WO2014081300A1 - Channel protein activatable liposomes - Google Patents

Abstract

Disclosed is a liposome, comprising a lipid bilayer enclosing a cavity, wherein the bilayer comprises a channel protein releasably linked to an eight-membered non-aromatic cyclic alkenylene group, preferably a cyclooctene group, and more preferably a trans-cyclooctene group. The liposomes are used in a kit comprising the liposome, the liposomal membrane of which comprises a channel protein linked to a Trigger, and an Activator for the Trigger, wherein the Trigger comprises the eight- membered non-aromatic cyclic alkenylene group, and the Activator comprises a diene.

Description

Title: CHANNEL PROTEIN ACTIVATABLE LIPOSOMES

Field of the invention

The invention pertains to activatable liposomes, the liposome membrane of which comprises a channel protein modified with a chemical Trigger.

Background of the invention The goal of drug delivery systems is to increase the efficacy and safety of both new and existing drugs. A number of drug compounds cannot be delivered safely and/or effectively by conventional routes or dosage forms such as oral tablets or injection. Alternative delivery methods can increase safety by sequestering drugs in carriers that reduce systemic exposure and decrease dose-limiting toxicity and side effects, or by providing sustained delivery so that therapeutic levels can be achieved with fewer and smaller doses. New delivery systems can also increase efficacy by several strategies, including: increasing stability of the drug; increasing the ability of the drug to reach its therapeutic target by prolonging the circulating half -life; and targeting delivery to the therapeutic site and effecting drug release in order to reduce the total circulating dose without diminishing efficacy. The most prominent delivery systems used in the clinic are based on liposomes (e.g. Doxil which are liposomes filled with doxorubicin). There are two liposomal targeting strategies: passive and specific. Passive targeting refers to the preferential accumulation of liposomes in tumours and at sites of infection and inflammation. Small sterically stabilized liposomes extravasate through leaky blood vessels that are formed through tumour angiogenesis or damaged by infection and inflammation. The liposomes accumulate in tumour interstices and at sites of infection and inflammation, where they gradually release their encapsulated drugs. This sequesters potentially toxic agents from susceptible non-target sites such as the brain, liver and heart. The mechanism of liposome accumulation may be a combination of the leakiness of the newly forming or damaged capillaries and enhanced vascular permeation by the coated liposomal particles themselves. Specific targeting involves the use of antibodies or ligands to tag liposomes so that they bind specifically to cells that express the appropriate cell-surface antigens or ligand receptors, respectively. In principle, liposomes can be targeted to any cell surface structure that can be recognized by a fragment of a specific antibody, or to any receptor for which a small and specific ligand can be produced. Hence, liposomes can be directed to specific classes of T and B lymphocytes or to tumor cells, preferentially expressing high levels of specific cell surface proteins. The goals of ligand targeting of liposomes are to concentrate them selectively at the therapeutic site, decrease the required dose by reducing non-specific losses, and reduce systemic exposure to drugs with toxic side effects.

There are two caveats, however: 1) At the target site, the drugs must be released from the liposomes efficiently enough to have a clinically significant effect, and 2) The target cells must be accessible.

With respect to liposome release, this can be governed by multiple processes and variables. Localization of passive or active targeted liposomes is usually followed by a relatively lengthy process, which can involve an internalization pathway followed by intracellular processing and drug release or expulsion back to the extracellular domain.

Alternatively, these Constructs stay in the extracellular domain and are slowly degraded, depending on the specific environment. When hposomes are taken up by cells through endocytosis, the liposome needs to be degraded and the drug has to be able to escape the endosomal or lysosomal compartment. In this respect it should be noted that endothelial cells do not possess a machinery to degrade liposomes and as a result these

Constructs are expulsed back to the extracellular environment. In addition, when liposomes are entrapped in macrophages, one needs a burst release of the drug to achieve an effective peak concentration which allows diffusion from the macrophage.

Currently the targeting and localization of liposomes can be controlled by passive or active methods, but there is only an indirect, unpredictable and non-universal control over the subsequent release from these liposomes. The release pathway, the extent of release, the release profile (slow or bolus), and the timing, to a large extent depend on the specifics and peculiarities of the target physiology and biology. To address this issue, liposomal delivery systems capable of release of their content under the influence of e.g. pH, thiols, and light have been developed.

For example, liposomes that destabilize under mildly acidic conditions, so-called "pH-sensitive liposomes, have been described as an approach to intracellular deliver an entrapped agent (Slepushkin et al., J. Biol. Chem., 272(4):2382 (1997); Wang et al., Proc. Natl. Acad. Sci.,

84:7851 (1987), Liu et al., Biochim. Biophys. Acta, 981:254 (1989)).

These liposomes are primarily composed of a lipid, such as

dioleoylphosphatidylethanolamine (DOPE), that forms a lipid bilayer in a defined pH range. Outside this pH range, the lipid bilayer destabilizes. After such liposomes enter cells via endocytosis, the acidic pH inside the endosomes causes the pH-sensitive liposomes to destabilize and release the entrapped agent.

Because pH-sensitive liposomes, like "conventional", "non-pH- sensitive liposomes", have short circulation lifetimes, addition of PEG- derivatized hpids to extend the blood circulation time has been proposed (Slepushkin et al.). However, addition of PEG-derivatized lipids

attenuates the pH-sensitivity of the liposomes, resulting in a loss of the desired rapid destabihzation of the liposome bilayer and accompanying rapid release of the entrapped agent into the cell. One approach to providing pH-sensitive liposomes having a long blood circulation lifetime and retaining the abihty of the liposome to rapidly destabilize is the use PEG-derivatized lipids where the PEG is attached to the lipid by a thiol- cleavable linkage for release at the target site, restoring the pH

sensitivity and effecting drug release (Kirpotin et al., FEBS Letters, 388: 115 (1996); and US7108863).

Also described in the art are liposomes capable of fusion with a target cell (U.S. Pat. No. 5,891,468). Fusogenic liposomes typically include a hydrophobic polymer extending from the liposomes' outer surfaces for penetration into a target cell membrane. The hydrophobic polymers are initially shielded by a hydrophilic polymer coating, and then exposed for fusion with the target membrane when the hydrophilic polymer coating is released by reaction with thiols.

An alternative liposome release technology is based on a well- studied bacterial channel protein "Mechanosensitive channel of large conductance", MscL, from E. coli. In its native form the channel creates a large non-selective pore of 3-4 nm in diameter in the membrane and allows the passage of ions, small molecules, peptides and smaller proteins (up to 7 kDa). In nature, MscL opens in response to the tension in the membrane. It has been shown that the hydrophilicity of the 22nd amino acid position of MscL affects the mechanosensitivity of the channel up to a point where it starts to open even in the absence of tension (Yoshimura et al. (1999) Biophys. J. 77, 1960-1972). Hydrophilic substitutions in this narrow pore constriction area of the channel cause hydration of the pore and

weakening of the hydrophobic van der Waals forces responsible for the close packing of the inner membrane helices in the closed state of the channel. The effect is reinforced if charged or bulky groups are introduced because of electrostatic repulsion and steric factors, respectively. This is reflected in the energetics of the gating transitions and leads to the opening of the channel even in the absence of tension. On the basis of this principle, the MscL protein was re-engineered to site-selectively- incorporate (masked) amine-functionalized molecules. A series of small modulators were designed, synthesized and specifically attached to an engineered Cysteine at position 22 in MscL. The working principle is that the protein-attached modulators would be charged only in response to a pre-defined stimulation (pH, light, etc) leading to hydration of the hydrophobic constriction zone of the pore and channel opening in the absence of the natural stimulus. In the case of light-activation, the masked reagents possess a nitrophenol moiety, which is removed upon

illumination. This affords a free amino moiety which, depending on the pH, can get protonated and trigger the opening of the channel. The abihty to control the release of liposome content with reversible channel opening and closing was demonstrated under the influence of UV and visible light, respectively (Kocer et al. (2005) Science 309, 755-758) and in response to a decrease in pH using channels modified to respond directly to pH as well as channels engineered (using masked reagents) to respond to pH only after illumination (Kocer et al. (2006) Angew. Chem. Int. Ed. 45, 3126- 3130). Rationally designed chemical modulators convert a bacterial channel protein into a pH-sensory valve. This methodology is also disclosed in PCT patent applications WO2005051902, WO03084508 and WO03000233. The activateable liposomal drug delivery systems discussed above allow an increased level of control over drug release from liposomes using light-, pH-, or thiol-mediated release. These mechanisms provide an additional selectivity for a specific local environment (e.g. low pH in certain tumors, high intracellular thiol concentration) or localized illumination. However, the drawback of this additional selectivity is that these tools cannot be universally applied. Due to the low penetration depth of light, the technology of controlled drug delivery is limited to disorders situated at or near the body surface or in combination with a catheter-light pipe. With respect to the pH-activateable liposomes, many potential targets do not have an extracellular pH that is significantly different from surrounding non-target tissue. Intracellular pH-mediated activation is possible, but does requires efficient cellular uptake, which may not be feasible as it depends on several variables, composition and targets. Likewise, thiol- based activation requires efficient cellular uptake of the liposome.

However, this requires that ideally most target cells can be reached by the administered liposome. Following intracellular release, the drug needs to be able to escape from the endosome or lysosome intact and, in the case of heterogeneous targeted tissue, it is advantageous if the drug can escape from the cell to target neighbouring cells that have not bound liposomes. Extracellular drug distribution may be of further importance in tumors that are difficult to penetrate due e.g. to elevated interstitial pressure, which impedes convectional flow. This is especially a problem for large Constructs like hposomes.

In addition, the approach to use Constructs that should be cleavable by endogenous elements in the target while stable to endogenous elements en route to the target may have limited applicabihty.

In order to avoid the drawbacks of current liposome activation, it has been proposed in WO2009144659 to make use of an abiotic, bio- orthogonal chemical reaction, viz. the Staudinger reaction, to provoke activation of a masked liposome. Briefly, in the introduced concept, the liposome membrane contains a channel protein modified with a chemical Trigger, and this channel protein-Trigger conjugate does not allow efflux of the liposome contents and is not activated endogeneously by e.g. thiols or a specific pH. Instead it is activated by a controlled administration of the Activator, i.e. a species that reacts with the Trigger moiety in the masked liposome, to induce release of the Trigger from the protein, leading to opening up of the channel protein and release of the liposome- entrapped drugs. In more detail, a cysteine in the channel protein was modified with moiety comprising an amine which was masked by an azide Trigger. Upon Staudinger reaction with a phosphine Activator, the Trigger is removed, unveiling the amine moiety, which due to its pKa is

protonated, leading to drug release from the liposome. The presented

Staudinger approach for this concept, however, has turned out not to work well, and its area of applicability is limited in view of the specific nature of the release mechanism imposed by the Staudinger reaction. Other drawbacks for use of Staudinger reactions are their limited reaction rates, and the oxidative instability of the phosphine components of these reactions. Therefore, it is desired to provide reactants for an abiotic, bio- orthogonal reaction that are stable in physiological conditions, that are more reactive towards each other, and that are capable of activating liposomes and inducing release of a entrapped drugs by means of a variety of mechanisms, thus offering a greatly versatile activated drug release method.

It is desirable to be able to activate liposomes selectively and predictably at the target site without being dependent on homogenous penetration and targeting, and on endogenous parameters which may vary en route to and within the target, and from indication to indication and from patient to patient. The use of a biocompatible chemical reaction that does not rely on endogenous activation mechanisms (eg pH, thiols) for selective liposome activation would represent a powerful new tool in cancer therapy. Selective activation of liposomes when and where required allows control over many processes within the body, including cancer. Therapies may thus be made more specific and effective, providing an increased therapeutic contrast between normal cells and tumour to reduce unwanted side effects.

Summary of the invention

In order to better address one or more of the foregoing desires, the invention presents, in one aspect, a hposome, comprising a lipid bilayer enclosing a cavity, wherein the bilayer comprises a channel protein releasably hnked to an eight-membered non-aromatic cyclic alkenylene group, preferably a cyclooctene group, and more preferably a trans- cyclooctene group. In another aspect, the invention provides a kit for the

administration and activation of an activatable liposome, the kit

comprising a liposome, the liposomal membrane of which comprises a channel protein linked to a Trigger, and an Activator for the Trigger, wherein the Trigger comprises an eight-membered non-aromatic cyclic alkenylene group as a dienophile, preferably a cyclooctene group, and more preferably a ircms-cyclooctene group, and the Activator comprises a diene.

In a further aspect, the invention is a hposomal composition, comprising:

(a) a Construct of a liposome comprising a lipid bilayer enclosing a cavity, wherein the bilayer comprises a channel protein and, optionally, a Targeting Agent;

(b) an eight-membered non-aromatic cyclic alkenylene group,

preferably a cyclooctene group, and more preferably a trans- cyclooctene group, linked to the Construct.

In a still further aspect, the invention resides in the use of an eight- membered non-aromatic cyclic alkenylene group as a dienophile,

preferably a cyclooctene group, and more preferably a ir n-s-cyclooctene group, as a Trigger on an activatable liposome, wherein the liposome comprises a channel protein linked to the Trigger, and wherein reaction of the dienophile with a diene results in activation of the liposome.

Detailed description of the invention

The invention, in a broad sense, is based on a judicious use the so- called inverse electron -demand Diels-Alder reaction (also referred to as the reiroDiels-Alder or rDA reaction), as a chemical tool in the activation of liposomes comprising a channel protein. As will be clear from the explanation that follows, the present use of the rDA reaction is

irrespective of the precise chemical components further present. Thus, e.g., liposomes are known, as is their use as a carrier for therapeutic agents. Also activatable liposomes comprising channel proteins are known, as outlined inter alia in WO2009144659.

The invention expressly is directed to providing a breakable linkage in an activatable hposome assembly. Breaking said linkage through the judiciously chosen rDA reaction, results in the liposome becoming activated. Or, alternatively, results in eliminating the deactivation of the liposome.

It should be noted that where, in this disclosure, it is spoken of "a cycloalkenylene group", "a cyclooctene group", or "a trans -cyclooctene group" these expressions refer to such a group by itself, or linked to a further chemical structure. In the art of chemistry, this is also indicated with the term "moiety". E.g., a cyclooctene group as present on the liposomes of the invention, can itself be attached to a further structure (e.g. polyethylene glycol), and can also be referred to as a "cyclooctene moiety."

Retro Diels-Alder reaction

The Retro Diels-Alder coupling chemistry generally involves a pair of reactants that couple to form an unstable intermediate, which intermediate ehminates a small molecule (depending on the starting compounds this may be e.g. N2, CO2 , RCN), as the sole by-product through a retro Diels-Alder reaction to form the retro Diels-Alder adduct. The paired reactants comprise, as one reactant (i.e. one Bio-orthogonal

Reactive Group), a suitable diene, such as a derivative of tetrazine, e.g. an electron-deficient tetrazine and, as the other reactant (i.e. the other Bio- orthogonal Reactive Group), a suitable dienophile, such as a strained trans -cyclooctene (TCO).

The exceptionally fast reaction of e.g. electron- deficient (substituted) tetrazines with a TCO moiety results in a ligation

intermediate that rearranges to a dihydropyridazine retro Diels-Alder adduct by eliminating N2 as the sole by-product in a [4+2] Retro Diels- Alder cycloaddition. In aqueous environment, the inititally formed 4,5- dihydropyridazine product may tautomerize to a 1,4-dihydropyridazine product.

The two reactive species are abiotic and do not undergo fast metabolism or side reactions in vivo. They are bio-orthogonal, e.g. they selectively react with each other in physiologic media. Thus, the compounds and the method of the invention can be used in a living organism. Moreover, the reactive groups are relatively small and can be introduced in biological samples or living organisms without significantly altering the size of biomolecules therein. References on the Inverse electron demand Diels Alder reaction, and the behavior of the pair of reactive species include: Thalhammer, F; Wallfahrer, U; Sauer, J, Tetrahedron Letters, 1990, 31 (47), 6851-6854; Wijnen, JW; Zavarise, S; Engberts, JBFN, Journal Of Organic Chemistry, 1996, 61, 2001-2005; Blackman, ML; Royzen, M; Fox, JM, Journal Of The American Chemical Society, 2008, 130 (41), 13518-19), R. Rossin, P. Renart Verkerk, Sandra M. van den Bosch, R. C. M. Vulders, 1. Verel, J. Lub, M. S. Robillard, Angew Chem Int Ed 2010, 49, 3375, N. K. Devaraj, R. Upadhyay, J. B. Haun, S. A. Hilderbrand, R. Weissleder, Angew Chem Int Ed 2009, 48, 7013, and Devaraj et al., Angew.Chem.Int.Ed., 2009, 48, 1-5.

It will be understood that, in a broad sense, according to the invention the aforementioned retro Diels-Alder coupling and subsequent liposome activation chemistry can be applied to basically any pair of molecules, groups, or moieties that are capable of being used in liposomal drug delivery. I.e. one of such a pair will comprise a Construct linked to a dienophile (the Trigger). The other one will be a complementary diene for use in reaction with said dienophile.

Trigger The activatable liposome according to the invention comprises a Construct comprising the liposome and the channel protein, denoted as D^D linked, directly or indirectly, but always via an amino group bound to the channel protein , to a Trigger moiety denoted as T^E, wherein the Trigger moiety is a dienophile. The dienophile, in a broad sense, is an eight-membered non-aromatic cyclic alkenylene moiety (preferably a cyclooctene moiety, and more preferably a ircms-cyclooctene moiety).

Optionally, the £r<ms-cyclooctene (TCO) moiety comprises at least two exocyclic bonds fixed in substantially the same plane, and/or it optionally comprises at least one substituent in the axial position, and not the equatorial position. The person skilled in organic chemistry will understand that the term "fixed in substantially the same plane" refers to bonding theory according to which bonds are normally considered to be fixed in the same plane. Typical examples of such fixations in the same plane include double bonds and strained fused rings. E.g., the at least two exocyclic bonds can be the two bonds of a double bond to an oxygen (i.e. C=0). The at least two exocyclic bonds can also be single bonds on two adjacent carbon atoms, provided that these bonds together are part of a fused ring (i.e. fused to the TCO ring) that assumes a substantially flat structure, therewith fixing said two single bonds in substantially one and the same plane. Examples of the latter include strained rings such as cyclopropyl and cyclobutyl. Without wishing to be bound by theory, the inventors believe that the presence of at least two exocyclic bonds in the same plane will result in an at least partial flattening of the TCO ring, which can lead to higher reactivity in the retro-Diels-Alder reaction.

In this invention, the TCO satisfies the following formula

(la)

A and P each independently are CR¾ or CR^aX^D provided that at least one is CR^aX^D. X^D is (0-C(0))_p-(LD)_n-(DD), S-C(0)-(I )_n-(DD), O-C(S)- (L^D)_n-(D^D), S-C(S)-(LD)_n-(DD), 0-S(0)-(L,D)n-(D^D), wherein p - 0 or 1.

Preferably, X^D is (0-C(0))_p-(LD)_n-(T)D), where p = 0 or 1, preferably 1, and n = 0 or 1.

In an interesting embodiment, Y,Z,X,Q each independently are selected from the group consisting of CR¾

S, SO, S0₂, O, NR^b, and SiR^c ₂, with at most three of Y, Z, X, and Q being selected from the group consisting of

wherein two R moieties together may form a ring, and with the proviso that no adjacent pairs of atoms are present selected from the group consisting of O-O, 0-NR^b, S-NR , O-S, O-S(O), 0-S(0)₂, and S-S, and such that Si is only adjacent to CR¾ or O.

In another interesting embodiment, one of the bonds PQ, QX, XZ, ZY, YA is part of a fused ring or consists of CR^a=CR^a, such that two exocyclic bonds are fixed in the same plane, and provided that PQ and YA are not part of an aromatic 5-or 6-membered ring, of a conjugated 7- membered ring, or of CR^a=CR^a; when not part of a fused ring P and A are independently CR^a2 or CR^aX^D provided that at least one is CR^aX^D ; when part of a fused ring P and A are independently CR^a or CX^D provided that at least one is CX^D; the remaining groups (Y,Z,X,Q) being independently from each other CR¾, C=CR^a ₂, C=0, C=S, C=NR^b, S, SO, S0₂, O, NR^b, SiR¾, such that at most 1 group is C=CR¾ C=0, C=S, C=NR^b, and no adjacent pairs of atoms are present selected from the group consisting of O-O, 0-NR^b, S-NR^b, O-S, O-S(O), 0-S(0)₂, and S-S, and such that Si, if present, is adjacent to CR^a2 or O, and the

bond, if present, is adjacent to CR¾ or C=CR^a ₂ groups; In some embodiments fused rings are present that result in two exocyclic bonds being fixed in substantially the same plane. These are selected from fused 3-membered rings, fused 4-membered rings, fused bicyclic 7-membered rings, fused aromatic 5-membered rings, fused aromatic 6-membered rings, and fused planar conjugated 7-membered rings as defined below:

Fused 3-membered rings are:

Therein E, G are part of the above mentioned 8-membered ring and can be fused to PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY, such that P, A are CR^a or CX^D, and such that CX^D can only be present in A and P.

E-G is CR^a-CR^a or CR^a-CX^D, and D is CR¾C=0, C=S, C=NR^b, NR^b, O, S; or E-G is CR^a-N or CX^D-N, and D is CR¾ C=0, C=S, C=NR^b, NR^bO, or S.

