group of the protected amino acid or amino acid derivative obtained in step a1); c1)purifying the deprotected amino acid or amino acid derivative obtained in step b1);d1) reacting the deprotected amino acid or amino acid derivative obtained in step c1) with another amino acid or amino acid derivative protected at the amine function, to obtain a peptide or a peptide derivative; e1) optionally purifying the peptide or peptide derivative obtained in step d1); f1) repeating steps b1), c1) d1) and e1) to add other amino acids or amino acid derivatives to the peptide chain, until the complete peptide chain is obtained; g1) isolating the protected peptide obtained in step f1); hi) removing the C-terminus protecting group and any other protecting groups from the chain to obtain the peptide.8) A method according to claim 7, wherein the amino function of the amino acids or amino acid derivatives that are added to the peptide chain is protected with a Fmoc group.9) A method according to any one of the claims 7 or 8, wherein the reaction in step d1) is carried out using TBTU as coupling agent and DIPEA as base in DCM as solvent.10) A method according to any one of the claims from 7 to 9, wherein in step e1) and / or in step g1) the purification and / or isolation is performed by dosing a solution of the peptide in a solvent selected from the group ethyl acetate, isopropyl acetate, acetonitrile, DCM or CPME into a solvent selected from the group n-hexane, n-heptane, diethyl ether, di-isopropyl ether, MTBE or MIBE, to obtain peptide precipitation.11) A method according to any one of the claims from 7 to 10, further comprising a step hi) of regeneration of the C-terminus protecting group obtained after deprotection in step g1) into a compound of formula (1) according to any one of the claims from (1) to (4) to be employed in step a1).12) A compound of formula (1):wherein n1, n2 and n3 are independently 0 or 1, X is OH, NH2 or halogen, and wherein, when n2 is 1 and n1 and n3 are 0, X is not OH.13) A compound of formula (1) according to claim 12, wherein n1, n2 and n3 are 0, or wherein n1 and n3 are 0 and n2 is 1, or wherein n1 and n2 are 0 and n3 is 1, or wherein n1 and n2 are 1 and n3 is 0, or wherein n2 and n3 are 1 and n1 is 0, or wherein n1, n2 and n3 are 1.14) A compound of formula (1) according to claim 12 or claim 13, wherein X is Br or NH2.15) A compound of formula (1) according to any one of the claims from 12 to 14 selected among the following compounds:(12) (13) (36).16) An amino acid or amino acid derivative protected with at least a compound of formula (1) according to any one of the claims from 1 to 4 as protecting group, or a peptide or a peptide derivative comprising at least a compound of formula (1) according to any one of the claims from 1 to 4 as protecting group.17) A kit for peptide synthesis comprising at least an amino acid or amino acid derivative protected with a compound of formula (1) according to anyone of the claims from 1 to 4, and at least a peptide synthesis coupling agent.18) A screening kit for peptide synthesis comprising at least two or a plurality of compound of formula (1) according to anyone of the claims from 1 to 4.19) Use of compound of formula (1) according to claims from 1 to 4 for the preparation of the pentapeptide Thymopentin, having formula H-Arg-Lys-Asp- Val-Tyr-OH.20) Use according to claim 19, wherein the compound of formula (1) has the formula of compound (8).

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