Fused 4-membered rings are:

\

E—D

I E-G is part of the above mentioned 8-membered ring and can be fused to PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY, such that P, A are C, CR^a or CX^D, and such that CX^D can only be present in A and P.

E, G are CR^a, CX^D or N, and D,M independently from each other are CR¾, C=0, C=S, C= NR^b, C=CR¾ S, SO, S0₂, O, NR^b but no adjacent 0-0 or S-S groups; or

E-D is C=CR^a and G is N, CR^a, CX^D and M is CR¾ S, SO, S0₂, O, NR^b; or E-D is C=N and G is N, CR^a, CX^D and M is CR¾ S, SO, S0₂, 0; or

D-M is CR^a=CR^a and E, G each independently are CR^a, CX^D or N; or D-M is CR^a=N and E is CR^a, CX^D, N, and G is CR^a or CX^D; or E is C, G is CR^a, CX^D or N, and D, M are CR¾ S, SO, S0₂, O, NR^b, or at most one of C=0, C=S, C= NR^b, C=CR¾, but no adjacent 0-0 or S-S groups; or E and G are C, and D and M independently from each other are CR¾, S, SO, S0₂, O, NR^b but no adjacent 0-0, or S-S groups.

Fused bicycli -membered rings are:

E-G is part of the above mentioned 8-membered ring and can be fused to PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY, such that P, A are C, CR^a or CX^D, and such that CX^D can only be present in A and P;

E,G are C, CR^a, CX^D or N; K, L are CR^a; D,M form a CR^a=CR^a or CR^a=N, or D,M independently from each other are CR¾, C=0, C=S, C= NR^b, C=CR^a ₂, S, SO, S0₂, O, NR^b but no adjacent 0-0, S-S, N-S groups; J is CR¾ C=0, C=S, C= NR^b, C=CR¾ S, SO, S0_¾ O, NR^b ; at most 2 N groups; or

E,G are C, CR^a, CX^D; K is N and L is CR^a; D,M form a

CR^a=CR^a bond or D,M independently from each other are CR¾, C=0, C=S, C= NR^b, C=CR¾ NR^b but no adjacent 0-0, S-S, N-S groups; J is CR¾, C=0, C=S, C= NR^b,

S, SO, S0₂, 0, NR^b ; at most 2 N groups; or E,G are C, CR^a, CX^D; K and L are N; D,M, J independently from each other are CR¾, C=0, C=S, C= NR^b, C=CR^a ₂ groups;

Fused aromatic 5-membered rings are

E, G are part of the above mentioned 8-membered ring and can be fused to QX, XQ, XZ, ZX, ZY, YZ.

E and G are C; one of the groups L, K, or M are O, NR^b, S and the remaining two groups are independently from each other CR^a or N; or E is C and G is N; L, K, M are independently from each other CR^a or N. Fused aromatic 6-membered rings are:

E, G are part of the above mentioned 8-membered ring and can be fused to QX, XQ, XZ, ZX, ZY, YZ.

E,G is C; L, K, D , M are independently from each other CR^a or N

Fused planar conjugated 7-membered rings are

are part of the above mentioned 8-membered ring can be fused to QX, XQ, XZ, ZX, ZY, YZ

E,G is C; L, K, D, M are CR^a; J is S, O, CR¾, NR^b.

(L^D)n is an optional linker, with n = 0 or 1, preferably linked to T^R via S, N, NH, or O, wherein these atoms are part of the linker, which may consist of multiple units arranged linearly and/or branched. D^D is a Construct, preferably linked via N, or NH, wherein these atoms stay with the Construct after release thereof.

T, F each independently denotes H, or a substituent selected from the group consisting of alkyl, F, CI, Br, or I.

Without wishing to be bound by theory, the inventors believe that in the foregoing embodiments, the rDA reaction results in a cascade-mediated release or elimination (i.e. cascade mechanism) of the Construct.

In several alternative embodiments, with reference to formula (la), said release or elimination is believed to be mediated by a strain release mechanism. Therein, in Embodiment 1, one of the bonds PQ, QP, QX, XQ, XZ, ZX, ZY, YZ, YA, AY consists of -CR^-CR^-, the remaining groups (from

A,Y,Z,X,Q,P) being independently from each other CR¾, S, O, SiR^c2, such that P and A are CR^a2, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si, if present, is adjacent to CR^a2 or O.

XD is 0-C(0)-(LD)_n-(DD), S-C(0)-(LD)_n-(E ), 0-C(S)-(LD)_n-(DD), S-C(S)-(L^D)_n-(D^D), NRd-C(0)-(L^D)_n-(D^D), NRd-C(S)-(L^D)„-(D^D), and then Y° is NHR^d, OH, SH; or X is C(0)-(LD)_n-(DD), C(S)-(LD)_n-(DD); and then YD is CR¾NHR^d, CR¾OH, CR¾SH, NH-NH₂, 0-NH₂, NH-OH.

Preferably X^D is NR^d-C(0)-(L^D)_n-(D^D), and Y^D is NHR^d.

In this Embodiment 1, the X^D and Y^D groups may be positioned cis or trans relative to each other, where depending on the positions on the TCO, cis or trans are preferred: if PQ, QP, AY or YA is - CR^aX^D-CR Y^D-, then X^D and Y^D are preferably positioned trans relative to each other; if ZX or XZ is -CR^aX^D-CR^aY^D -, then X^D and Y^D are preferably positioned cis relative to each other.

In Embodiment 2, A is CR^aX^D and Z is CR^aY^D, or Z is CR^aX^D and A is CR^aY°, or P is CR^aX^D and X is CR^aY^D, or X is CR^aX^D and P is CR^aY⁰, such that X^D and Y^D are positioned in a trans conformation with respect to one another; the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR¾, S, O, SiR¾ such that P and A are CR^a2, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si, if present, is adjacent to CR¾ or O; X° is 0-C(0)-(LD)_n-(DD), S-C(0)-(LD)_n-<T>D), 0-C(S)-(LD)„-(D^D), S-C(S)-(LD)_n-(DD), NRd-C(0)-(LD)_n-(DD)_; NRd-C(S)-(LD)„-(I ), and then Y° is NHR^d, OH, SH, CR^d ₂NHR^d CR^d ₂OH, CR^d ₂SH, NH-NH₂, 0-NH₂, NH- OH; or X^D is CR^d ₂-0-C(0)-(L^D)„-(D^D), CR^d ₂-S-C(0)-(L^D)_n-(D^D), CR^d ₂-0-C(S)- (L^D)_n-(D^D), CR^d ₂-S-C(S)-(LD)_n-(DD), CR^d ₂-NR^d-C(0)-(LD)_n-(DD), CR^d2-NR^d- C(S)-(L^D)_n-(D^D); and then Υ° is NHR^d, OH, SH; or X^D is C(0)-(L^D)_n-(D^D), C(S)-(L^D)_n-(D^D); and then Y° is CR^d ₂NHR<J, CR¾OH, CR¾SH, NH-NH₂, O- NH₂, NH-OH.

Preferably X^D is NR^<i-C(O)-(LD)_n-(D^D), and Y^D is NHR^d.

In Embodiment 3, A is CR^aY° and one of P, Q, X, Z is CR^aX^D, or P is CR^aY° and one of A, Y, Z, X is CR^aX^D, or Y is CR^aY° and X or P is CR^aX^D, or Q is CR^aY° and Z or A is CR^aX^D, or either Z or X is CR^aY^D and A or P is CR^aX^D, such that X^D and Y° are positioned in a trans conformation with respect to one another; the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR^a ₂, S, O, SiR¾ such that P and A are CR¾, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si, if present, is adjacent to CR¾ or O.

X^D is (O-C(O))_P-(L^D)n-(D^D), S-C(O)-(L^D)_n-(D^D), O-C(S)-(L^D)„- (D^D), S-C(S)-(LD)_n-(DD); Y is CR¾NHR^d CR¾OH, CR¾SH, NH-NH₂, O- NH₂, NH-OH; p = 0 or 1.

Preferably X^D is (O-C(O))_p-(LD)_n-(DD), with p=l, and Y^D is

CR¾NHRd.

In Embodiment 4, P is CR^aY° and Y is CR^aX^D, or A is CR^aY° and Q is CR^aX^D, or Q is CR^aY^D and A is CR^aX^D, or Y is CR^aY^D and P is CR^aX^D, such that X^D and Y⁰ are positioned in a trans conformation with respect to one another; the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR^a _¾ S, O, SiR¾ such that P and A are CR^a ₂, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si, if present, is adjacent to CR^a ₂ or O.

X^D is (O-C(O))_p-(LD)_n-(DD), S-C(O)-(L^D)n-(DD), O-C(S)-(LD)_n- (D^D), S-C(S)-(L^D)_n-(D^D); Y^D is NHR^d, OH, SH; p = 0 or 1.

Preferably X^D is (O-C(O))_p-(L^D)n-(D^D), with p=l, and Y^D is

NHR^d.

In Embodiment 5, Y is Y^D and P is CR^aX^D, or Q is Y° and A is

CR^aX^D; the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR^a ₂, S, O, SiR¾ such that P and A are CR^a ₂, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si, if present, is adjacent to CR¾ or O.

XD is (0-C(0))_p-(LD)_n-(DD), S-C(0)-(LD)_n-(DD), 0-C(S)-(LD)_n- (D^D), S-C(S)-(LD)_n-(DD), NR^d-C(0)-(LD)_n-(DD), NR^d-C(S)-(LD)_n-(D^D), C(O)- (L^D)_n-(D^D), C(S)-(LD)_n-(DD); YD is NH; p - 0 or 1.

Preferably X^D is NRd-C(0)-(LD)_n-(DD) or (0-C(0))_p-(LD)_n-(E ), with p = 0 or 1.

In Embodiment 6, Y is Y^D and P or Q is X^D, or Q is Y¹³ and A or Y is X^D; the remaining groups (from A,Y,Z,X,Q,P) being independently from each other CR¾, S, O, SiR¾ such that P and A are CR^a2, and no adjacent pairs of atoms are present selected from the group consisting of O-O, O-S, and S-S, and such that Si, if present, is adjacent to CR¾ or O.

X^D is N-C(0)-(L^D)n-(D^D), N-C(S)-(L^D)_n-(D^D); Y° is NH;

Preferably X^D is N-C(0)-(L^D)„-(D^D)

Also herein, (L^D)_n is an optional linker, with n = 0 or 1, preferably linked to T^Rvia S, N, NH, or O, wherein these atoms are part of the linker, which may consist of multiple units arranged linearly and/or branched. D^D is a Construct, preferably linked via N, or NH, wherein these atoms stay with the Construct after release thereof.

T, F each independently denotes H, or a substituent selected from the group consisting of alkyl, F, CI, Br, or I. It is preferred that when D^D is bound to T^R or L^D via NH, this

NH is a primary amine (-NH2) residue from D^D, and when D^D is bound via N, this N is a secondary amine (-NH-) residue from D^D.

It is further preferred that said N or NH comprised in D^D are bound to an aliphatic or aromatic carbon of D^D. In a preferred embodiment said N or NH comprised in D^D, which is bound to the Trigger, is protonated in physiological environment after release of D^D from the Trigger.

It is preferred that when L^D is bound to T^R via NH, this NH is a primary amine (-NH2) residue from L^D, and when L^D is bound via N, this N is a secondary amine (-NH-) residue from L^D. Similarly, it is preferred that when L^D is bound via O or S, said O or S are, respectively, a hydroxyl (-OH) residue or a sulfhydryl (-SH) residue from L^D.

It is further preferred that said S, N, NH, or O moieties comprised in L^D are bound to an aliphatic or aromatic carbon of L^D.

Where reference is made in the invention to a linker L^D this can be self-immolative or not, or a combination thereof, and which may consist of multiple self-immolative units. It will be understood that if L^D is not self-immolative, the linker equals a spacer S^p.

By way of further clarification, if p=0 and n=0, the species D^D directly constitutes the leaving group of the elimination reaction, and if p=0 and n=l, the self-immolative linker constitutes the leaving group of the elimination. The position and ways of attachment of linkers L^D and construct D^D are known to the skilled person (see for example Papot et al, Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 618-637).

Nevertheless, typical but non-limiting examples of self-immolative linkers L^D are benzyl-derivatives, such as those drawn below. On the right, an example of a self-immolative linker with multiple units is shown; this linker will degrade not only into CO2 and one unit of 4-aminobenzyl alcohol, but also into one l,3-dimethylimidazolidin-2-one unit.

X = O or S or NH or NR with R = alkyl or aryl

By substituting the benzyl groups of aforementioned self-immolative linkers L^D , preferably on the 2- and/or 6-position, it may be possible to tune the rate of release of the construct D^D, caused by either steric and/or electronic effects on the intramolecular elimination reaction. Synthetic procedures to prepare such substituted benzyl-derivatives are known to the skilled person (see for example Greenwald et al, J. Med. Chem., 1999, 42, 3657-3667 and Thornthwaite et al, Polym. Chem., 2011, 2, 773-790). Some examples of substituted benzyl-derivatives with different release rates are drawn below.

In a preferred embodiment, the TCO of formula (la) is an all-carbon ring. In another preferred embodiment, the TCO of formula (la) is a

heterocyclic carbon ring, having of one to two oxygen atoms in the ring, and preferably a single oxygen atom. Each R^a as above-indicated can independently be H, alkyl, aryl, OR', SR', S(=0)R"', S(=0)₂R"', S(=0)₂NR'R", Si-R'", Si-O-R'", OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', F, CI, Br, I, N₃, S0₂H, S0₃H, S0₄H, PO3H, P0₄H, NO, N0₂, CN, OCN, SCN, NCO, NCS, CF₃, CF₂-R', NR'R", C(=0)R', C(=S)R', C(=0)0-R', C(=S)0-R', C(=0)S-R', C(=S)S-R\ C(=0)NR'R", C(=S)NR'R", NR'C(=0)-R"', NR'C(=S)-R"', NR'C(=0)0-R"', NR'C(=S)0-R"', NR'C(=0)S-R"', NR'C(=S)S-R'", OC(=0)NR'-R"', SC(=0)NR'-R"',

OC(=S)NR'-R"', SC(=S)NR'-R"', NR'C(=0)NR"-R", NR'C(=S)NR"-R", CR'NR", with each R' and each R" independently being H, aryl or alkyl and R'" independently being aryl or alkyl;

Each R^b as above indicated is independently selected from the group consisting of H, alkyl, aryl, O-aryl, O-alkyl, OH, C(=0)NR'R" with R' and R" each independently being H, aryl or alkyl, R'CO-alkyl with R' being H, alkyl, and aryl;

Each R^c as above indicated is independently selected from the group consisting of H, alkyl, aryl, O-alkyl, O-aryl, OH; Each R^d as above indicated is independently selected from H, Ci-6 alkyl and Ci-6 aryl;

wherein two or more Ra^,b^,c^,d moieties together may form a ring;

Preferably, each R^a is selected independently from the group consisting of H, alkyl, O-alkyl, O-aryl, OH, C(=0)0-R', C(=0)NR'R", NR'C(=0)-R"', with R' and R" each independently being H, aryl or alkyl, and with R'" independently being alkyl or aryl.

The synthesis of TCO's as described above is well available to the skilled person. This expressly also holds for TCO's having one or more heteroatoms in the strained cycloalkene rings. References in this regard include Cere et al. Journal of Organic Chemistry 1980, 45, 261 and Prevost et al. Journal of the American Chemical Society 2009, 131, 14182.

In a preferred embodiment, the TCO of formula (la) is an all-carbon ring. In another preferred embodiment, the TCO of formula (la) is a

heterocyclic carbon ring, having of one to two oxygen atoms in the ring, and preferably a single oxygen atom.

In a preferred embodiment, the frcms-cyclooctene moiety satisfies formula (lb):

(lb)

wherein, in addition to the optional presence of at most two exocyclic bonds fixed in the same plane, each R^a independently denotes H, or, in at most four instances, a substituent selected from the group consisting of alkyl, aryl, OR', SR', S(=0)R"', S(=0)₂R'", S(=0)₂NR'R", Si-R'", Si-O-R'", OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', F, CI, Br, I, N₃, S0₂H, SO3H, SO4H, PO3H, PO4H, NO, N0₂, CN, OCN, SCN, NCO, NCS, CF₃, CF₂-R', NR'R", C(=0)R', C(=S)R', C(=0)0-R', C(=S)0-R', C(=0)S-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'C(=0)-R"', NR'C(=S)-R"', NR'C(=0)0-R"', NR'C(=S)0-R"', NR'C(=0)S-R"', NR'C(=S)S-R"', OC(=0)NR'-R"',

SC(=0)NR'-R"', OC(=S)NR'-R"', SC(=S)NR'-R"', NR'C(=0)NR"-R",

NR'C(=S)NR"-R", CR'NR", with each R' and each R" independently being H, aryl or alkyl and R'" independently being aryl or alkyl;

Each R^e as above indicated is independently selected from the group consisting of H, alkyl, aryl, OR', SR', S(=0)R"', S(=0)₂R"', Si-R'", Si-O-R'", OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', F, CI, Br, I, N₃, S0₂H, SO3H, PO3H, NO, NO¾ CN, CF₃, CF2-R', C(=0)R', C(=S)R', C(=0)0-R', C(=S)0-R', C(=0)S-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'C(=0)-R"', NR'C(=S)- R'", NR'C(=0)0-R'", NR'C(=S)0-R"\ NR'C(=0)S-R"', NR'C(=S)S-R"', NR'C(=0)NR"-R", NR'C(=S)NR"-R", CR'NR", with each R' and each R" independently being H, aryl or alkyl and R'" independently being aryl or alkyl;

wherein two R^{a θ} moieties together may form a ring;

and wherein T and F each independently denote H, or a substituent selected from the group consisting of alkyl, F, CI, Br, and I, and X^D is (O- C(0))_p-(LD)_n-(DD), S-C(0)-(LD)_n-(DD), 0-C(S)-(LD)_n-(DD), S-C(S)-(LD)_n-(DD), 0-S(0)-(LD)_n-(DD), wherein p = 0 or 1. Preferably, X^D is (0-C(0))_p-(LD)_n- (D^D), where p = 0 or 1, preferably 1, and n = 0 or 1.

Preferably, each R^a and each R^e is selected independently from the group consisting of H, alkyl, O-alkyl, O-aryl, OH, C(=0)0-R', C(=0)NR'R", NR'C(=0)-R"', with R' and R" each independently being H, aryl or alkyl, and with R'" independently being alkyl or aryl.

In the foregoing dienophiles, it is preferred that the at least two exocyclic bonds fixed in the same plane are selected from the group consisting of (a) the single bonds of a fused cyclobutyl ring, (b) the hybridized bonds of a fused aromatic ring, (c) an exocyclic double bond to an oxygen, and (d) an exocyclic double bond to a carbon.

The TCO, containing one or two X^D moieties, may consist of multiple isomers, also comprising the equatorial vs. axial positioning of substituents, such as X^D, on the TCO. In this respect, reference is made to Whitham et al. J. Chem. Soc. (C), 1971, 883-896, describing the synthesis and characterization of the equatorial and axial isomers of iran-s-cyclo-oct- 2-en-ol, identified as (IRS, 2RS) and (1SR, 2RS), respectively. In these isomers the OH substituent is either in the equatorial or axial position.

In a preferred embodiment, with reference to formula (la), for structures where the substituents of A and/or P, such as X^D and Υ° can be either in the axial or the equatorial position, the substituentis in the axial position.

Preferred dienophiles, which are optimally selected for D^D release believed to proceed via a cascade elimination mechanism, are selected from the following structures:

= rest of attached D^D, L^D-D^D

— = rest of attached D^D, L^D-D^D

= rest of attached D^D, L -D^D

Preferred dienophiles, which are optimally selected for D^D release beheved to proceed via a strain release mechanism, are selected from the following structures:

^■~^w = rest of attached D^D, L^D-D

In a further preferred embodiment, the dienophile is a compound selected from the following structures:

^•~^>~ = rest of attached D^D, L^D-D^D

In alternative embodiments, the dienophile is a compound selected from the following structures:

= rest of attached D^D, L^D-D^D

The dienophile of formula (la) and the diene are capable of reacting in an inverse electron- demand Diels-Alder reaction. Activation of the Liposome by the retro Diels-Alder reaction of the Trigger with the Activator leads to release of the Drug.

Below a reaction scheme is given for a [4+2] Diels-Alder reaction between the (3,6)-di-(2-pyridyl)-s-tetrazine diene and a trans- cyclooctene dienophile, followed by a retro Diels Alder reaction in which the product and dinitrogen is formed. The reaction product may

tautomerize, and this is also shown in the scheme. Because the trans- cyclooctene derivative does not contain electron withdrawing groups as in the classical Diels Alder reaction, this type of Diels Alder reaction is distinguished from the classical one, and frequently referred to as an "inverse electron demand Diels Alder reaction". In the following text the sequence of both reaction steps, i.e. the initial Diels-Alder cyclo-addition (typically an inverse electron demand Diels Alder cyclo-addition) and the subsequent retro Diels Alder reaction will be referred to in shorthand as "retro Diels Alder reaction" or "retro-DA". It will sometimes be abbreviated as "rDA" reaction. The product of the reaction is then the retro Diels-Alder

In a general sense, the invention is based on the recognition that a Construct can be released from frcms-cyclooctene derivatives satisfying formula (la) upon cyclooaddition with compatible dienes, such as tetrazine derivatives. The dienophiles of formula (la) have the advantage that they react (and effectuate Construct release) with substantially any diene.

Without wishing to be bound by theory, the inventors believe that the molecular structure of the retro Diels-Alder adduct is such that a spontaneous elimination reaction within this rDA adduct releases the Construct. Particularly, with reference to the aforementioned cascade- mediated mechanism, the inventors believe that appropriately modified rDA components lead to rDA adducts wherein the bond to the Construct on the dienophile is destabilized by the presence of a lone electron pair on the diene. Alternatively, with reference to the aforementioned strain release mechanism, the inventors beheve that the molecular structure of the retro Diels-Alder adduct is such that a spontaneous elimination or cyclization reaction within this rDA adduct releases the Construct.

Particularly, the inventors believe that appropriately modified rDA components, i.e. according to the present invention, lead to rDA adducts wherein the bond to the Construct on the part originating from the dienophile is broken by the reaction with a nucleophile on the part originating from the dienophile, while such an intramolecular reaction within the part originating from the dienophile is precluded prior to rDA reaction with the diene.

The general concept of using the retro-Diels Alder reaction in the activation of liposomes comprising channel proteins is illustrated in Scheme 1. Scheme 1: general scheme of activation of a masked liposome according to this invention.

Tri er

Trigger-Construct conjugate ene

Activation, retro Diels-Alder reaction (-N2)

vator retro Diels-Alder adduct

D release

Herein the "Construct" is a chemical assembly of a liposome having a lipid membrane (typically a 1 lipid bilayer enclosing a cavity), with a channel protein being present in said bilayer, particularly present in an outer bilayer thereof, the linkage of said channel protein to the Trigger always comprising an amino group.

In this scheme "TCO" stands for irans-cyclooctene. The term ircwis-cyclooctene is used here as possibly including one or more hetero- atoms, and particularly refers to a structure satisfying formula (la). In a broad sense, the inventors have found that - other than the attempts made on the basis of the Staudinger reaction - the selection of a TCO as the trigger moiety for a masked liposome Construct, provides a versatile tool to render unstable drug containing liposomes into stable drug containing liposomes, wherein the drug release occurs through a powerful, abiotic, bio -ortho onal reaction of the dienophile (Trigger) with the diene (Activator), viz the aforementioned retro Diels-Alder reaction, and wherein the masked liposome Construct is a Construct-dienophile conjugate.

It will be understood that in Scheme 1 in the retro Diels- Alder adduct as well as in the end product, the indicated TCO group and the indicated diene group are the residues of, respectively, the TCO and diene groups after these groups have been converted in the retro Diels- Alder reaction.

A requirement for the successful application of an abiotic bio- orthogonal chemical reaction is that the two participating functional groups have finely tuned reactivity so that interference with coexisting functionality is avoided. Ideally, the reactive partners would be abiotic, reactive under physiological conditions, and reactive only with each other while ignoring their cellular/physiological surroundings (bio-orthogonal). The demands on selectivity imposed by a biological environment preclude the use of most conventional reactions.

The inverse electron demand Diels Alder reaction, however, has proven utility in animals at low concentrations and semi-equimolar conditions (R. Rossin et al, Angewandte Chemie Int Ed 2010, 49, 3375- 3378). The reaction partners subject to this invention are strained trans- cyclooctene (TCO) derivatives and suitable dienes, such as tetrazine derivatives. The cycloaddition reaction between a TCO and a tetrazine affords an intermediate, which then rearranges by expulsion of dinitrogen in a retro-Diels— Alder cycloaddition to form a dihydropyridazine

conjugate. This and its tautomers is the retro Diels-Alder adduct.

Reflecting the suitability of the rDA reaction, the invention provides, in one aspect, the use of a tetrazine as an activator for the release, in a physiological environment, of a Construct (i.e. as defined above) hnked to a ir n-s-cyclooctene. In connection herewith, the invention also pertains to a tetrazine for use as an activator for the release, in a physiological environment, of a substance hnked to a iran-s-cyclooctene, and to a method for activating, in a physiological environment, the release of a substance linked to a iran-s-cyclooctene, wherein a tetrazine is used as an activator.

The present inventors have come to the non-obvious insight, that the structure of the TCO of formula (la), par excellence, is suitable to provoke the release of a Construct linked to it, as a result of the reaction involving the double bond available in the TCO dienophile, and a diene. The features believed to enable this are (a) the nature of the rDA reaction, which involves a re-arrangement of double bonds, which can be put to use in provoking an elimination cascade; (b) the nature of the rDA adduct that bears a dihydro pyridazine group that is non-aromatic (or another non- aromatic group) and that can rearrange by an ehmination reaction to form conjugated double bonds or to form an (e.g. pyridazine) aromatic group, (c) the nature of the rDA adduct that may bear a dihydro pyridazine group that is weakly basic and that may therefore catalyze elimination reactions. Alternatively, the feature believed to enable this is the change in nature of the eight membered ring of the TCO in the dienophile reactant as compared to that of the eight membered ring in the rDA adduct. The eight membered ring in the rDA adduct has significantly more conformational freedom and has a significantly different conformation as compared to the eight membered ring in the highly strained TCO prior to rDA reaction. A nucleophilic site in the dienophile prior to rDA reaction is locked within the specific conformation of the dienophile and is therefore not properly positioned to react intramolecularly and to thereby release the Construct. In contrast, and due to the changed nature of the eight membered ring, this nucleophilic site is properly positioned within the rDA adduct and will react intramolecularly, thereby releasing the Construct. According to the above, but without being limited by theory, we beheve that the Construct release is mediated by strain-release of the TCO-dienophile after and due to the rDA reaction with the diene Activator.

In a broad sense, the invention puts to use the recognition that the rDA reaction, using a dienophile of formula (la), as well as the rDA adduct embody a versatile platform for enabling provoked release of a Construct (i.e. based on a liposome as defined above) in a bioorthogonal reaction.

It is to be emphasized that the invention is thus of a scope well beyond specific chemical structures. In a broad sense, the invention puts to use the recognition that the rDA reaction using a dienophile of formula (la) as well as the rDA adduct embody a versatile platform for enabling provoked Construct release in a bioorthogonal reaction.

Reflecting this, the invention also presents the use of the inverse electron-demand Diels-Alder reaction between a ircms-cyclooctene and a tetrazine as a chemical tool for the release, in a physiological environment, of a bound substance. The fact that the reaction is bio-orthogonal, and that many structural options exist for the reaction pairs, will be clear to the skilled person. E.g., the rDA reaction is known in the art of pre-targeted medicine. Reference is made to, e.g., WO 2010/119382, WO 2010/119389, and WO 2010/051530. Whilst the invention presents an entirely different use of the reaction, it will be understood that the various structural possibilities available for the rDA reaction pairs as used in pre-targeting, are also available in the field of the present invention. The dienophile trigger moiety used in the present invention comprises a ircms-cyclooctene ring, the ring optionally including one or more hetero-atoms. Hereinafter this eight-membered ring moiety will be defined as a ir ns-cyclooctene moiety, for the sake of legibility, or abbreviated as "TCO" moiety. It will be understood that the essence resides in the possibility of the eight-membered ring to act as a dienophile and to be released from its conjugated Construct upon reaction. The skilled person is familiar with the fact that the dienophile activity is not necessarily dependent on the presence of all carbon atoms in the ring, since also heterocyclic monoalkenylene eight-membered rings are known to possess dienophile activity.

Thus, in general, the invention is not limited to strictly Construct-substituted ircms-cyclooctene. The person skilled in organic chemistry will be aware that other eight-membered ring-based dienophiles exist, which comprise the same endocyclic double bond as the trans- cyclooctene, but which may have one or more heteroatoms elsewhere in the ring. I.e., the invention generally pertains to eight-membered non- aromatic cychc alkenylene moieties, preferably a cyclooctene moiety, and more preferably a iran-s-cyclooctene moiety, comprising a conjugated Construct.

Other than is the case with e.g. medicinally active substances, where the in vivo action is often changed with minor structural changes, the present invention first and foremost requires the right chemical reactivity combined with an appropriate design of the Construct -conjugate. Thus, the possible structures extend to those of which the skilled person is familiar with that these are reactive as dienophiles.

It should be noted that, depending on the choice of nomenclature, the TCO dienophile may also be denoted E-cyclooctene. With reference to the conventional nomenclature, it will be understood that, as a result of substitution on the cyclooctene ring, depending on the location and molecular weight of the substituent, the same cyclooctene isomer may formally become denoted as a Z-isomer. In the present invention, any substituted variants of the invention, whether or not formally Έ" or "Z," or "cis" or "trans" isomers, will be considered derivatives of unsubstituted iran,s-cyclooctene, or unsubstituted E- cyclooctene. The terms "trans -cyclooctene" (TCO) as well as E-cyclooctene are used interchangeably and are maintained for all dienophiles according to the present invention, also in the event that substituents would formally require the opposite nomenclature. I.e., the invention relates to cyclooctene in which carbon atoms 1 and 6 as numbered below are in the E entgegen) or trans position.

Formula (1)

The present invention will further be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-hmiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.

It is furthermore to be noticed that the term "comprising", used in the description and in the claims, should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. Thus, the scope of the expression "a device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that with respect to the present invention, the only relevant components of the device are A and B.

In several chemical formulae below reference is made to "alkyl" and "aryl." In this respect "alkyl", each independently, indicates an aliphatic, straight, branched, saturated, unsaturated and/or or cyclic hydrocarbyl group of up to ten carbon atoms, possibly including 1-10 heteroatoms such as O, N, or S, and "aryl", each independently, indicates an aromatic or heteroaromatic group of up to twenty carbon atoms, that possibly is substituted, and that possibly includes 1-10 heteroatoms such as O, N, P or S. "Aryl" groups also include "alkylaryl" or "arylalkyl" groups (simple example: benzyl groups). The number of carbon atoms that an "alkyl", "aryl", "alkylaryl" and "arylalkyl" contains can be indicated by a designation preceding such terms (i.e. Ci-io alkyl means that said alkyl may contain from 1 to 10 carbon atoms). Certain compounds of the invention possess chiral centers and/or tautomers, and all enantiomers, diasteriomers and tautomers, as well as mixtures thereof are within the scope of the invention. In several formulae, groups or substituents are indicated with reference to letters such as "A", "B", "X", "Υ', and various (numbered) "R" groups. The definitions of these letters are to be read with reference to each formula, i.e. in different formulae these letters, each independently, can have different meanings unless indicated otherwise.

In all embodiments of the invention as described herein, alkyl is preferably lower alkyl (C1.4 alkyl), and each aryl preferably is phenyl.

The TCO is preferably an all-carbon TCO.

Activator-induced release

The Activator comprises a Bio -orthogonal Reactive Group, wherein this Bio-orthogonal Reactive Group of the Activator is a diene. This diene reacts with the other Bio-orthogonal Reactive Group, the

Trigger, and that is a dienophile (vide supra). The diene of the Activator is selected so as to be capable of reacting with the dienophile of the Trigger by undergoing a Diels-Alder cycloaddition followed by a retro Diels-Alder reaction, giving the Retro Diels-Alder adduct. This intermediate adduct then releases the Construct or Constructs, where this Construct release can be caused by various circumstances or conditions that relate to the specific molecular structure of the retro Diels-Alder adduct.

Without wishing to be bound by theory, the inventors believe that the Activator, in one embodiment, is selected such as to provoke Construct release via an ehmination or cascade elimination (via an intramolecular elimination reaction within the Retro Diels-Alder adduct). This ehmination reaction can be a simple one step reaction, or it can be a multiple step reaction that involves one or more intermediate structures. These intermediates may be stable for some time or may immediately degrade to the thermodynamic end product or to the next intermediate structure. When several steps are involved, one can speak of a cascade reaction. In any case, whether it be a simple or a cascade process, the result of the elimination reaction is that the Construct gets released from the retro Diels-Alder adduct. Without wishing to be bound by theory, the design of both components (i.e. the diene Activator and the dienophile Trigger) is such that the distribution of electrons within the retro Diels- Alder adduct is unfavorable, so that a rearrangement of these electrons must occur. This situation initiates the intramolecular (cascade) elimination reaction to take place, and it therefore induces the release of the Construct or Constructs. Occurrence of the elimination reaction in and Trigger release from the Construct is not efficient or cannot take place prior to the Retro Diels-Alder reaction, as the Trigger-Construct itself is relatively stable as such. Elimination can only take place after the Activator and the Trigger-Construct have reacted and have been assembled in the retro Diels-Alder adduct.

Without wishing to be bound by theory, the above two examples illustrate how the unfavorable distribution of electrons within the retro Diels-Alder adduct can be relieved by an elimination reaction, thereby releasing the Construct. In one scenario, the elimination process produces end product A, where this product has a conjugation of double bonds that was not present in the retro Diels-Alder adduct yet. Species A may tautomerize to end product B, or may rearrange to form end product C. Then, the non-aromatic dihydro pyridazine ring in the retro Diels-Alder adduct has been converted to the aromatic pyridazine ring in the end product C. The skilled person will understand that the distribution of electrons in the retro Diels-Alder adduct is generally unfavorable relative to the distribution of the electrons in the end products, either species A or B or C. Thus, the formation of a species more stable than the retro Diels- Alder adduct is the driving force for the (cascade) elimination reaction. In any case, and in whatever way the process is viewed, the Construct (here the amine Construct-NEh') is effectively expelled from the retro Diels- Alder adduct, while it does not get expelled from the Trigger-Construct alone.

The below scheme depicts a possible alternative release mechanism for the cascade elimination, in addition to the two discussed above. Without wishing to be bound by theory, the below examples illustrates how the unfavorable distribution of electrons within the retro Diels-Alder adduct may be relieved by an elimination reaction, thereby releasing the Construct. This process may evolve via various

tauromerisations that are all equilibria. Here, the rDA reaction produces tautomers A and B, which can interchange into one and other. Tautomer B can lead to the elimination into product C and thereafter into D.

As discussed above, in this invention, the releasing effect of the rDA reaction is, in one embodiment, caused by an intramolecular cyclization/elimination reaction within the part of the Retro Diels-Alder adduct that originates from the TCO dienophile. A nucleophilic site present on the TCO (i.e. the dienophile, particularly from the Y⁰ group in this Trigger, vide supra) reacts with an electrophihc site on the same TCO (particularly from the X^D group in this Trigger, vide supra) after this TCO reacts with the Activator to form an rDA adduct. The part of the rDA adduct that originates from the TCO, i.e. the eight membered ring of the rDA adduct, has a different conformation and has an increased conformational freedom compared to the eight membered ring in the TCO prior to the rDA reaction, allowing the nucleophilic reaction to occur, thereby releasing the Construct as a leaving group. The intramolecular cyclization/elimination reaction takes place, as the nucleophilic site and the electrophilic site have been brought together in close proximity within the Retro Diels-Alder adduct, and produce a favorable structure with a low strain. Additionally, the formation of the cyclic structure may also be a driving force for the intramolecular reaction to take place, and thus may also contribute to an effective release of the leaving group, i.e. release of the Construct. Reaction between the nucleophilic site and the electrophilic site does not take place or is relatively inefficient prior to the Retro Diels- Alder reaction, as both sites are positioned unfavorably for such a reaction, due to the relatively rigid, conformationally restrained TCO ring. The Liposomal composition itself is relatively stable as such and elimination is favored only after the Activator and the Liposomal composition have reacted and have been assembled in a retro Diels-Alder adduct that is subject to intramolecular reaction. In a preferred embodiment the TCO ring is in the crown conformation. The example below illustrates the release mechanism pertaining to this invention.

r gger con uga e

Trigger conjugate rDA adduct

Activator

The above example illustrates how the intramolecular cyclization/elimination reaction within the retro Diels-Alder adduct can result in release of a Construct. The rDA reaction produces A, which may tautomerize to product B and C. Structures B and C may also tautomerize to one another (not shown). rDA products A, B, and C may intramolecularly cyclize, releasing the bound moiety, and affording structures D, E, and F, which optionally may oxidise to form product G. As the tautomerization of A into B and C in water is very fast (in the order of seconds) it is the inventors' belief, that release occurs predominantly from structures B and C. It may also be possible that the nucleophilic site assists in expelhng the Construct species by a nucleophilic attack on the electrophilic site with subsequent release, but without actually forming a (stable) cyclic structure. In this case, no ring structure is formed and the nucleophilic site remains intact, for example because the ring structure is shortlived and unstable and breaks down with reformation of the nucleophilic site.

Without wishing to be bound by theory, the above example illustrates how the conformational restriction and the resulting

unfavorable positioning of the nucleophilic and electrophilic site in the TCO trigger is relieved following rDA adduct formation, leading to an elimination/cyclization reaction and release.

With respect to the nucleophilic site on the TCO, one has to consider that the site must be able to act as a nucleophile under conditions that may exist inside the (human) body, so for example at physiological conditions where the pH = ca. 7.4, or for example at conditions that prevail in malignant tissue where pH-values may be lower than 7.4. Preferred nucleophiles are amine, thiol or alcohol groups, as these are generally most nucleophilic in nature and therefore most effective.

The Activator is a diene. The person skilled in the art is aware of the wealth of dienes that are reactive in the Retro Diels-Alder reaction. The diene comprised in the Activator can be part of a ring structure that comprises a third double bond, such as a tetrazine (which is a preferred Activator according to the invention).

Generally, the Activator is a molecule comprising a heterocyclic moiety comprising at least 2 conjugated double bonds.

Preferred dienes are given below, with reference to formulae

(2)-(4).

In formula (2) R¹ is selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R\ OR', SR', C(=0)R', C(=S)R', C(=0)0-R', C(=0)S-R', C(=S)0-R', C(=S)S-R", C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=0)OR", NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR", NR'C(=0)NR"R", NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl; A and B each independently are selected from the group consisting of alkyl-substituted carbon, aryl substituted carbon, nitrogen, N⁺0-, N⁺R with R being alkyl, with the proviso that A and B are not both carbon; X is selected from the group consisting of O, N-alkyl, and C=0, and Y is CR with R being selected from the group consisting of H, alkyl, aryl, C(=0)OR', C(=0)SR', C(=S)OR', C(=S)SR', C(=0)NR'R" with R' and R" each independently being H, aryl or alkyl.

(3) A diene particularly suitable as a reaction partner for cyclooctene is given in formula (3), wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2-R', NO2, OR', SR', C(=0)R', C(=S)R', OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', S(=0)R', S(=0)2R"', S(=0)₂NR'R", C(=0)0-R', C(=0)S-R', C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=0)OR", NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR", OC(=0)NR'R", SC(=0)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=0)NR"R",

NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl; A is selected from the group consisting of N-alkyl, N-aryl, C=0, and CN-alkyl; B is O or S; X is selected from the group consisting of N, CH, C -alkyl, C-aryl, CC(=0)R', CC(=S)R\ CS(=0)R', CS(=0)₂R"', CC(=0)0-R', CC(=0)S-R', CC(=S)0-R', CC(=S)S-R', CC(=0)NR'R", CC(=S)NR'R", R' and R" each independently being H, aryl or alkyl and R'" independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and N⁺O.

(4)

Another diene particularly suitable as a reaction partner for cyclooctene is diene (4), wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', NO, NO2, OR, SR', CN, C(=0)R, C(=S)R', OC(=0)R", SC(=0)R"', OC(=S)R"', SC(=S)R"', S(=0)R', S(=0)₂R"', S(=0)₂OR, POsR'R", S(=0)₂NR'R", C(=0)0-R', C(=0)S- R, C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NRC(=0)OR", NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR", OC(=0)NR'R", SC(=0)NR'R", OC(=S)NRR", SC(=S)NR'R",

NR'C(=0)NR"R", NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl; A is selected from the group consisting of N, C-alkyl, C-aryl, and N⁺O-; B is N; X is selected from the group consisting of N, CH, C-alkyl, C-aryl, CC(=O)R', CC(=S)R', CS(=0)R', CS(=O)₂R'", CC(=0)0-R', CC(=0)S-R', CC(=S)0-R', CC(=S)S-R', CC(=O)NR'R", CC(=S)NR'R", R' and R" each independently being H, aryl or alkyl and R'" independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and N⁺O".

(5) (6) (7)

According to the invention, particularly useful dienes are 1,2- diazine, 1,2,4-triazine and 1,2,4,5-tetrazine derivatives, as given in formulas (5), (6) and (7), respectively.

The 1,2-diazine is given in (5), wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2-R', NO₂, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', S(=O)R', S(=O)₂R"', S(=O)₂NR'R", C(=O)O-R', C(=O)S-R',

C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=O)SR", NR'C(=S)SR", OC(=O)NR'R", SC(=O)NR'R", OC(=S)NR'R", SC(=S)NR'R",

NR'C(=O)NR"R", NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R" independently being aryl or alkyl; X and Y each independently are selected from the group consisting of O, N-alkyl, N-aryl, C=O, CN-alkyl, CH, C-alkyl, C-aryl, CC(=O)R', CC(=S)R\ CS(=O)R', CS(=O)₂R"', CC(=O)O-R', CC(=O)S-R', CC(=S)0-R', CC(=S)S-R',

CC(=O)NR'R", CC(=S)NR'R", with R' and R" each independently being H, aryl or alkyl and R'" independently being aryl or alkyl, where X-Y may be a single or a double bond, and where X and Y may be connected in a second ring structure apart from the 6-membered diazine. Preferably, X-Y represents an ester group (X = O and Y = C=O; X-Y is a single bond) or X- Y represents a cycloalkane group (X = CR' and Y = CR"; X-Y is a single bond; R' and R" are connected), preferably a cyclopropane ring, so that R' and R" are connected to each other at the first carbon atom outside the 1,2-diazine ring.

The 1,2,4-triazine is given in (6), wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2-R', N0₂, OR', SR'_; C(=0)R', C(=S)R', OC(=0)R"', SC(=0)R", OC(=S)R"'_; SC(=S)R"', S(=0)R', S(=0)₂R"', S(=0)₂NR'R", C(=0)0-R', C(=0)S-R', C(=S)0-R, C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=0)OR", NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR", OC(=0)NR'R", SC(=0)NR'R", OC(=S)NR'R", SC(=S)NR'R",

NR'C(=0)NR"R", NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R " independently being aryl or alkyl; X is selected from the group consisting of CH, C-alkyl, C-aryl, CC(=0)R', CC(=S)R\ CS(=0)R', CS(=0)₂R'", CC(=0)0-R', CC(=0)S-R', CC(=S)0-R', CC(=S)S-R', CC(=0)NR'R", CC(=S)NR'R", R' and R" each independently being H, aryl or alkyl and R'" independently being aryl or alkyl.

The 1,2,4,5-tetrazine is given in (7), wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF3, CF2-R', NO, N0₂, OR', SR', CN, C(=0)R', C(=S)R', OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', S(=0)R', S(=0)₂R'", S(=0)₂OR', P0₃R*R",

S(=0)₂NR'R", C(=0)0-R', C(=0)S-R', C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=0)OR",

NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR", OC(=0)NR'R", SC(=0)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=0)NR"R", NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl.

Electron -deficient 1,2-diazines (5), 1,2,4-triazines (6) or 1,2,4,5-tetrazines (7) are especially interesting as such dienes are generally more reactive towards dienophiles. Di- tri- or tetra-azines are electron deficient when they are substituted with groups or moieties that do not generally hold as electron-donating, or with groups that are electron-withdrawing. For example, R¹ and or R² may denote a substituent selected from the group consisting of H, alkyl, NO2, F, CI, CF3, CN, COOR, CONHR, CONR2, COR, SO2R, SO2OR, SO2NR2, PO3R2, NO, 2-pyridyl, 3- pyridyl, 4-pyridyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,4-pyrimidyl, 2,4 imidazyl, 2,5 imidazyl or phenyl, optionally substituted with one or more electron- withdrawing groups such as N0₂, F, CI, CF₃, CN, COOR, CONHR, CONR, COR, SO2R, SO2OR, SO2NR2, PO3R2, NO, Ar, wherein R is H or Ci-Ce alkyl, and Ar stands for an aromatic group, particularly phenyl, pyridyl, or naphthyl.

The 1,2,4,5-tetrazines of formula (7) are most preferred as

Activator dienes, as these molecules are most reactive in retro Diels-Alder reactions with dienophiles, such as the preferred TCO dienophiles, even when the R¹ and/or R² groups are not necessarily electron withdrawing, and even when R¹ and/or R² are in fact electron donating. Electron donating groups are for example OH, OR', SH, SR', NH₂, NHR', NR'R", NHC(=0)R", NR'C(=0)R", NHC(=S)R", NR'C(=S)R", NHSO2R", NR'S0₂R" with R' and R" each independently being alkyl or aryl groups. Examples of other electron donating groups are phenyl groups with attached to them one or more of the electron donating groups as mentioned in the list above, especially when substituted in the 2-, 4- and/or 6-position(s) of the phenyl group.

According to the invention, 1,2,4,5-tetrazines with two electron withdrawing residues, or those with one electron withdrawing residue and one residue that is neither electron withdrawing nor donating, are called electron deficient. In a similar way, 1,2,4,5-tetrazines with two electron donating residues, or those with one electron donating residue and one residue that is neither electron withdrawing nor donating, are called electron sufficient. 1,2,4,5-Tetrazines with two residues that are both neither electron withdrawing nor donating, or those that have one electron withdrawing residue and one electron donating residue, are neither electron deficient nor electron sufficient. The 1,2,4,5-tetrazines can be asymmetric or symmetric in nature, i.e. the R¹ and R² groups in formula (7) may be different groups or may be identical groups, respectively. Symmetric 1,2,4,5-tetrazines are more convenient as these Activators are more easily accessible via synthetic procedures.

We have tested several 1,2,4,5-tetrazines with respect to their ability as Activator to release a model Construct (e.g. benzyl amine, phenol) from a Trigger-Construct conjugate, and we have found that tetrazines that are electron deficient, electron sufficient or neither electron deficient nor electron sufficient are capable to induce the Construct release.

Furthermore, both symmetric as well as asymmetric tetrazines were effective.

Electron deficient 1,2,4,5 tetrazines and 1,2,4,5-tetrazines that are neither electron deficient nor electron sufficient are generally more reactive in retro Diels-Alder reactions with dienophiles (such as TCOs), so these two classes of 1,2,4,5-tetrazines are preferred over electron sufficient 1,2,4,5-tetrazines, even though the latter are also capable of inducing Trigger release in Trigger-Construct conjugates.

In the following paragraphs specific examples of 1,2,4,5- tetrazine Activators according to the second embodiment of this invention will be highlighted by defining the R¹ and R² residues in formula (7).

Symmetric electron deficient 1,2,4,5-tetrazines with electron withdrawing residues are for example those with R¹ = R² = H, 2-pyridyl, 3- pyridyl, 4-pyridyl, 2,4-pyrimidyl, 2,6-pyrimidyl, 3,5-pyrimidyl, 2,3,4-triazyl or 2,3,5-triazyl. Other examples are those with R¹ = R² = phenyl with COOH or COOMe carboxylate, or with CN nitrile, or with CONH₂,

CONHCH3 or CON(CH₃)₂ amide, or with SO3H or S0₃Na sulfonate, or with SO2NH2, SO2NHCH3 or S0₂N(CH₃)₂ sulfonamide, or with PO3H2 or POsNa2 phosphonate substituents in the 2-, 3- or 4- position of the phenyl group, or in the 3- and 5-positions, or in the 2- and 4-positions, or in the 2,- and 6-positions of the phenyl group. Other substitution patterns are also possible, including the use of different substituents, as long as the tetrazine remains symmetric. See below for some examples of these structures.

Symmetric electron sufficient 1,2,4,5-tetrazines with electron donating residues are for example those with R¹ = R² = OH, OR', SH, SR', NH₂, NHR', NR'2, NH-CO-R', NH-SO-R', NH-SO2-R', 2-pyrryl, 3-pyrryl, 2- thiophene, 3-thiophene, where R' represents a methyl, ethyl, phenyl or tolyl group. Other examples are those with R¹ = R² = phenyl with OH, OR', SH, SR', NH₂, NHR', NR'₂, NH-CO-R', NR"-CO-R', NH-SO-R' or NH-SO2-R' substituent(s), where R' represents a methyl, ethyl, phenyl or tolyl group, where R" represents a methyl or ethyl group, and where the substitution is done on the 2- or 3- or 4- or 2- and 3- or 2- and 4- or 2- and 5- or 2- and 6- or 3- and 4- or 3- and 5- or 3-, 4- and 5-position(s). See below for some examples of these structures.

Symmetric 1,2,4,5-tetrazines with neither electron withdrawing nor electron donating residues are for example those with R = R² = phenyl, methyl, ethyl, (iso)propyl, 2,4-imidazyl, 2,5-imidazyl, 2,3- pyrazyl or 3,4-pyrazyl. Other examples are those where R¹ = R² = a hetero(aromatic) cycle such as a oxazole, isoxazole, thiazole or oxazoline cycle. Other examples are those where R¹ = R² = a phenyl with one electron withdrawing substituent selected from COOH, COOMe, CN, CONH₂, CONHCH3, CON(CH₃)₂, SO3H, SOsNa, S0₂NH₂, SO2NHCH3, S02N(C1¾)2, PO3H2 or POsNa2 and one electron donating subsituent selected from OH, OR', SH, SR\ NH₂, NHR', NR'₂, NH-CO-R', NR"-CO-R', NH-SO-R' or NH-SO2-R' substituent(s), where R' represents a methyl, ethyl, phenyl or tolyl group and where R" represents a methyl or ethyl group. Substitutions can be done on the 2- and 3-, 2- and 4-, 2,- and 5-, 2- and 6, 3- and 4-, and the 3- and 5-positions. Yet other examples are those where R¹ = R² = a pyridyl or pyrimidyl moiety with one electron donating subsituent selected from OH, OR', SH, SR\ NH₂, NHR', NR'₂, NH-CO-R', NR"-CO-R', NH-SO-R' or NH-SO2-R' substituents, where R' represents a methyl, ethyl, phenyl or tolyl group and where R" represents a methyl or ethyl group. See below for some examples.

In case asymmetric 1,2,4,5-tetrazines are considered, one can choose any combination of given R¹ and R² residues that have been highlighted and listed above for the symmetric tetrazines according to formula (7), provided of course that R¹ and R² are different. Preferred asymmetric 1,2,4,5-tetrazines are those where at least one of the residues R¹ or R² is electron withdrawing in nature. Find below some example structures drawn.

Further considerations regarding the Activator

Preferred Activators are 1,2-diazines, 1,2,4-triazines and 1,2,4,5-tetrazines, particularly 1,2,4,5-tetrazines, are the preferred diene Activators. In the below, some relevant features of the Activator will be highlighted, where it will also become apparent that there are plentiful options for designing the right Activator formulation for every specific application.

According to the invention, the Activator, e.g. a 1,2,4,5- tetrazine, has useful and beneficial pharmacological and ph arm aco -kinetic properties, implying that the Activator is non-toxic or at least sufficiently low in toxicity, produces metabolites that are also sufficiently low in toxicity, is sufficiently soluble in physiological solutions, can be applied in aqueous or other formulations that are routinely used in pharmaceutics, and has the right log D value where this value reflects the

hydrophilic/hydrophobic balance of the Activator molecule at physiological pH. As is known in the art, log D values can be negative (hydrophilic molecules) or positive (hydrophobic molecules), where the lower or the higher the log D values become, the more hydrophilic or the more hydrophobic the molecules are, respectively. Log D values can be predicted fairly adequately for most molecules, and log D values of Activators can be tuned by adding or removing polar or apolar groups in their designs. Find below some Activator designs with their corresponding calculated log D values (at pH = 7.4). Note that addition of methyl, cycloalkylene, pyridine, amine, alcohol or sulfonate groups or deletion of phenyl groups modifies the log D value, and that a very broad range of log D values is accessible.

log D =

- 0 50 - 0.10 - 3.07 - 1.33 - 0.09 - 3.42

3.02 1.33 0.58 - 2 22 0.69 - 2.85 1.18

The given log D numbers have been calculated from a weighed method, with equal importance of the 'VG' (Viswanadhan, V. N.; Ghose, A. K.; Revankar, G. R.; Robins, R. K., J. Chem. Inf. Comput. Sci., 1989, 29, 163-172), 'KLOP* (according to Klopman, G.; Li, Ju-Yun.; Wang, S.; Dimayuga, M.: J.Chem.Inf.Comput.Sci., 1994, 34, 752) and HYS' (according to the PHYSPROP© database) methods, based on an aqueous solution in 0.1 M in Na⁺/K⁺ CI".

The Activator according to the invention has an appropriate reactivity towards the Trigger-Construct, and this can be regulated by making the diene, particularly the 1,2,4,5-tetrazines, sufficiently electron deficient. Sufficient reactivity will ensure a fast retro Diels-Alder reaction with the Trigger-Construct as soon as it has been reached by the Activator.

The Activator according to the invention has a good bio- availability, implying that it is available inside the (human) body for executing its intended purpose: effectively reaching the Trigger-Construct at the Target. Accordingly, the Activator does not stick significantly to blood components or to tissue that is non-targeted. The Activator may be designed to bind to albumin proteins that are present in the blood (so as to increase the blood circulation time, as is known in the art), but it should at the same time be released effectively from the blood stream to be able to reach the Trigger-Construct. Accordingly, blood binding and blood releasing should then be balanced adequately. The blood circulation time of the Activator can also be increased by increasing the molecular weight of the Activator, e.g. by attaching polyethylene glycol (PEG) groups to the Activator ('pegylation'). Alternatively, the PKPD of the activator may be modulated by conjugating the activator to another moiety such as a polymer, protein, (short) peptide, carbohydrate.

The Activator according to the invention may be multimeric, so that multiple diene moieties may be attached to a molecular scaffold, particularly to e.g. multifunctional molecules, carbohydrates, polymers, dendrimers, proteins or peptides, where these scaffolds are preferably water soluble. Examples of scaffolds that can be used are (multifunctional) polyethylene glycols, poly (propylene imine) (PPI) dendrimers, PAMAM dendrimers, glycol based dendrimers, heparin derivatives, hyaluronic acid derivatives or serum albumine proteins such as HSA.

Depending on the position of the Trigger-Construct (e.g.

inside the cell or outside the cell; specific organ that is targeted) the

Activator is designed to be able to effectively reach this Trigger-Construct. Therefore, the Activator can for example be tailored by varying its log D value, its reactivity or its charge. The Activator may even be engineered with a targeting agent (e.g. a protein, a peptide and/or a sugar moiety), so that the target can be reached actively instead of passively. In case a targeting agent is applied, it is preferred that it is a simple moiety (i.e. a short peptide or a simple sugar).

According to the invention, a mixture of different Activators can be applied. This may be relevant for regulation of the release profile of the drug.

The Activator that according to the invention will cause and regulate drug release at the target may additionally be modified with moieties giving extra function(s) to the Activator, either for in-vitro and/or for in-vivo studies or applications. For example, the Activator may be modified with dye moieties or fluorescent moieties (see e.g. S. Hilderbrand et al., Bioconjugate Chem., 2008, 19, 2297-2299 for 3-(4-benzylamino)- 1,2,4,5-tetrazine that is amidated with the near-infrared (NIR) fluorophore VT680), or they may be functionalized with imaging probes, where these probes may be useful in imaging modalities, such as the nuclear imaging techniques PET or SPECT. In this way, the Activator will not only initiate drug release, but can also be localized inside the (human) body, and can thus be used to localize the Trigger- Construct inside the (human) body. Consequently, the position and amount of drug release can be monitored. For example, the Activator can be modified with DOTA (or DTP A) ligands, where these ligands are ideally suited for complexation with ^mIn³⁺-ions for nuclear imaging. In other examples, the Activator may be linked to ¹²³I or ¹⁸F moieties, that are well established for use in SPECT or PET imaging, respectively. Furthermore, when used in combination with e.g. beta-emitting isotopes, such as Lu-177, or Y-90, liposome activation can be combined with localized radiotherapy in a pretargeted format. Preferred activators for use in the rDA believed to function via cascade-mediated release (i.e. cascade mechanism) are:

The 1,2,4,5-tetrazine given in Formula (8a) and (8b), wherein each R¹ and each R² independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', N0₂, OR', SR\ C(=0)R', C(=S)R', OC(=0)R"\ SC(=0)R"', OC(=S)R"\ SC(=S)R"', S(=0)R', S(=0)₂R"', S(=0)₂NR'R", C(=0)0-R', C(=0)S-R\ C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=0)OR", NR'C(=S)OR",

NR'C(=0)SR", NR'C(=S)SR", OC(=0)NR'R", SC(=0)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=0)NR"R", NR'C(=S)NR"R" with each R' and each R^: independently being H, aryl or alkyl, and R'" independently being aryl or alkyl.

Other preferred activators for use in the rDA believed to function cascade-mediated release (i.e. cascade mechanism) are:

Preferred activators for use in rDA believed to function via strain- release mediation, are:



The Activator can have a hnk to a desired moiety such as a peptide, protein, carbohydrate, PEG, or polymer. Preferably, these Activators for use in the cascade mechanism satisfy one of the following formulae: R = (link to) peptide, protein,

carbohydrate, PEG, polymer

Preferably, these Activators for use in the strain-release mechanism, satisfy one ofthe following formulae:

, protein,

polymer

Synthesis routes to the above activators are readily available to the skilled person, based on standard knowledge in the art. References to tetrazine synthesis routes include Lions et al, J. Org. Chem., 1965, 30, 318-319; Horwitz et al, J. Am. Chem. Soc, 1958, 80, 3155-3159; Hapiot et al, New. J. Chem., 2004, 28, 387-392, Kaim et al, Z. Naturforsch., 1995, 50b, 123-127.

The combination of and reaction between the TCO-Trigger and the Activator

Note that in cases of release of amine functional Constructs these can be e.g. primary or secondary amine, aniline, imidazole or pyrrole type of moieties, so that the Construct is varying in leaving group character. The drawn fused ring products may or may not tautomerize to other more favorable tautomers.

Hereunder, some nonlimiting model combinations of TCO-Constuct conjugates and tetrazine Activators illustrate the possibihties for cascade elimination induced model Construct release from the retro Diels-Alder adduct. The Construct, whether or not via a linker, is preferably attached to a carbon atom that is adjacent to the double bond in the TCO ring.

L^D = self-immolatoive linker

The above example of urethane (or carbamate) substituted TCOs gives release of an amine functional Construct from the adduct. The tetrazine Activator is symmetric and electron deficient.

L^D = self-immolatoive linker H, and R₂ = Bn-NH₂, or R, = Bn-NH₂ and R₂ = H

The above examples of urethane (or carbamate) substituted TCOs gives release of an amine functional Construct from the adduct. The tetrazine Activator is asymmetric and electron deficient. Note that use of an asymmetric tetrazine leads to formation of retro Diels-Alder adduct regiomers, apart from the stereo-isomers that are already formed when symmetric tetrazine are employed.

L^D = self-immolaloive linker

The above example of urethane (or carbamate) TCOs gives release of an amine functional Construct from the adduct. The tetrazine Activator is symmetric and electron sufficient.

The following schemes depict non-limiting examples illustrative for the various strain release release mechanisms that can be made to apply on the basis of the choice for the rDA reaction for activating a Trigger- Construct conjugate.

embodiment 1 tetrazine

embodiment 2

embodiment 4

tetrazine

embodiment 5

embodiment 6

L^D = self-immolatoive linker Masked liposomes

A masked liposome comprises one or more conjugates of the construct D^D and the Trigger T^R and comprises a liposome formulation that is capable of release of entrapped drugs after its release from the Trigger. Such a masked liposome may optionally have specificity for disease targets. It will be understood that the Construct can optionally be attached to the TCO derivative through a linker L^D or a self-immolative linker L^D, or a combination thereof, and which may consist of multiple (self-immolative, or non immolative) units. In the Trigger- Construct conjugate, the Construct D^D and the Trigger T^R - the TCO derivative- can be directly linked to each other. They can also be bound to each other via a linker or a self-immolative linker L^D. It will be understood that the invention encompasses any conceivable manner in which the dienophile Trigger is attached to the Construct.

It will be understood that formula la and lb describe the Trigger and describe how the Trigger is attached to D^D, L^D, S^p, but that species D^D, L^D, S^p are not part of the Trigger and should be viewed as seperate entities, as can be seen in e.g. Scheme 1.

It will be understood that the Construct is linked to the TCO in such a way that the Construct is eventually capable of being released after formation of the retro Diels-Alder adduct. Generally, this means that the bond between the Construct and the TCO, or in the event of a hnker, the bond between the TCO and the linker L^D, or in the event of a self- immolative hnker L^D, the bond between the linker and the TCO and between the Construct and the hnker, should be cleavable. Predominantly, the Construct is linked via N or NH, and the optional linker is linked via a hetero-atom, preferably via O, N, NH, or S. The cleavable bond is preferably selected from the group consisting of carbamate, thiocarbamate, carbonate, ether, ester, amine, amide, thioether, thioester, sulfoxide, and sulfonamide bonds.

The invention involves the manipulation of liposome membrane-embedded channel proteins, resulting in opening up of the protein channel and drug release from the liposome.

This aspect of the invention provides compositions comprising a vesicle, particularly a lipid vesicle, comprising a channel protein or functional fragment thereof, characterized in that the channel protein or fragment thereof comprises, in a side chain of an amino acid of said protein or fragment thereof, a Trigger. According to particular embodiments, vesicles are provided wherein a Trigger is hnked to a channel protein or fragment thereof through a sulphur, and always (as mentioned before) via an amino group. According to further embodiments, vesicles are provided wherein the Trigger is hnked to a cysteine in a channel protein or fragment thereof, always via an amino group. According to particular embodiments, the sulphur to which the Trigger is linked corresponds to a modified thiol function of a cysteine in the peptide or protein.

According to further embodiments modified peptides and proteins are provided wherein a cysteine side chain, carrying a Trigger as described herein, is located in a region of the peptides or proteins which upon introduction of a charge modulates the functionality of the peptide or protein. In further particular embodiments, vesicles are provided wherein a channel protein, which is a cysteine mutant of a wild-type channel protein is provided with a Trigger. In particular embodiments, vesicles are provided comprising a channel protein which is a mechanosensitive channel of large conductance (MscL), more particularly E. coli MscL comprising a Gly22Cys mutation, which is provided with a Trigger.

According to particular embodiments, vesicles are provided with a channel protein comprising a substituted amino acid, the side chain of the amino acid having the structure depicted in formula (10).

formula (10).

Yet another aspect of the present invention provides compounds for modifying thiol groups on a protein or peptide, which compounds have the general formula (11) :

TR-L^Dn-NR3-CRlR2-XS-S^p-ZS (11)

- wherein R3 is selected from the group consisting of H, alkyl, aryl, OR', C(=0)NR'R", R'CO-R", C(=0)-R', C(=0)0-R', C(=S)S-alkyl, C(=S)S-aryl,

C(=S)0-alkyl, C(=S)0-aryl, C(=0)S-alkyl, C(=0)S-aryl, NR'R", with R' and R" each independently being H, aryl or alkyl.

- wherein L^D is an optional self-eliminating linker with n= 0 or 1.

- wherein Rl and R2 are independently selected from the group consisting of H, alkyl, aryl, OR', SR', S(=0)R"', S(=0)₂R'", S(=0)₂NR'R", Si-R'", Si-O- R"\ OC(=0)R"', SC(=0)R"', OC(=S)R"\ SC(=S)R"', F, CI, Br, I, N₃, S0₂H, S0₃H, SO4H, PO3H, PO4H, NO, N0₂, CN, OCN, SCN, NCO, NCS, CF₃, CF2-R', NR'R", C(=0)R', C(=S)R', C(=0)0-R', C(=S)0-R', C(=0)S-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'C(=0)-R"', NR'C(=S)-R"',

NR'C(=0)0-R"', NR'C(=S)0-R"', NR'C(=0)S-R"', NR'C(=S)S-R"',

OC(=0)NR'-R"', SC(=0)NR'-R"', OC(=S)NR'-R"', SC(=S)NR'-R"',

NR'C(=0)NR"-R", NR'C(=S)NR"-R", CR'NR", with each R' and each R" independently being H, aryl or alkyl and R"' independently being aryl or alkyl; preferably Rl and R2 are independently selected from H, Ci-6 alkyl

- wherein X^s is selected from the group consisting of oxygen, sulfur, nitrogen, carbonyl, aromate, alkene, alkyn, ester, ether, thioester, thioether, amide, amine, imine, alkane, carbamate, carbonate, carboxylic acid ester, sulfate, sulfonate, aminooxy, disulfide, (oligo)dimethylsiloxane, sulfoxide, phosphate and phosphite

- wherein S^p is a spacer, and

- wherein Z^s is a thiol reactive group.

In a particular embodiment, X^s comprises a carbonyl group (e.g. the carboxylic acid ester CO-O) or consists of a carbonyl group.

Optionally CR1R2 represents an aromatic ring with or without further substituents. In particular embodiments the nature of the Rl and R2 group is chosen such that the compound after reaction with an Activator has a hydrophilic or polar nature

In particular embodiments of the compounds provided herein, Rl and R2 are independently selected from the group consisting of H, and a substituted or unsubstituted alkyl chain with a length of 1, 2, 3 or 4 carbons.

In further particular embodiments of compounds according to the invention having formula (11) described above, X^s is CO-O.

In further particular embodiments of compounds according to the invention having formula (11) described above, S^p is selected from the group consisting of an oxygen, sulfur, nitrogen, carbonyl, aromate, alkene, alkyn, ester, ether, thioester, thioether, amide, amine, imine, alkane, alkene, alkyne, carbamate, carbonate, carboxyhc acid ester, aromate, sulfate, sulfonate, aminooxy, disulfide, (ohgo)dimethylsiloxane, sulfoxide, phosphate and phosphite

Examples of spacers S^p are:

Examples of thiol reactive groups Z^s are:

Particular embodiments of the above-described compounds are compounds wherein S^p corresponds to a structure selected from one of the spacer formulae described herein.

Further particular embodiments of the above compounds are compounds wherein Z^s is a thiol reactive group selected from the group consisting of an acrylate, maleimide, halogen, disulfide, thioester and thiosulfonate. Further particular embodiments of the above compounds are compounds wherein Z^s is a thiol reactive group selected from one of the formulae described herein. in preferred embodiments the Trigger is linked to the channel protein according to one of the follo win g formulae.

in preferred embodiments the linkage of the Trigger to the protein satisfies one of the following formulae

In particular embodiments of the compounds of the invention

corresponding to formula (11) described above, Rl and R2 are H.

In further particular embodiments S^p is -CH2-CH2-.

In further particular embodiments X^s is CO-O.

In further particular embodiments Z^s is -S-S02-CH3.

In further particular embodiments n = 0

Det ailed descript ion The polypeptide can be of any length. Typically, the polypeptide has a length of between 25 and 100 amino acids, more typically, less than 70 amino acids. The polypeptide can be isolated from a natural source, can be a recombinant protein or can be a synthetic protein produced by chemical peptide synthesis. The polypeptide can correspond to a naturally occurring wild-type polypeptide or can contain one or more mutations. These mutations can correspond to naturally occurring mutations or can be mutations not encountered in nature and introduced by recombinant technology or during chemical peptide synthesis. The polypeptides used in the methods of the present invention may comprise a cysteine residue located in an appropriate position in the wild-type protein, and may thus be ready for modification according to methods of the present invention. In these embodiments, the thiol of the side chain of the cysteine amino acid present within the polypeptide is reacted with the thiol-reactive agent corresponding to formula (11). Alternatively, where the polypeptide does not naturally comprise a cysteine in the appropriate position, a cysteine can be introduced by recombinant technology or can be introduced during peptide synthesis. In further embodiments of the methods of the invention a thiol function is incorporated in the polypeptide of interest via modification of an NH2 group (Lys, Nterminus), a COOH group (Asp, Glu, C-terminus) or an imidazole group (His) using protein modification reagents such as available from e.g. Pierce (Rockville, IL). In further embodiments, a thiol function is incorporated into the polypeptide via peptide synthesis using less frequent natural amino acids such as for example homocysteine or mercaptovaline or using synthetically prepared non-naturally occurring amino acids with a thiol function.

According to particular embodiments of the modification methods of the present invention, the polypeptide is a channel protein and modification methods of the present invention are used to introduce into the

polypeptide a group with a manipulate able charge. More particularly, the polypeptide is a Large-conductance mechanosensitive channel polypeptide or MscL polypeptide, of which the structure/function is charge sensitive. Introducing a manipulateable charge onto an MscL polypeptide makes it possible to manipulate the structure/function of the MscL, i.e. the opening or closing of the channel. The modification of an MscL on cysteine with compound with formula (11) and the subsequent generation of a primary amine in the reaction with the Activator is shown in the scheme directly below. The Trigger group on the modified polypeptides of the present invention carries no charge. Upon reaction with an Activator, the Trigger is removed, leading to the formation of a primary amine, which is protonated at a pH value below its pKa (7,75), which in turn leads to opening of the pore and drug release.

In particular embodiments, of the present invention the modified polypeptides are polypeptides wherein the introduction of a charge changes the function of the polypeptide.

Particular embodiments of the modified polypeptides of the present invention are channel proteins or functional fragments thereof. Channel proteins are proteins which form channels in membrane structures which channels allow the selective passage of certain molecules and compounds. Fragments of these channel proteins, i.e. truncated at N- and/or C- terminus can maintain the channel protein function of allowing selective passage. In a more particular embodiment the present invention provides modified large-conductance mechanosensitive channel proteins (MscL). Members of the MscL family show considerable variation in amino acid sequence identity over their complete protein sequences (up to 60%) but all have the conserved sequence motif of Protein Family 01741 [Large- conductance mechanosensitive channel, MscL family]. Particular examples are E. coli MscL [Genbank Accession AAA58088 ] or L. lactis MscL

[Genbank Accession 5 NP_268258].

MscL proteins do not naturally comprise cysteines or thiol side chains. However, different amino acids in MscL proteins can be modified into cysteine for the purposes of the present invention, i.e. introducing a manipulateable charge which affects the conformation of the protein and thus the opening/closing of the channel. These amino acids are located in the regions corresponding to amino acids 1 to 14, 15-45, 46-75 and 76-100 of E coli. MscL.

In a particular embodiment of the present invention, the modified channel protein is E. coli MscL with the mutation Gly22Cys, L. lactis MscL with the mutation Gly20Cys or another protein of the MscL family mutated at the amino acid corresponding to Gly22 of E. coli MscL, and comprising a Trigger conjugate as described in formula (11). It has been demonstrated that modification of this residue into a charged derivative results in the opening of the protein (Yoshimura et al., 1999, cited above).

In further particular embodiments, the modified polypeptide corresponds to the E. coli MscL with the double mutation Gly22Cys, Val23Cys or another MscL protein with this double cysteine mutation at the

corresponding amino acids comprising a Trigger conjugate as described in formula (11).

In particular embodiments, the invention provides modified MscLs which, in addition to the Trigger, comprise genetically engineered changes in the outside loop, such as receptor recognising domains (e.g. RGD) or antigen binding parts of an antibody to that loop to obtain target binding of the MscL to a particular tissue organ or cell type.

In yet other embodiments the modified channel protein is a Shaker K+ potassium channel protein comprising a Trigger conjugate as described in formula (11).. Sukhareva et al. (2004) in J. Gen. Physiol. 122, 541-566, disclose that the introduction of a charged amino acid at position 475 in Shaker H4 results in the opening of the channel. Accordingly, a particular embodiment of a modified channel protein according to the invention is a Shaker protein comprising a Trigger conjugate as described in formula (11). linked to a thiol side chain. More particularly, the Shaker protein is a protein comprising mutation Pro475Cys in Shaker H4 (Accession P08510) or equivalent positions in proteins of the Shaker family such as Pro406Cys in Kv2.1, Gly229Cys in KvAP, Alal08Cys in KcsA, Glu92Cys in MthK, and Glyl43Cys in KirBac.

The compositions and methods making use of the vesicles and/or

compositions described herein are particularly suitable for the delivery of small hydrophilic molecules. These can be released from liposomes upon opening of channel proteins comprised within the liposome membrane. Loading of the lipid vesicle can be accomplished in many ways as long as the small molecules are dissolved in a hydrophilic solvent, which is separated from the surrounding hydrophilic solvent by a lipid bilayer.

Liposomes are known as convenient delivery vehicles for biologically active molecules. In a preferred embodiment use is made of long circulating liposomes , which are typically 150 nm or smaller, neutral and have a specific composition : cholesterol-containing with either

phosphatidylcholine and an hydrophilic polymer (e.g. PEG) or

sphingomyelin etc. In particular embodiments, the vesicles of the present invention further comprise, in addition to one or more modified channel proteins, one or more Targeting agents T^T. Suitable liposome formulations for use as drug delivery vehicles are known in the art, some aspects of which are also described below. Methods to incorporate channel proteins in liposomes are described in detail e.g. in WO2005/051902.

The vesicles and compositions of the present invention are particularly suitable for the delivery of hydrophilic small molecules that are small enough to pass through the pore of a channel protein. Typically these small molecule have a diameter of no more than 60 Angstrom, more particularly no more than 50 Angstrom and more particularly no more than 40 Angstrom. Example hereof are peptides and also proteins with a length of about 60 to 70 amino acids.

Vesicle-Forming" Lipid Component

The liposome composition of the present invention, is composed primarily of vesicle-foriniiig lipids. Such a vesicle-forming lipid is one which (a) can form spontaneously into bilayer vesicles in water, as exemplified by the phospholipids, or (b) is stably incorporated into lipid bilayers, with its bydropbobic moiety in contact with the interior, hydrophobic region of the bilayer membrane, and its head group moiety oriented toward the exterior, polar surface of the membrane.

The vesicle-forming lipids of this type are preferably ones having two hydrocarbon chains, typically acyl chains, and a head group, either polar or nonpolar. There are a variety of synthetic vesicle-forming lipids and naturally-occurring vesicle-forming lipids, including the phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phospliatidic acid, phosphatidyls ositol, and sphingomyelin, where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of imsaturation. The above-described lipids and

phospholipids whose acyl chains have varying degrees of saturation can be obtained commercially or prepared according to published methods. Other suitable lipids include glycolipids and sterols such as cholesterol.

Additionally, the vesicle-forming- lipid is selected to achieve a specified degree of fluidity or rigidity, to control the stability of the liposome in. serum and to control the rate of release of the entrapped agent in the liposome.

Liposomes having a more rigid, lipid, bilayer, or a liquid, crystalline bilayer, are achieved by incorporation of a relatively rigid lipid, e.g., a lipid having a relatively high phase transition temperature, e.g., up to 60 °C. Rigid, i.e., saturated., lipids contribute to greater membrane rigidity in the lipid bilayer. Other lipid components, such as cholesterol, are also known to contribute to membrane rigidity n lipid bilayer st uctures. On the other hand, lipid fluidity is achieved by incorporation of a relatively fluid lipid, typically one having a. lipid phase with a relatively low liquid to liquid-crystalline phase transition temperature, e.g., at or below room temperature. In one embodiment of the invention, the liposom es are prepared, with a relatively rigid lipid to impart rigidity to the lipid bilayer. In this embodiment, the lipids forming the liposomes have a phase transition temperature of between about 37-70 °C. In a preferred embodiment, the vesicle forming lipid is distearyi phosphatidylcholine (DSPC), which has a phase transition temperature of 62 °C.

Hydrophilic polymers

Suitable hydrophilic polymers for use in the conjugates, where the polymers are also intended to extend liposome-circulation time, include polyvinylpyrrolidone, poly inylmeihylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline,

polyhydroxypropylmethaerylamide, polymethacrylamide,

poi dmiet ylacrylamide, polyhydroxypropylmethacrylate,

polyhydroxyethylaerylate, hydroxymethylcellulose, liydroxyethylceilulose, polyethyleneglycol, and polyaspartamide.

In a preferred embodiment, the hydrophilic polymer i.s polyethyleneglycol, preferably as a PEG chain having a molecular weight between 500-10,000 daltons, typically between 1,000-5,000 daltons.

The surface coating on the liposome provided by the hydrophilic polymer chains provides colloidal stability and, at a sufficient polymer surface density, serves to protect the liposomes from uptake b the reticulo- endothelial system,

an extended blood circulation lifetime for the liposomes to reach the target cells. The extent of enhancement of blood circulation time is preferably severalfold over that achieved in the absence of the polymer coating, as described in U.S. Pat. No. 5,013,556.

Targeting agents T^T

The kits and method of the invention are very suitable for use in targeted delivery of drugs.

A " target" as used in the present invention relates to a target for a targeting agent for therapy. For example, a target can be any molecule, which is present in an organism, tissue or cell. Targets include cell surface targets, e.g. receptors, glycoproteins, peptides, carbohydrates, monosacharides,

polysaccharides; structural proteins, e.g. amyloid plaques; abundant

extracellular targets such as stroma, extracellular matrix targets such as growth factors, and proteases; enzymes; and/or foreign bodies, e.g. pathogens such as viruses, bacteria, fungi, yeast or parts thereof. Examples of targets include compounds such as proteins of which the presence or expression level is correlated with a certain tissue or cell type or of which the expression level is up regulated or down-regulated in a certain disorder. According to a particular embodiment of the present invention, the target is a protein such as a

[internalizing or non-internalizing] receptor.

Examples of targets include somatostatin receptor, transferrin receptor, monoamine oxidase, muscarinic receptors, myocardial sympatic nerve system, leukotriene receptors, e.g. on leukocytes, urokinase plasminogen activator receptor (uPAR), folate receptor, apoptosis marker, (anti-Jangiogenesis marker, gastrin receptor, GPIIb/IIIa receptor and other thrombus related receptors, fibrin, calcitonin receptor, tuftsin receptor, integrin receptor, fibronectin, VEGF/EGF and VEGF/EGF receptors, TAG72, CEA, CD19, CD20,CD22, CD40, CD45, CD74, CD79, CD105, CD138, CD174, CD227, CD326, CD340, MUC1, MUC16, GPNMB, PSMA, Cripto, Tenascin C, Melanocortin-1 receptor, CD44v6, G250, HLA DR, ED-B, TMEFF2 , EphB2, EphA2, FAP, Mesothelin, GD2, CAIX, 5T4, matrix metalloproteinase (MMP), VCAM-1, ICAM-1, PECAM-1, P/E/L-selectin receptor, LDL receptor, P-glycoprotein, neurotensin receptors, neuropeptide receptors, substance P receptors, NK receptor, CCK receptors, sigma receptors, interleukin receptors, insulin receptor, liver hepatocytes receptor, herpes simplex virus tyrosine kinase, human tyrosine kinase.

In order to allow specific targeting of the above-listed targets, the targeting agent T^T can comprise compounds including but not limited to antibodies, antibody fragments, e.g. Fab2, Fab, scFV, diabodies, triabodies, VHH, antibody (fragment) fusions (eg bi-specific and trispecific mAb fragments), proteins, peptides, e.g. octreotide and derivatives, VIP, MSH, LHRH, chemotactic peptides, bombesin, elastin, peptide mimetics, carbohydrates, monosacharides, polysaccharides, viruses, whole cells, (e.g. bone marrow stem cells), drugs, polymers, chemotherapeutic agents, receptor agonists and antagonists, cytokines, hormones, steroids, vitamins. Examples of organic compounds envisaged within the context of the present invention are, or are derived from, estrogens, e.g.

estradiol, androgens, progestins, corticosteroids, methotrexate, folic acid, and cholesterol. In a preferred embodiment, the targeting agent T^T is an antibody. According to a particular embodiment of the present invention, the target is a receptor and a targeting agent is employed, which is capable of specific binding to the target. Suitable targeting agents include but are not limited to, the ligand of such a receptor or a part thereof which still binds to the receptor, e.g. a receptor binding peptide in the case of receptor binding protein ligands. Other examples of targeting agents of protein nature include interferons, e.g. alpha, beta, and gamma interferon, transferrin, interleukins, and protein growth factor, such as tumor growth factor, e.g. alpha, beta tumor growth factor, platelet- derived growth factor (PDGF), uPAR targeting protein, apolipoprotein, LDL, annexin V, endostatin, and angiostatin. Alternative examples of targeting agents include DNA, RNA, PNA and LNA.

According to a further particular embodiment of the invention, the target and targeting agent are selected so as to result in the specific or increased targeting of a tissue or disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme. This can be achieved by selecting targets with tissue-, cell- or disease- specific expression. For example, membrane folic acid receptors mediate intracellular accumulation of folate and its analogs, such as methotrexate. Expression is limited in normal tissues, but receptors are overexpressed in various tumor cell types.

Preparation

Attachment of a lipid, to a Hydrophilic Polymer such, as PEG is known in the art. As described above, in one embodiment of the invention, the liposomes include a ligand or T^T for targeting the liposomes to a selected, cell type or another liposome containing the proper receptor. The T^T is bound, to the liposome by covalent attachment to the free distal end. of a lipid-anchored hydrophilic polymer chain. in one embodiment^" of the invention, the hydiOphilie polymer chain is PEG, and several methods for attachment of ligands to the distal ends of PEG chains have been described (see e.g. US5891468 and refs therein). In these methods, the inert terminal methoxy group of mPEG is replaced with a reactive functionality suitable for conjugation reactions, such as an amino or hyd.razi.de group. The end functionalized. PEG is attached to a lipid, typically DSPE. The functionalized PEG-DSPE derivatives are employed in liposome formation and the desired ligand is attached, to the reactive end of the PEG chain before or after liposome formation. For example, where the targeting moiety is an antibody or an antibody fragment, the polymer chains are functionalized to contain reactive groups suitable for coupling with, for example, sulfhydryls, amino groups, and aldehydes or ketones (typically derived, from mild, oxidation of carbohydrate portions of an antibody) present in the antibody. Examples of such PEG-terminal reactive groups include maleimide (for reaction with sulfhydryl groups), N- hydroxysuccinimide (NHS) or NHS-carbonate ester (for reaction with primary amines), hydxazide or hydrazine (for reaction with, aldehydes or ketones), iodoacetyl (preferentially reactive with sulfhydryl groups) and dithiopyridme (t ol-reactive). Another example is the attachment of folic acid to a DSPE-PEG conjugate as described in US6936272. Folic acid is mixed with amino-PEG-DSPE and reacted in the presence of N-hydroxy-s- norbornene-2,3-(hcarboxyhc acid imide (HONB) and

dicyclohexylcarbodiimide (DCC) to form a folic acid-PEG-DSPE conjugate. This conjugate is included in the lipid mixture during liposome

preparation to form liposomes including a folic acid targeting ligand.

Preferably, in attaching a targeting agent T^T to a PEG-functionalized lipid, the T^T does not suffer any loss of activity. It is also possible to incorporate the T^T-polymer -lipid conjugate into preformed liposomes by insertion, where the ligand-polymer-lipid conjugate is incubated with the preformed liposomes under conditions suitable to allow the conjugate to become incorporated into the liposome lipid bilayer. The insertion technique has been described in the art, for example in U.S. Pat. No. 6,056,973. As described above, the liposomes optionally contain a T^T bound to the surface of the lipid by attachment to surface lipid components. Generally, such a ligand is coupled to the polar head group of a vesicle-forming lipid and various methods have been described for attachment of iigands to lipids. In one preferred method, the T is coupled to the lipid, by a coupling reaction described below, to form a lipid conjugate. This conjugate is added to a solution of lipi ds for formation, of liposomes, as will be described. In another method, a vesicle -forming' lipid activated for covalent attachment of T^T is incorporated into liposomes. The formed liposomes are exposed to the T^T to achieve attachment of the T^T to the activated lipids.

A variety of methods are available for preparing a conjugate composed of an T^T arid a vesicle-forming lipid. For example, water-soluble, amine- containing T^T can be covalently attached to lipids, such as

phosphatidylethanolamine, by reacting the aniine-eori aming moiety with a lipid which has been derivatized to contain an activated ester of N- hy diOxy-succinimide . As another example, biomolecules, and in particular large biomolecules such as proteins, can be coupled to lipids according to reported, methods. One method involves Schiff-base formation between an aldehyde group on a lipid, typically a phospholipid, and. a primar amino acid on the T^T. The aldehyde group is preferably formed by periodate oxidation of the lipid. The coupling reaction, after removal of the oxidant, is carried out in the presence of a reducing agent, such as dithiothreitoi, as described by Heath, (1981). Typical aldehyde-lipid precursors suitable in the method include ia.ctosjdceramide, trihexosylceramine, galacto cerebrosicie,

phosphatidylglycerol, phosphatidylinositol and gangliosides.

A second general coupling method is applicable to thiol-T^T, and involves formation of a disulfide or thioether bond between a lipid and. the T^T. In the disulfide reaction, a lipid amine, such as phosphatidyl-ethanolamine, is modified to contain a pyridyldithio derivative which can react wit an exposed thiol group in the T^T. Reaction conditions for such a method can be found, in Martin (1981). The thioether coupling method, described by Martin (1982), is carried out by forming a sulf ydryl-reactive phospholipid, such as N-(4)P~nialeimid.oplienyl(¾uiyry])phospbatidyletb.anolamine, and reacting the lipid with the thiol-containing T^T.

Another method for reacting an T^T with a lipid involves reacting the affinity moiety with a lipid, which has bee derivatized to contain an activated ester of -hydroxysiicdnimide. The reaction is typically carried out in the presence of a mild detergent, such as deoxycholate. Like the reactions described, above, this coupling- reaction is preferably performed prior to incorporating the lipid into the liposome,

The above-described coupling techniques are exemplary and it will be appreciated that other suitable methods are known in the art and have been described, for example in U.S. Pat. Nos. 4,605,630, 4,731,324,

4,429,008, 4,622,294 and 4,483,929.

The liposomes of the invention are preferably prepared to have

substantially homogeneous sizes in a selected size range, typically between about 0.01 to 0.5 microns, more preferably between 0.03-0.40 microns. One effective sizing method for RE Vs and MLVs involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size in the range of 0.03 to 0.2 micron, typically 0.05. 0.08, 0.1, or 0.2 microns. The pore size of the membrane corresponds roughly to the largest sizes of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane. Homogenization methods are also useful for down-sizing liposomes to sizes of 100 am or less (Martin, 1990). In a preferred embodiment of the present invention , the liposomes are extruded through a series of polycarbonate filters with pore sizes ranging from 0.2 to 0.08 micron resulting' in liposomes having diameters in the approximate ra ge of 120+/-10 nm.

Administration

In the context of the invention, the liposome is administered first, and it will take a certain time period before the liposome has reached the Target. This time period may differ from one application to the other and may be minutes, days or weeks. After the time period of choice has elapsed, the Activator is administered, will find and react with the liposome and will thus activate Drug release at the Target. The reaction between the Trigger and the Activator may occur extracellularly or intracellulary, or both.

The compositions of the invention can be administered via different routes including subcutaneous, intramuscular, interlesional (to tumors), ntertracheal by inhalation, topical, internasal, intraocular, via direct injection into organs and intravenous. Formulations suitable for these different types of administrations are known to the skilled person. Liposomes or Activators according to the invention can be administered together with a pharmaceutically acceptable carrier. A suitable

pharmaceutical carrier as used herein relates to a carrier suitable for medical or veterinary purposes, not being toxic or otherwise unacceptable. Such carriers are well known in the art and include sahne, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. The preferred mode of administration is intravenous injection.

It will be understood that the chemical entities administered, viz. the liposome and the activator, can be in a modified form that does not alter the chemical functionality of said chemical entity, such as salts, hydrates, or solvates thereof.

After administration of the liposome, and before the administration of the Activator, it is possible to remove excess Liposome by means of a Clearing Agent in cases when Liposome activation in circulation is undesired and when natural Liposome clearance is insufficient. A Clearing Agent is an agent, compound, or moiety that is administered to a subject for the purpose of binding to, or complexing with, an administered agent (in this case the Liposome) of which excess is to be removed from circulation. The Clearing Agent is capable of being directed to removal from circulation. The latter is generally achieved through hver receptor-based mechanisms, although other ways of secretion from circulation exist, as are known to the skilled person. In the invention, the Clearing Agent for removing circulating Liposome, preferably comprises a diene moiety, e.g. as discussed above, capable of reacting to the TCO moiety of the Liposome.

The Trigger and Activator can be selected such to achieve a specific release kinetics, which is a feature that can advantageously utilized to vary and tailor the release rate of an entrapped agent. In this manner one can choose to effect a slow drug release or a burst release.

Drugs

Entrapped in the liposomes is a therapeutic agent or drug for delivery to the target. A variety of therapeutic agents can be entrapped in lipid vesicles, including water-soluble agents that can be stably encapsulated in. the aqueous compartment of the vesicles, lipophilic compounds that stably partition in the lipid phase of the vesicles, or agents that can be stably attached, e.g., by electrostatic attachment to the outer vesicle surfaces. Exemplary water-soluble compounds include small, water-soluble organic compounds, peptides, proteins, DNA plasmids, oligonucleotides and gene fragments. The liposome-entrapped compound may also be an imaging agent for tracking progression of a disease. The entrapped agent may also be a reporter molecule, such as an enzyme or a fluorophore. for use in assays. The drug or agent to be delivered may be a polynucleotide capable of expressing a selected protein, when taken up by a target cell, an

oligonucleotide or oligonucleotide analog designed for binding to a. specific- sequence nucleic acid in the target cells (e.g. siRNA, antisense

oligonucleotide), or any other therapeutic polymer or small-molecule therapeutic or diagnostic agent.

Liposomes can contain an entrapped gene (cDNA plasmid) to be delivered to target cells, for gene therapy, A variety of gen es for treatment of various conditions have been described, and coding sequences for specific genes of interest can be retrieved from DNA sequence databanks, such as GenBank or EMBL. The selected coding sequences may encode any of a variety of different types of proteins or polypeptides, depending on the particular application. For example, the fusogenic liposome may be used to introduce sequences encoding enzymes into, e.g., stem cells or lymphocytes of individuals suffering from an enzyme deficiency. For instance, in the case of individuals with adenosine deaminase (ADA) deficiency, sequences encoding ADA may be transfected into stem cells or lymphocytes of such individuals.

In related applications, the liposomes may contain genes encoding any of a variety of circulating proteins, such as ai-aniitrypsin, clotting factors (e.g.. Factor VIII, Factor IX) and globins (e.g., β-globrn,

hemoglobin), for the treatment of hemophilia, sickle-cell, anemia and other blood-related diseases. Other examples of gene coding sequences suitable for use with the present invention include sequences encoding structural proteins; receptors, such as low density lipoprotein receptor (LDL-R) for transfection of hepatocytes to treat LDL-defieient patients, human CD4 and soluble forms thereof, and the like; transmembrane proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) for treatment of cystic fibrosis patients; signalling molecules; cytokines, such as various growth factors (e.g., TGF-alpha_., TGF-beta, EGF, FGF, IGF, NGF, PDGF, CGF, CSF, SCF), interleukins, interferons, erythropoietin, and the like, as well as receptors for such cytokines; antibodies including chimeric antibodies: genes useful in targeting malignant tumors (e.g., malignant melanoma by transformation of, e.g., tumor-infiltrating lymphocytes, TIL), tumor suppressor genes such as p53 or RB genes that regulate apoptosis such as Be 1-2 gene for thymidine kinase followed by ganciclovir gene for cytosine deaminase followed by 5-fluorocytosine gene for over expression of MDR-1 gene product to protect normal cells from cytotoxic chemotherapy, with genes deleterious to tumors, such as tumor necrosis factor, leukemia inhibitory factor, or various other toxic genes; hormones, such as insulin and growth hormone; transcriptional and translational regulatory elements; and the like. The liposomes may also encode enzymes to convert a noncytotoxic prodrug into a cytotoxic drug in tumor cells or tumor-adjacent endothelial cells.

In one embodiment of the invention, the liposomes contain a polynucleotide designed to be incorporated into the genome of the target cell or designed for autologous replication within the cell. In another embodiment, the compound entrapped in the lipid vesicles is an

oligonucleotide segment designed, for sequence-specific binding to cellular RNA or DNA.

Other drugs relevant to this invention include but are not limited to: antibodies, antibody derivatives, antibody fragments, e.g. Fab2, Fab, scFV, diabodies, triabodies, antibody (fragment) fusions (eg bi-specific and trispecific mAb fragments), proteins, aptamers, oligopeptides, oligosaccharides, as well as peptides, peptoids, steroids, organic drug compounds, toxins (e.g. ricin A, diphtheria toxin, cholera toxin), hormones, viruses, antiproliferative/antitumor/cytotoxic agents, antibiotics, cytokines, anti-inflammatory agents, anti-viral agents, antihypertensive agents, chemosensitizing and radiosensitizing agents. Some embodiments use auristatins, maytansines, cahcheamicin, duocarmycins, maytansinoids DM1 and DM4, auristatin MMAE, CC1065 and its analogs, camptothecin and its analogs, SN-38 and its analogs. Exemplary classes of cytotoxic agents include antimetabolites, natural products and their analogs, enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA alkylators, radiation sensitizers, DNA intercalators, DNA cleavers, anti-tubulin agents, topoisomerases inhibitors, platinum-based drugs, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, taxanes, lexitropsins, the pteridine family of drugs, diynenes, the podophyllotoxins, dolastatins,

maytansinoids, differentiation inducers, and taxols. Particularly useful members of those classes include, for example, duocarmycin ,

methotrexate, methopterin, dichloromethotrexate, 5-fLuorouracil DNA minor groove binders, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, camptothecin,

cahcheamicin, esperamicin, ene-diynes, and their analogues.

Exemplary drugs include the dolastatins and analogues thereof including: dolastatin A ( U.S. Pat No. 4,486,414), dolastatin B

(U.S. Pat No. 4,486,414), dolastatin 10 (U.S. Pat No. 4,486,444, 5,410,024, 5,504,191, 5,521,284, 5,530,097, 5,599,902, 5,635,483, 5,663, 149,

5,665,860, 5,780,588, 6,034,065, 6,323,315),dolastatin 13 (U.S. Pat No. 4,986,988), dolastatin 14 (U.S. Pat No. 5,138,036), dolastatin 15 (U.S. Pat No. 4,879,278), dolastatin 16 (U.S. Pat No. 6,239, 104), dolastatin 17 (U.S. Pat No. . 6,239,104), and dolastatin 18 (U.S. Pat No. . 6,239,104), each patent incorporated herein by reference in their entirety. In exemplary embodiments of the invention, the drug moiety is a mytomycin, vinca alkaloid, taxol, anthracycline, a calicheamicin, maytansinoid or an auristatin. Examples of hydrophilic small molecules that are envisaged to be provided in the liposomes of the present invention include, but are not- limited to, peptides and proteins that modulate the immune response such as interleukins; potent inhibitors of protein synthesis in human cells such as Diphteria toxin (fragment); activators of immune system for

macrophage-mediated destruction of tumour cells such as muramyl dipeptide; drugs for the treatment of lung fibrosis such as Cis-4- hydroxyproline; compounds for cancer treatment such as Cisplatin and derivatives thereof, cytosine a abinose, carboplatin, methotrexate, 1-SD- arabino-furanyl-cytosine (ara-C),5-fluoro-uraciL iloxuridine, and geintitabine; antibacterial agents such as phosphonopeptides; activator of prodrugs such as Glucuronidase for the activation of e.g. epirubicin- glucuroni.de; small therapeutic proteins and peptides such as insulin, growth factors and chemokines. According to a further particular embodiment of the invention, the drug is selected so as to target and or address a disease, such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, angiogenesis, an organ, and reporter gene/enzyme.

In one embodiment, the compound is useful for treatment of a plasma cell disorder, such as multiple myeloma, which is characterized by neoplasms of B-lymphocyte lineage cells. Therapeutic agents preferred for treatment of multiple myeloma include melphalan, cyclophosphamide, prednisone, chlorambucil, carmustine, dexamethasone, doxorubicin, cisplatin, paclitaxel, vincristine, lomustine, and interferon. Also contemplated is irstracytoplasmic delivery of plasmids, antisense oligonucleotides, and ribozymes for the treatment of cancer and viral infections.

In some embodiments the released drug is in. fact a prodrug designed to release a further drug. Drugs optionally inelude a membrane translocation moiety (adamantine, poly-lysine/argine, TAT) and or a targeting agent (against eg a tumor eel receptor) optionally linked through a stable or labile linker.

Additional Embodiment 1

With reference to formula (la) and (lb) for Triggers that function via cascade-mediated release or elimination (i.e. cascade mechanism), when p = 1 and n = 1 it is preferred that L^D is hnked to T^R via N or NH or an aliphatic or aromatic carbon, wherein these atoms are part of the linker. It is further preferred that said N and NH moieties comprised in L^D are bound to an aliphatic or aromatic carbon.

With reference to formula (la) and (lb) for Triggers that function via cascade-mediated release or elimination (i.e. cascade mechanism), when p = 0 and n = 1 it is preferred that L^D is hnked to T^R via S or O, wherein these atoms are part of the linker. It is further preferred that said S and O moieties comprised in L^D are bound to an aliphatic or aromatic carbon or carbonyl or thiocarbonyl of L^D.

With reference to formula (la) and (lb) for Triggers that function via cascade-mediated release or elimination (i.e. cascade mechanism), in particular embodiments when X^D is S-C(0)-(L^D)_n-(D^D), 0-C(S)-(L^D)_n-(D^D), S-C(S)-(L^D)_n-(D^D) and n = 1 it is preferred that L^D is linked to T^R via N or NH or an aliphatic or aromatic carbon, wherein these atoms are part of the linker. It is further preferred that said N and NH moieties comprised in L^D are bound to an aliphatic or aromatic carbon.

Additional Embodiment 2

Further preferred activators for use with Triggers based on the cascade mechanism are:

The 1,2,4,5-tetrazine given in Formula (8c-g), wherein each R¹ and each R² independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', N0₂, OR', SR'_; C(=0)R', C(=S)R', OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', S(=0)R', S(=0)₂R"', S(=0)₂NR'R", C(=0)0-R', C(=0)S-R', C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=0)OR", NR'C(=S)OR",

NR'C(=0)SR", NR'C(=S)SR", OC(=0)NR'R", SC(=0)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=0)NR"R", NR'C(=S)NR"R" with each R' and each R' independently being H, aryl or alkyl, and R'" independently being aryl or alkyl.

Other preferred activators for use with Triggers based

cascade mechanism are:

Other preferred activators for use with Triggers based on the strain release mechanism are:

The Activator can have a hnk to a Masking Moiety M^M such as a peptide, protein, carbohydrate, PEG, or polymer. Preferably, these

Activators for use with Triggers based on the cascade mechanism satisfy one of the following formulae:

R = (link to) peptide, protein, carbohydrate, PEG, polymer

Additional embodiment 3:

Some embodiments satisfy the one of the following formulas:

= rest of attached L^D-D Additional embodiment 4:

Preferred liposomal formulations which can be used in combination with a channel protein of this invention include:

1- Azolectin

2- DOPC:cholesterol:DSPE-PEG2000 (70:20: 10 mol%)

3- DPhPC:POPG:cholesterol (70:25:5 weight %)

4- DOPG:DOPC (75:25 weight %)

The incorporation of a channel protein of this invention into such liposomal formulations is established; reference is made

Ref: Kocer A, Walko M, Feringa BL, Synthesis and utilization of reversible and irreversible light-activated nanovalves derived from the channel protein MscL. Nat Protoc. 2007;2(6): 1426-37

EXAMPLES

The following examples demonstrate the invention or aspects of the invention, and do not serve to define or limit the scope of the invention or its claims.

Methods. 1H-NMR and ¹³C-NMR spectra were recorded on a Varian Mercury (400 MHz for ^!H-NMR and 100 MHz for ¹³C-NMR) spectrometer at 298 K. Chemical shifts are reported in ppm downfield from TMS at room temperature. Abbreviations used for splitting patterns are s = singlet, t = triplet, q = quartet, m = multiplet and br = broad. IR spectra were recorded on a Perkin Elmer 1600 FT-IR (UATR). LC-MS was performed using a Shimadzu LC-10 AD VP series HPLC coupled to a diode array detector (Finnigan Surveyor PDA Plus detector, Thermo Electron Corporation) and an Ion-Trap (LCQ Fleet, Thermo Scientific). Analyses were performed using a Alltech Alltima HP C₁₈ 3μ column using an injection volume of 1-4 μί, a flow rate of 0.2 niL min ¹ and typically a gradient (5% to 100% in 10 min, held at 100% for a further 3 min) of CH₃CN in H₂0 (both containing 0.1% formic acid) at 25 °C. Preparative RP-HPLC (CH₃CN / H₂0 with 0.1% formic acid) was performed using a Shimadzu SCL-IOA VP coupled to two Shimadzu LC-8A pumps and a Shimadzu SPD-10AV VP UV-vis detector on a Phenomenex Gemini 5μ Ci8 110A column. Size exclusion (SEC) HPLC was carried out on an Agilent 1200 system equipped with a Gabi radioactive detector. The samples were loaded on a Superdex-200 10/300 GL column (GE Healthcare Life Sciences) and eluted with 10 mM phosphate buffer, pH 7.4, at 0.35-0.5 mL/min. The UV wavelength was preset at 260 and 280 nm. The concentration of antibody solutions was determined with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) from the absorbance at 322 nm and 280 nm, respectively.

Materials. All reagents, chemicals, materials and solvents were obtained from commercial sources, and were used as received: Biosolve, Merck and Cambridge Isotope Laboratories for (deuterated) solvents; and Aldrich, Acros, ABCR, Merck and Fluka for chemicals, materials and reagents. All solvents were of AR quality. 2-((methylsulfonyl)thio)ethyl 2-aminoacetate hydrochloride was prepared following Kocer et al. Angew. Chem. Int. Ed. 2006, 45, 3126 -3130.

General examples

The invention can be exemplified with the same combinations of TCO and diene as included in applications WO2012156919A1 (e.g. Examples 9 - 14) and WO2012156920A1 (e.g. Examples 8 - 11), except that a Construct as defined hereinbefore is taken in lieu of a drug as disclosed therein.

Example 1

Synthesis of tetrazine Activators

For previously synthesized tetrazines see WO2012156919A1 and WO2012156920A1. Bis-pyridyl-tetrazine-NHS derivative was described in J. Nucl. Med. 2013, 54,1989- 1995.

3, 6-dibenzyl-l, 2,4, 5 -tetrazine (4)

Hydrazine hydrate (2.43 mL, 50.0 mmol) was added to a solution of benzyl cyanide (1.16 mL, 10.0 mmol) and Znl₂ (160 mg, 0.5 mmol) in DMF (20 mL) and the solution was stirred overnight at 60°C under argon. NaN0₂ (3.45 g, 50.0 mmol in 10 mL H₂0) was added dropwise to the suspension at room temperature. 1M HCl (ca. 80 mL) was added until gas formation stopped and pH=2. The mixture was extracted with CH₂C1₂ (3x80 mL) and the combined organic fractions were dried with Na₂S0₄ and concentrated. 4 was obtained after silica gel column chromatography (EtOAc/heptanes, 1/20) as purple oil. Yield: 0.64 g (2.44 mmol, 45%). ^XH NMR (400 MHz, CDCls): δ 7.38-7.26 (m, 8H), 3.75 (s, 4H) ppm. ¹³C NMR (400 MHz, CDCI3): δ 129.9, 129.1, 128.0, 127.9, 23.6 ppm. No MS data available due to poor ionization.

S nthesis of 3, 6-diisopropyl- 1,2,4,5 -tetrazine (5)

Hydrazine hydrate (13.2 mL, 312 mmol) was added to isobutyronitrile (3.59 mL, 40.0 mmol) and Znl₂ (0.64 g, 2.0 mmol) and the mixture was stirred overnight at 60°C under argon. NaN0₂ (13.45 g, 200 mmol in 200 mL H₂0) was added dropwise to the light colored suspension at room temperature over a cold-water bath. 1M HCl (ca. 400 mL) was added to the pink solution until gas formation stopped and pH=2. The mixture was extracted with CH₂C1₂ (4x 100 mL) and the combined organic fractions were dried with Na₂S0₄ and concentrated. 5 was obtained after silica gel column chromatography (EtOAc/hexanes, 1/9) as volatile purple oil. Yield: 3.6 g (21.4 mmol, quantitative yield). R_f: 0.25 (EtOAc/hexanes, 1/9). ^XH NMR (400 MHz, CDC1₃): δ 3.63 (sep, 7=7.2 Hz, 2H), 1.52 (d, =7.0 Hz, 12H) ppm. ¹³C NMR (400 MHz, CDC1₃): δ 173.8, 34.2, 21.3 ppm. ESI-MS [M+H⁺] : calc: 167.13 Da, found: 167.08 Da.

3,6-dimethyl-l,2,4,5-tetrazine (8)

Acetamidine hydrochloride (3.97 mg; 42.0 mmol) was dissolved in water (20 mL), and hydrazine hydrate (4.0 mL; 84.0 mmol) was added. The mixture was stirred at 20°C under an atmosphere of argon for 5 h. Water (20 mL) was added, followed by sodium nitrite (14.4 g; 210 mmol). The reaction mixture was cooled on an icebath and acidified to pH=3 by careful addition of acetic acid (15.0 g; 250 mmol). The dark pink, aqueous solution was extracted with dichloromethane (2 times 50 mL), and the combined organic layers were washed with 1 M hydrochloric acid (50 mL), dried over magnesium sulfate, and the solvent was removed by evaporation. The product was obtained as dark red crystals (750 mg; 33%). ^!H-NMR (CDCI₃): δ = 3.04 (s, 6H) ppm. ¹³C-NMR (CDCI₃): δ = 167.2, 21.0 ppm. GC-MS: m/z = +110 M⁺ (calcd 110.06 for C₄H₆N₄). -methyl-6-(pyridin-3-yl)-l,2,4,5-tetrazine (10)

Hydrazine hydrate (2.68 mL, 55.2 mmol) was added to 3-cyanopyridine (500 mg, 4.8 mmol), acetamidine hydrochloride (2.00 g, 21.2 mmol) and sulfur (78 mg, 2.4 mmol) and the mixture was stirred overnight under argon at room temperature. The reaction mixture was concentrated and suspended in a mixture of THF (10 mL) and AcOH (12 mL) over a cold-water bath. NaN0₂ (2.76 g, 40.0 mmol in 10 mL H₂0) was added dropwise and the mixture was stirred for another 5 minutes. H₂0 (80 mL) and CHCI₃ (100 mL) were added and the layers were separated. The organic layer was washed with H₂0 (2x100 mL), dried with Na₂S0₄ and concentrated. Silica gel column chromatography (acetone/hexanes, 1/4) yielded 10 contaminated with a small amount of the bis-pyridyl side product. Recrystallization from EtOAc yielded 10 as long needles (70 mg, 0.40 mmol, 8%). Concentration of the EtOAc filtrate yielded another crop (170 mg) of almost pure 10. ^XH NMR (400 MHz, CDC1₃): δ 9.80 (dd, /₁₌0.8 Hz, 2=1.9 Hz), 8.88-8.84 (m, 2H), 7.55 (m, 1H), 3.14 (s, 3H) ppm. ¹³C NMR (400 MHz, CDCI₃): δ 168.0, 163.1, 153.2, 149.3, 135.1, 127.9, 123.9, 21.3 ppm. ESI-MS [M+H⁺] calc: 174.08 Da, found: 174.08 Da.

3-methyl-6-(pyridin-4-yl)-l,2,4,5-tetrazine (11)

Hydrazine hydrate (2.68 mL, 55.2 mmol) was added to 4-cyanopyridine (500 mg, 4.8 mmol), acetamidine hydrochloride (2.00 g, 21.2 mmol) and sulfur (78 mg, 2.4 mmol) and the mixture was stirred overnight under argon at room temperature. The reaction mixture was concentrated and suspended in a mixture of THF (10 mL) and AcOH (12 mL) over a cold-water bath. NaN0₂ (2.76 g, 40.0 mmol in 10 mL H₂0) was added dropwise and the mixture was stirred for another 5 minutes. H₂0 (80 mL) and CHC1₃ (100 mL) were added and the layers were separated. The organic layer was washed with H₂0 (2x100 mL), dried with Na₂S0₄ and concentrated. Silica gel column chromatography (acetone/hexanes, 1/4) yielded 11 with a ca. 20% contamination of a thiadiazole compound. The crude material (220 mg) was recrystallized from diisopropylether to yield 11 as pink crystals. Yield: 135 mg (0.78 mmol, 16%). R_f: 0.07 (acetone/hexanes, 1/4). ^lH NMR (400 MHz, CDCI₃): δ 8.91 (dd, J₁₌ 1.5 Hz, J₂= 4.7 Hz, 2H), 8.44 (dd, /i=1.8 Hz, J₂=A.5 Hz, 2H), 3.17 (s, 3H) ppm. ¹³C NMR (400 MHz, CDCI₃): δ 168.5, 163.0, 151.1, 139.2, 121.2, 21.4 ppm. ESI-MS [M+H⁺]: calc: 174.08 Da, found: 174.08 Da. -methyl-6-(3-methylpyridin-2-yl)-l, 2,4,5 -tetrazine (12)

Hydrazine hydrate (2.76 mL, 56.2 mmol) was added to 3-methylpicolinonitrile (0.57 g, 4.8 mmol), acetamidine hydrochloride (2.00, 21.2 mmol) and sulfur (155 mg, 4.8 mmol) and the mixture was stirred under argon at room temperature for 40 hours. EtOH (10 mL) was added and the mixture was filtered. The filtrate was concentrated and suspended in a mixture of THF (10 mL) and AcOH (12 mL) over a cold-water bath. NaN0₂ (2.76 g, 40.0 mmol in 10 mL H₂0) was added dropwise and the mixture was stirred for another 5 minutes. H₂0 (80 mL) and CHCI₃ (100 mL) were added and the layers were separated. The organic layer was washed with H₂0 (2x100 mL), dried with Na₂S0₄ and concentrated. Silica gel column chromatography (acetone/hexanes, 1/4) yielded 12 as purple liquid. Yield: 110 mg (0.59 mmol, 12%). ^!H NMR (400 MHz, CDCI₃): δ 8.73 (dd, Ji=0.8 Hz, /₂=4.6 Hz, 1H), 7.76 (ddd, /₁₌0.8 Hz, J₂=L5, /₃= 7.8 Hz, 1H), 7.43 (dd, 7₁₌4.7 Hz, ₂=7.8 Hz, 1H), 3.17 (s, 3H), 2.60 (s, 3H) ppm. ¹³C NMR (400 MHz, CDC1₃): δ 167.3, 166.0, 149.8, 148.0, 139.7, 134.5, 125.2, 21.4, 19.8 ppm. ESI-MS [M+H⁺] calc: 188.09 Da, found: 188.08 Da.

3-meth -6-phenyl-l,2,4,5-tetrazine (14)

Hydrazine hydrate (3.24 mL, 66.7 mmol) was added to benzonitrile (600 mL, 5.8 mmol), acetamidine hydrochloride (2.41 g, 25.5 mmol) and sulfur (94 mg, 2.9 mmol) and the mixture was stirred overnight under argon at room temperature. The reaction mixture was concentrated and suspended in a mixture of THF (10 mL) and AcOH (12 mL) over a cold-water bath. NaN0₂ (3.33 g, 28.3 mmol in 10 mL H₂0) was added dropwise and the mixture was stirred for another 5 minutes. H₂0 (50 mL) and CHC1₃ (100 mL) were added and the layers were separated. The organic layer was washed with H₂0 (2x70 mL), dried with Na₂S0₄ and concentrated. Silica gel column chromatography (acetone/hexanes, 1/4) yielded 14 with some contamination (ca. 75 mg). The crude product could not be purified by recrystallization from numerous solvents. ^!H NMR (400 MHz, CDC1₃): δ 8.59 (dd, ₁₌1.6 Hz, J₂=8.2 Hz, 2H), 7.68- 7.60 (m, 2H), 7.50-7.46 (m, 1H), 3.10 (s, 3H) ppm. ¹³C NMR (400 MHz, CDC1₃): δ 167.3, 164.2, 132.6, 131.8, 129.3, 127.9, 21.2 ppm. MALDI-TOF-MS: [M+H⁺] : calc: 173.08 Da, found 173.30 Da.

Example 2

TCO synthesis

The following TCO constructs have been prepared according to WO2012156920A1

3-PNP-TCO was synthesized following WO2012156919A1.

Axial-TCO-1 -Doxorubicin

The synthesis of Axial- TCO-1 -Doxorubicin is described in WO2012156919A1.

3-PNP-TCO (41.9 mg; 1.44*10^"4 mol) was dissolved in dichloromethane (1.5 mL), and DIPEA (55.7 mg; 4.32* 10^"4 mol) and 1-naphthylmethylamine (27.2 mg; 1.73* 10^{" 4} mol) were added. The reaction mixture was stirred at 20°C under and atmosphere of argon and slowly turned yellow. After 20 h the solvent was removed by evaporation in vacuo, and the mixture was redis solved in dichloromethane and washed with subsequently, 1 M aqueous sodium hydroxide (5 times 2.5 mL) and 1 M aqueous citric acid (2 times 1.5 mL). The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness. The product was further purified by preparative RP-HPLC, and isolated by lyophilization, to yield a white powder (32.0 mg; 72%). 1H-NMR (CDC1₃): δ = 8.04 (d, IH), 7.89 (d, IH), 7.81 (d, IH), 7.54 (m, 2H), 7.45 (m, 2H), 5.79 (m, IH), 5.56 (d, IH), 5.40 (m, IH), 5.03 (br. s, IH), 4.85 (m, 2H), 2.44 (m, IH), 2.2 - 1.6 (br. m, 6H), 1.43 (m, IH), 1.02 (m, IH), 0.79 (m, IH) ppm. ¹³C-NMR (CDCI₃): δ = 131.7, 131.4, 128.8, 128.5, 126.5, 126.4, 125.9, 125.4, 123.5, 74.2, 43.2, 40.7, 35.9, 29.1, 29.0, 24.1 ppm. FT-IR (ATR): v = 3322, 2927, 2857, 1692, 1533, 1258, 1070, 1025, 987 cm^"1. LC-MS: m/z = +310.25 [M+H]⁺ (calcd 309.17 for C₂oH₂₃N0₂).

Axial-(E)-cyclooct-2 -en-1 -yl 4-nitrobenzoate

Axial-(£)-cyclooct-2-en-l-ol (152 mg, 1.20 mmol) was dissolved in 10 mL dichloromethane. 4-(N,N-dimethylamino)pyridine (306 mg, 2.50 mmol) was added and the solution was cooled in an ice-bath. A solution of 4-nitrobenzoyl chloride (201 mg, 1.08 mmol) in 5 mL dichloromethane was added in portions over a 5 min period. The solution was stirred for 3 days. The solvent was partially removed by rotary evaporation. The remaining solution (a few mL) was chromatographed on 19 g silica, using dichloromethane as the eluent. The product fractions were rotary evaporated yielding a colourless solid (144 mg, 0.52 mmol, 48%).

¹ H-NMR (CDCI₃): δ 8.4-8.2 (m, 4H), 5.9 (m, IH), 5.6 (m, 2H), 2.2 (dd, IH), 2.5 (m, IH), 2.15-1.7 (m, 6H), 1.55 (m, IH), 1.2 (dt, IH), 0.9 (dt, IH).

Equatorial-(E)-cy

Equatorial-(£^')-cyclooct-2-en- l-ol (154 mg, 1.22 mmol) was dissolved in 10 mL dichloromethane. 4-(N,N-dimethylamino)pyridine (300 mg, 2.46 mmol) was added and the solution was cooled in an ice-bath. A solution of 4-nitrobenzoyl chloride (268 mg, 1.44 mmol) in 5 mL dichloromethane was added in portions over a 5 min period. The solution was stirred for 4 days. The solvent was removed by rotary evaporation and the residue was chromatographed on 19 g silica, using dichloromethane as the eluent. The product fractions were rotary evaporated yielding a colourless solid.

1H-NMR (CDC1₃): δ 8.4-8.1 (m, 4H), 5.9 (m, 1H), 5.7 (m, 1H), 5.4 (m, 1H), 2.5 (m, 1H), 2.3 (m, 1H), 2.1-1.8 (m, 3H), 2.8-2.4 (m, 4H), 1.8-1.4 (m, 4H), 1.0-0.8 (m, 1H).

(E)-3

Cyclooct-2-en-l-ol (5.002 g, 39.64 mmol) was dissolved in 100 mL THF. Phenol (3.927 g, 41.78 mmol ) was added to the solution. Triphenylphosphine (10.514 g, 40.01 mmol) was added and the resulting solution was cooled in an ice-bath. A solution of diethyl azodicarboxylate (6.975 g, 40.01 mmol) in 50 mL THF was added over a 30 min period. The reaction mixture was stirred for 24 h and then rotary evaporated. The residue was stirred with heptane, the mixture was filtered and the filtrate was rotary evaporated. The residue was chromatographed on 50 g silica, using heptane as eluent. Product fractions were rotary evaporated and the residue was stirred with methanol until homogeneous, then filtered, and rotary evaporated. The residue was purified by Kugelrohr distillation to yield the product as an oil (3.5 g, 17.33 mmol, 44%).

3-phenoxycyclooctene (5.5 g, 27.23 mmol) was dissolved in heptane - ether (ca.1/2). The solution was irradiated for 7 days while the solution was continuously flushed through a 42 g silver nitrate impregnated silica column (containing ca. 4.2 g silver nitrate). The column was rinsed twice with TBME, then with TBME containing 5% methanol, then with TBME containing 10% MeOH. The product fractions were washed with 100 mL 15% ammonia (the same ammonia being used for each fraction), then dried and rotary evaporated. The column material was stirred with TBME and 15% ammonia, then filtered, and the layers were separated. The organic layer was dried and rotary evaporated. The first two TBME fractions were combined, and all other fractions were separately rotary evaporated, then examined for the presence of the product (none of the fractions contained a pure trans-cyclooctene isomer, however). The product fractions were combined and chromatographed on 102 g silica, using heptane as the eluent. The first fractions yielded the pure minor (believed to be axial) isomer as an oil (144 mg, 0.712 mmol, 2.6%). The next fractions contained a mixture of minor and major isomer. Pure major (believed to be equatorial) isomer was eluted last, yielding a colourless solid (711 mg, 3.52 mmol, 13%).

(Z)-3-phenoxycyclooct-l-ene: ^!H-NMR (CDC1₃): δ 7.25 (m, 2H), 6.9 (m, 3H), 5.7 (m,

1H), 5.5 (m, 1H), 5.1 (m, 1H), 2.5-2.0 (m, 3H), 1.3-1.9 (m, 7H).

(£)-3-phenoxycyclooct-l-ene (axial isomer): ^-NMR (CDC1₃): δ 7.25 (m, 2H), 6.9 (m, 3H), 5.9 (m, 1H), 5.6 (m, 1H), 4.9 (s, 1H), 2.4 (m, 1H), 2.2 (m, 1H), 2.0-0.8 (m, 8H)

(£)-3-phenoxycyclooct-l-ene (equatorial isomer): ^-NMR (CDC1₃): δ 7.25 (m, 2H), 6.9 (m, 3H), 5.9 (m, 1H), 5.55 (m, 1H), 4.8 (m, 1H), 2.45-2.25 (m, 2H), 2.05-1.4 (m, 6H), 1.0-0.8 (m, 2H) -cyclooct-2-en-l-yl 2-phenylacetate

by product

Axial (£')-cyclooct-2-en-l-ol (102 mg, 0.81 mmol) was dissolved in 7.5 mL dichloromethane with 4-(N,N-dimethylamino)pyridine (303 mg, 2.70 mmol). A solution of phenylacetyl chloride (155 mg, 1.00 mmol) in 2.5 mL dichloromethane was added in portions over a 5 min period to the ice-cooled solution. The reaction mixture was stirred for 4 days, then washed with water. The aqueous layer was extracted with 10 mL dichloromethane. The combined organic layers where dried and rotary evaporated, followed by chromatography yielding a colourless powder (22 mg) which was identified as the depicted byproduct.

Axial-(E)-3-(benzy

Axial (ZTl-cyclooct-2-en- l-ol (131 mg, 1.04 mmol) was dissolved in 5 mL THF. Sodium hydride (60 % dispersion in oil, 80 mg, 2 mmol) was added. The mixture was stirred for 5 min, then heated at 55 °C for 1 h, and then stirred at rt for 4 h. Benzyl bromide (210 iL, 300 mg, 1.9 mmol ) was added in 5 small portions. The reaction mixture was stirred for 4 days, after which 10 mL water was added carefully. The mixture was extracted with 2 x 10 mL dichloromethane and the successive organic layers were washed with 10 mL water, dried and rotary evaporated. The residue was heated at ca. 40°C under high vacuum in order to remove most of the benzyl brormde. The residue was purified by chromatography on 20 g silica using heptane as eluent, followed by elution with toluene. The latter solvent eluted the product. The product fractions were rotary evaporated, leaving a colourless oil, which contained traces of dibenzyl ether (69 mg, 0.32 mmol, 31%).

'H-NMR (CDC1₃): δ 7.4-7.2 (m, 5H), 6.0 (m, 1H), 5.45 (d, 1H), 4.7-4.4 (dd, 2H), 4.2 (s, 1H), 2.5 (m, 1H), 2.2-1.8 (m, 4H), 1.7-1.5 (m, 3H), 1.3-1.1 (m, 1H), 0.8 (m, 1H)

Axial-(E)-2,5-dioxopyrrolidin-l-yl 5-((((2,5-dioxopyrrolidin-l-yl)oxy)carbonyl)oxy)- 1 -methylcyclooct-3-enecarboxylate TCO-2

( -5-bromocyclooct-l-ene

1,5-cyclooctadiene (225 mL, 1.83 mol) was added to ice-cooled 310 mL 33% hydrogen bromide in acetic acid over a 30 min period at ca. 10°C. The mixture was stirred for 2 days, then 300 mL water was added, and the mixture was extracted with 2 x 300 mL pentane containing some TBME. The successive organic layers were washed with 75 mL water and with 75 mL sodium bicarbonate solution. Drying and rotary evaporation left 325 g residue which was used as such in the next step. (Z)-cyclooct-4-enecarbonitrile

A mixture of 700 mL DMSO and sodium cyanide (117.3 g, 2.39 mol) was heated to 90°C. The bromide obtained above was added over a 4 h period at 90 - 96°C. The mixture was subsequently heated at 98°C for 16 h, then it was cooled and 200 mL water was added during this cooling process. The mixture was extracted with 3 x 300 mL pentane containing some TBME. Washing with 50 mL water, drying and rotary evaporation resulted in 170 g residue which was used as such in the next step. See J. Org. Chem. 1988, 53, 1082 for a similar procedure.

(Z) -cyclooct-4-enecarboxylic acid

The product obtained above was treated with 100 mL ethanol, 160 mL 35% hydrogen peroxide, and 400 mL 30% sodium hydroxide solution, via the method described by D. Hartley in J. Chem. Soc. 1962, 4722. After acidification, further workup and

Kugelrohr distillation, the distillate (94.4 g) appeared to be mainly the starting nitrile. This distillate, combined with ca. 25 g of the solid residue from the Kugelrohr distillation, was stirred with 400 mL ethanol. Potassium hydroxide (155 g, 2.35 mol) was added, and the mixture was cooled with cold water (reaction mixture attained 40°C). When the temperature had dropped to 25°C, 35 mL water was added, followed by the portion-wise addition of 140 mL 35% hydrogen peroxide (foaming,

temperature around 30°C). After the addition was complete and the temperature had dropped, the cooling-bath was removed and replaced by a heating mantle. The mixture was warmed up slowly, resulting in an exothermal reaction and foaming. Hereby the temperature gradually reached 63°C (some cooling was necessary). When the temperature had decreased to 55 C, 100 mL 30% sodium hydroxide solution was added. The mixture was then heated for 4 h, while distilling off ca. 350 mL of solvent. Another 30 mL 30% sodium hydroxide solution was added and the mixture was heated under reflux for 10 h. The reaction mixture was cooled to rt, 400 mL heptane was added and the layers were separated. The organic layer was washed with a small amount of water. The combined aqueous layers were acidified with cone,

hydrochloric acid and extracted with 3 x 250 mL TBME. Drying, rotary evaporation and Kugelrohr distillation gave 109.77 g of the desired acid (0.713 mol, 39% yield based on 1,5-cyclooctadiene).

(Z)-l -methylcyclooct-4-enecarboxylic acid

A mixture of diisopropylamine (90.2 g, 0.893 mol) and 300 mL THF was cooled below -20°C. n-Butyllithium in hexanes (2.5 N, 360 mL, 0.900 mol) was added in a slow stream, keeping the temperature below -20°C. The solution was stirred for 15 min, then cooled to -50°C. (Z)-cyclooct-5-enecarboxylic acid (54.0g, 0.351 mol), dissolved in 150 mL THF, was added over a 20 min period at temperatures between - 50 and -25°C. The mixture was stirred for an additional 40 minutes, allowing the temperature to rise to -5°C. The mixture was subsequently heated for 3 h at 50°C, then cooled to -50°C. lodomethane (195.5 g, 1.377 mol) was added over a 20 min period at temperatures between -50 and -30°C. The mixture was stirred overnight, heated for 1 h at 40°C, then rotary evaporated in order to remove most of the solvents. Toluene (250 mL) was added to the residue, followed by 500 mL dilute hydrochloric acid. The layers were separated and the organic layer was washed with 100 mL 2 N

hydrochloric acid. The successive aqueous layers were extracted with 2 x 250 mL toluene. The organic layers were dried and rotary evaporated. The residue was purified by Kugelrohr distillation to yield 59.37 g of the methylated acid (0.353 mol, 100%), which was sufficiently pure to be used as such in the next step.

'H-NMR (CDC1₃): δ 5.75-5.60 (m, 1H), 5.55-5.40 (m, 1H), 2.4-1.5 (m, 10H), 1.27 (s, 3H). C-NMR (CDCI₃): δ 185.5 (C=0), 131.9 (=CH), 126.5 (=CH), 46.2, 35.3, 32.3, 27.1 (CH₃), 26.1, 24.8, 24.7. -5-methyl-9-oxabicyclo[3.3.2]dec-7-en-10-one

To a mixture of the methylated acid (42.0 g, 0.25 mol), 300 mL dichloromethane, and 300 mL water sodium bicarbonate was added (68.9 g, 0.82 mol). The mixture was stirred for 10 min, then it was cooled in ice. A mixture of potassium iodide (125.2 g, 0.754 mol) and iodine (129 g, 0.508 mol) was added over a 1 h period in 6 equal portions. The reaction mixture was stirred for 3½ h. Sodium bisulfite was added slowly, until the dark colour had disappeared. The layers were separated and the cloudy aqueous layer was extracted with 2 x 250 mL dichloromethane. Drying and rotary evaporation gave the desired iodolactone.

¹ H-NMR (CDC1₃, product signals): δ 5.65-5.5 (m, 2H), 4.8 (dt, IH), 3.95 (dt, IH), 2.6-1.95 (m, 8H).

The iodolactone was dissolved in 250 mL toluene, and DBU (65.2 g, 0.428 mol) was added. The mixture was allowed to stand overnight, after which it was heated under reflux for 75 min (NMR indicated full conversion). After cooling the reaction mixture, it was washed with 150 and 100 mL water. The successive aqueous layers were extracted with 250 mL toluene. The organic layers were dried and rotary evaporated and the residue was purified by Kugelrohr distillation to yield 38.86 g of the bicyclic olefin (0.234 mol, 94%, containing a trace of toluene).

¹ H-NMR (CDCI₃): δ 5.95-5.85 (m, IH), 5.45-5.35 (dm, IH), 5.05 (bs, IH), 2.5-2.3 (m,

IH), 2.2-2.0 (m, 2H), 1.95-1.6 (m, 5H), 1.27 (s, 3H).

1³C-NMR (CDCI₃): δ 177.2 (C=0), 129.1 (=CH), 127.9 (=CH), 79.2 (CH), 45.2, 43.0, 31.9, 29.5 (CH₃), 26.6, 24.0.

(Z)-methyl 5 -hydroxy- 1 -methylcyclooct-3-enecarboxylate

The bicyclic olefin obtained above (38.86 g, 0.234 mol), plus another batch of 1.5 g bicyclic olefin, was stirred for 64 h at 25 - 30°C with 250 mL methanol and potassium bicarbonate (100.0 g, 1.0 mol). Another 50.0 g potassium bicarbonate (0.5 mol) was added because NMR indicated the presence of ca. 35% starting olefin. The mixture was stirred for an additional 64 h, but the amount of starting material remained unchanged. Filtration, washing with methanol and rotary evaporation of the filtrate gave a residue, which was chromatographed on 200 g silica using dichloromethane as the eluent. The starting olefin eluted first, then a mixture of starting olefin and product eluted. Further elution with dichloromethane / methanol gave 6.69 g of product, contaminated with ca. 15% of starting olefin, and then 17.53 g of pure product (total 0.117 mmol, 48%).

1H-NMR (CDC1₃): δ 5.6-5.5 (m, 1H), 5.35-5.25 (m, 1H), 5.0-4.85 (m, 1H), 3.63 (s, 3H), 2.90 (d, 1H, OH), 2.35-1.90 (m, 5H), 1.75-1.45 (m, 3H), 1.20 (s, 3H).

1³C-NMR (CDCI₃): δ 178.8 (C=0), 132.7 (=CH), 129.0 (=CH), 68.0 (CH), 52.0 (CH₃), 46.1, 35.9, 33.7, 30.4, 26.8, 24.7 (CH₃).

(E)-methyl 5-hydroxy-l -methylcyclooct-3-enecarboxylate

The two portions of hydroxy ester obtained above, plus 2.29 g of hydroxy ester from another experiment (total amount 26.51 g, 133.8 mmol) were mixed with 25.0 g methyl benzoate and heptane / ether (ca. 4/1). The solution was irradiated, the irradiated solution being continuously flushed through a silver nitrate impregnated silica column (213.6 g, containing ca. 126 mmol silver nitrate). During the irradiation process some solvent was lost due to evaporation; this solvent was replaced by ether. The irradiation and flushing were stopped when the irradiated solution contained hardly any starting material. The silica column was successively flushed with 600 mL TBME, 500 mL TBME / 5% methanol, 500 mL TBME / 10% methanol, and 500 mL TBME / 20% methanol. The first 3 eluates were rotary evaporated. The first eluate contained methyl benzoate and the starting hydroxy ester in a ca. 2/3 ratio. The fourth eluate was washed with 300 mL 10% ammonia solution, then dried and rotary evaporated (axial/equatorial ratio of the trans-cycloctene was ca. 5/4). The residues from the second and third eluate were combined, dissolved in TBME and washed with the ammonia layer of above. Drying and rotary evaporation gave a residue which consisted of the axial /equatorial isomers of the trans-cycloctene in a ratio of ca. 5/4. The residual column material was stirred with TBME, 100 mL water and the ammonia layer of above. Filtration, layer separation, drying and rotary evaporation gave a residue. The process was repeated twice to give a residue which consisted of the axial/equatorial isomers of the trans cycloctenes in a ratio of ca. 1/7. All fractions of the trans cyclooctenes were combined to give a total yield of 19.1 g (96.5 mmol, 72%).

Note: The axial/equatorial assignment is based on the the stereochemistry of the hydroxy group, in similar fashion as for trans-cycloocten-2-ol. In both isomers the hydroxy and methylester substituents are positionsed cis relative to each other. In the axial isomer, these czs-positioned subsituents are both in the axial position.

1 H-NMR (CDC1₃) (mixture of isomers): axial isomer: δ 5.8 (m, IH), 5.35 (m, IH), 4.2 (m, IH), 3.72 (s, 3H), 2.7 (m, IH), 2.3-1.7 (m, 6H), 1.5 (m, IH), 1.3 (m, IH), 1.19 (s, 3H). ¹³C-NMR (CDCI₃): δ 177.6 (C=0), 136.1 (=CH), 132.3 (=CH), 74.8 (CH), 51.5 (CH₃), 47.5, 46.0, 39.9, 38.9, 34.8 (CH₃), 31.0.

^LH-NMR (CDC1₃) (mixture of isomers): equatorial isomer: δ 6.05 (m, IH), 5.6 (dd, IH), 4.45 (bs, IH), 3.62 (s, 3H), 2.35-1.7 (m, 8H), 1.5 (m, IH), 1.08 (s, 3H). ¹³C- NMR (CDC1₃): δ 180.7 (C=0), 135.2 (=CH), 130.3 (=CH), 69.6 (CH), 52.1 (CH₃), 44.9, 44.7, 38.3, 30.9, 29.8, 18.3 (CH₃).

Axial-(E)-5 -hydroxy- 1 -methylcyclooct-3-enecarboxylic acid

A solution of 1.60 g potassium hydroxide in 5 mL water was added over a 5 min period to a water-cooled solution of the trans-cyclooctene ester isomer mixture (0.49 g, 2.47 mmol, ratio of the axial/equatorial isomer ca. 2½/l) in 11 mL methanol. The solution was stirred for 18 h at 28°C. 15 mL water was added and the mixture was extracted with 2 x 30 mL TBME. The combined organic layers were washed with 10 mL water, then dried and rotary evaporated to give the non-hydrolyzed equatorial ester. The combined aqueous layers were treated with 30 mL TBME, and then with 4.5 g citric acid. The layers were separated and the aqueous layer was extracted with 30 mL TBME. The organic layers were dried and rotary evaporated at 55°C to afford 0.34 g (1.85 mmol, 75%) of the pure axial isomer of the trans-cyclooctene acid.

'H-NMR (CDCI₃): δ 6.15-5.95 (m, 1H), 5.6 (d, 1H), 4.45 (bs, 1H), 2.4-1.7 (m, 7H), 1.6 (dd, 1H), 1.18 (s, 3H).

¹³C-NMR (CDC1₃): δ 185.4 (C=0), 134.8 (=CH), 130.7 (=CH), 69.8 (CH), 44.8, 38.2, 31.0, 29.8 (CH₂), 18.1 (CH₃).

Note: The hydrolysis of the axial/equatorial ester appears to be extremely selective. Whereas the axial isomer hydrolyzes surprisingly easily at rt, the major isomer remains unaffected, thus enabling an straightforward separation between both isomers (the equatorial isomer hydrolyzes upon overnight heating at ca. 60°C). In both isomers the hydroxy and carboxylic substituents are positionsed cis relative to each other. In the axial isomer, these ds-positioned subsituents are both in the axial position.

Axial-(E)-2,5-dioxopyrrolidin-l-yl 5-(((( 2,5-dioxopyrrolidin-l - carboxylate TCO-2

To a solution of Axial-(£)-5-hydroxy-l-methylcyclooct-3-enecarboxylic acid obtained above (375 mg, 2.04 mmol) in 10.1 g acetonitrile there was added N,N- diisopropylethylamine (1.95 g, 15.07 mmol), followed by N,N'-disuccinimidyl carbonate (2.25 g, 8.79 mmol). The mixture was stirred for 3 days at rt, and subsequently rotary evaporated at 55 °C. The residue was chromatographed on 20 g silica, elution being done with dichloromethane, followed by elution with

dichloromethane containing some TBME. The latter solvent mixture eluted the product. The product fractions were combined and rotary evaporated. The resulting residue was stirred with TBME until a homogeneous suspension was obtained.

Filtration and washing gave 400 mg of product.

'H-NMR (CDCI₃): δ 6.15-6.0 (m, 1H), 5.6 (dd, 1H), 5.25 (bs, 1H), 2.8 (2s, 8H), 2.5- 1.85 (m, 8H), 1.25 (s, 3H). Axial-TCO-2-Doxorubicin

mol) were dissolved in DMF (5 mL), and DIPEA (148 mg; 1.15* 10^~3 mol) was added. The solution was stirred under an atmosphere of argon at 20°C for 18 h. Acetonitrile (6.5 mL), formic acid (0.2 mL), and water (6.5 mL), were added and the suspension was filtered. The filtrate was purified by preparative RP-HPLC (50 v% acetonitrile in water, containing 0.1 v% formic acid). The product was isolated by lyophilization, dissolved in chloroform (3 mL), and precipitated in diethyl ether (20 mL), to yield 134 mg of an orange powder (68%). 1H-NMR (CDC1₃): δ - 13.97 (s, 1H), 13.22 (s, 1H), 8.03 (d, 7=7.9 Hz, 1H), 7.78 (t, 7=8.0 Hz, 1H), 7.38 (d, 7=8.6 Hz, 1H), 5.85 (m, 1H), 5.59 (m, 1H), 5.51 (s, 1H), 5.29 (s, 1H). 5.16 (d, 7=8.4 Hz, 1H), 5.12 (s, 1H), 4.75 (d, 7=4.8 Hz, 2 H), 4.52 (d, 7=5.8 Hz, 1H), 4.15 (q, 7=6.5 Hz, 1H), 4.08 (d, 7=3.6 Hz, 3H), 3.87 (m, 1H), 3.69 (m, 1H), 3.26 (d, 7=18.8 Hz, 1H), 3.00 (m, 2H), 2.81 (s, 4H), 2.4 - 1.7 (br. m, 13H), 1.62 (s, 2H), 1.30 (d, 7=6.5 Hz, 3H), 1.23 (s, 3H) ppm. ¹³C-NMR (CDCI₃): δ = 213.89, 187.07, 186.68, 174.30, 169.27, 161.03, 156.15, 155.64, 154.66, 135.73, 135.49, 133.58, 131.70, 131.10, 120.88, 119.83, 118.43, 111.58, 111.40, 100.73, 72.09, 69.65, 67.28, 65.54, 56.67, 46.87, 44.38, 35.75, 34.00, 30.49, 30.39, 30.20, 25.61, 17.92, 16.84 ppm. LC-MS: m/z = +873.42 [M+Na]⁺ , - 849.58 [M-H]^" (calcd 850.28 for C42H46N2O17).

Axial-(E)-2-((methylsulfonyl)thio)ethyl 2-(((cyclooct-2-en-l- yloxy)carbonyl)amino)acetate TCO 4

N,N-diisopropylethylamine (755 mg, 5.85 mmol) was added to a solution of (£)- cyclooct-2-en-l-yl (4-nitrophenyl) carbonate (3-PNP-TCO; 248 mg, 0.852 mmol; axial isomer) in 18.5 g dichloromethane, followed by the addition of 2- ((methylsulfonyl)thio)ethyl 2-aminoacetate hydrochloride (435 mg, 1.74 mmol) and 9 mg 4-(N,N-dimethylamino)pyridine. After stirring for 4 days at 35°C and 3 days at rt there was still a small amount of the starting 4-nitrophenylcarbamate left. Therefore, an additional 50 mg of 2-((methylsulfonyl)thio)ethyl 2-aminoacetate hydrochloride was added, followed by overnight stirring and rotary evaporation at 50°C. Toluene (150 mL) was added to the residue. The solution was extracted with 15 x 5 mL water in order to remove almost all of the 4-nitrophenol. Drying and rotary evaporation at 50°C left a residue which was dissolved in 5 mL toluene. Heptane (75 mL) was added gradually and the mixture was stirred for 2 h, then decanted. The precipitate was dissolved in 5 mL toluene. Heptane (75 mL) was added gradually and the mixture was stirred for 1 h, then decanted. The precipitate constitutes the nearly pure product. 'H-NMR (CDCI₃): δ 5.9-5.75 (m, 1H), 5.52 (dd, 1H), 5.32 (bs, 1H), 5.25 (bs, 1H), 4.46 (t, 2H), 4.00 (d, 2H), 3.42 (t, 2H), 3.37 (s, 3H), 2.45 (m, 1H), 0.7 - 2.1 (m, 9H). ¹³C-NMR (CDC1₃): δ 170.1 and 156.0 (C=0), 132.2 (=CH), 131.3 (=CH), 74.8 (CH), 63.4 (CH₂), 51.1 (CH₃), 42.8, 40.9, 36.2, 36.1, 35.0, 29.3, 24.3 (all CH₂).

Example 3

Tetrazine induced release of doxorubicin from TCO-l-doxorubicin

The tetrazines featured in Figure 1 were tested with respect to their ability to release doxorubicin from TCO-2-doxorubicin. It shall be understood that the tetrazine- induced release in this experiment can be considered representative of the cleavage of lipid-TCO-PEG constructs. The relative release yield for each tetrazine is given in Figure 1 (+++ = highest). PBS/MeCN (1 mL, 3/1), preheated at 37°C and TCO-2-doxorubicin (10 μL of a 2.5 mM solution in DMSO, 1 eq.) were added to a preheated injection vial. Tetrazine (10 μΐ. of a 25 mM solution in DMSO, 10 eq.) was added and the vial was vortexed. After incubation for 1 hour at 37°C, the vial was placed in LC-MS autosampler at 10°C. LC- MS analysis was performed using a 5% to 100% ¾0/MeCN gradient over 11 minutes with a C18 reverse-phase column at 35°C. A control sample containing only TCO-2-doxorubicin (1 eq), as well as a sample containing only doxorubicin (1 eq.), was analyzed under the same conditions. All tetrazine containing samples were measured twice and the doxorubicin control sample was run after every three other samples during an overnight program. The peak area of released dox was divided by the peak area of TCO-2-doxorubicin or doxorubicin reference signals and multiplied by 100 to calculate the percentage of release. The calculated percentage of release was corrected when it was observed that the TCO-2-doxorubicin was not fully converted to inv-DA adduct(s). This was done by quantification of remaining dox-TCO, but full conversion was almost always observed. Peak areas (used for doxorubin quantification) were determined at λ^470-500 nm where characteristic doxorubicin absorption takes place and peak integration was done by hand.

Example 4

In a similar fashion as Example 3, the release of doxorubicin from TCO-1- doxorubucin as induced by tetrazines 1,8,9 in PBS/ACN and in serum was measured. From Table 1 it is clear that tetrazine 8 affords the highest release and that the release yields are retained when testing in serum. Serum experiments were conducted as followes:

TCO-l-doxorubucin (6.25 xlO^"8 mol) was dissolved in DMSO (0.050 mL), and PBS (0.475 mL) was added slowly in aliquots of 0.010 mL, followed by mouse serum (0.475 mL). A portion of this mixture (0.200 mL) was equilibrated at 37 °C, and a solution of tetrazine (1.25 xlO^"7 mol) in DMSO (0.005 mL) was added, and the solution was thoroughly mixed and incubated at 37 °C in the dark for 4 h.

Subsequently, cold MeCN (0.200 mL) was added, followed by centrifugation at 13400 rpm for 5 min. The supernatant was used for further analysis by

HPLCMS/PDA analysis to determine the release of doxorubicin.

Table 1. Doxorubicin release (%) from Axial-TCO-l-Doxorubicin following addition of 10 equiv. tetrazine 1,9,8 in 25% MeCN in PBS or 50% serum at 37 °C; measured with LCMS at 4 h (n=3).

Probe PBS/MeCN (3/1) Serum

Ϊ 7+3 Ϊ2+Ϊ

9 55+4 46+3

8 79+3 75+4

-^[a] 0 0

[a] no release of doxorubicin from Axial- TCO-l-Doxorubicin at 37 °C in PBS (72 h) and serum (24 h).

Example 5

Versatility of the TCO mask

To demonstrate the versatility of the TCO mask, the stability of a range of TCO derivatives as model compounds was tested under various conditions. In addition, the tetrazine-induced TCO activation was studied under the same conditions. The results in Tables 2-4 support the versatility of the TCO mask and at the same time demonstrate that in addition to aromatic and aliphatic carbamates also carbonates and aromatic and aliphatic esters and ethers are effectively cleaved from the TCO upon tetrazine reaction. In addition to amines, also hydroxy and carboxylic acids form stable conjugates with TCO and can subsequently be cleaved in a range of conditions.

Typical example for testing the stability of a TCO compound

The TCO stock solution (10 iL 25 mM; 2.5* 10^"7 mol) was added to a solution of the specific condition (100 iL). The mixture was stirred at the specific condition for a certain amount of time, and then the fate of the TCO compound was monitored by HPLC-MS/PDA analysis and/or GC-MS analysis, and an estimation of its stability was made.

Typical example for testing the feasibility of the deprotection

The TCO stock solution (10 μί, 25 mM in acetonitrile; 2.5* 10^"7 mol) was added to a solution of the specific condition (100 μί.). A solution of 3, 6-dimethyl- 1,2,4,5- tetrazine (8, 20 uL 25 mM in acetonitrile; 5.0* 10^"7 mol) was added, and the mixture was stirred at the specific condition for a certain amount of time. The reaction was monitored by HPLC-MS/PDA analysis and/or GC-MS analysis, and the percentage of deprotection was estimated. Conditions:

A) in acetonitrile with 5 equivalents of pyridine per TCO at 20°C

B) in acetonitrile with 5 equivalents of DIPEA per TCO at 20°C

C) in acetonitrile with 5 equivalents of piperidine per TCO at 20°C

D) in acetonitrile with 5 equivalents of n-butylamine per TCO at 20°C

E) in acetonitrile with 5 equivalents of 2-mercaptoethanol per TCO at 20°C

F) in tetrahydrofuran with 5 equivalents of triphenylphosphine per TCO at 20°C

G) in acetonitrile with 5 equivalents of DCC per TCO at 20°C

H) in acetonitrile with 5 equivalents of PyBOP per TCO at 20°C

I) in acetonitrile with 1 v% of formic acid at 20°C

J) in chloroform at 20°C

) in chloroform with 1 v% of formic acid at 20°C

L) in chloroform with 1 v of trifluoroacetic acid at 20°C

M) in chloroform with 10 v% of trifluoroacetic acid at 20°C

N) in chloroform with 33 v% of trifluoroacetic acid at 20°C Z) in 25% acetonitrile in water at 20°C

Table 2

Table 3

Table 4

d i f d 20°C l d h i Example 6

Preparation of a liposome comprising TCO modified channel protein and the tetrazine-induced release of liposomal content

Liposomes containing a quenched dye in the interior and TCO-protein in the membrane were prepared. Subsequently, the tetrazine-induced cleavage of TCO- protein and the resulting release of the liposomal contents was demonstrated by the de-quenching of the released dye.

Protein expression and purification

G22C MscL was expressed and purified as described previously (Kocer et al, 2007, Nat. Prot.). In short, all mutant constructs were transformed into CaCl₂-competent E. coli PB104 cells and were grown in LB medium in the presence of 10 μg/mL chloramphenicol and 100 μg/mL ampicillin. Cells were grown in a bioreactor with pH 7.5, temperature 37 °C, and oxygen control (dissolved oxygen >70%), using a complex medium [12 g/L Bacto-Tryptone (BD), 24 g/L yeast extract (BD), potassium phosphate (17 mM KH2P04 and 72 mM K2HP04) (pH 7), supplemented with chloramphenicol and ampicillin). 40% (vol/vol) glycerol/L medium was used as additional carbon source and 0.1% (w/v) L-arabinose to induce the protein expression. Cells were harvested after 120 minutes.

Membrane vesicles were prepared as described elsewhere (Kocer et al, 2007, Nat. Prot.). Briefly, cells were broken using a cell disrupter (Type TS/40; Constant Systems) at 1.7 kbar and 5 °C. After two subsequent centrifugation steps, membrane vesicles were resuspended and homogenized in ice-cold 25 mM Tris-HCl (pH 8.0) to 7 g (wet weight)/mL, and frozen in liquid nitrogen and stored at -80 °C.

Protein modification and Isolation

Protein was isolated as described by Kocer et. al. (Kocer et al, 2007, Nat Prot).

Briefly, membrane vesicles were solubilized by solubilization buffer (10 mM NaPi pH:8.0, 300 mM NaCl, l%(v/v) Triton and 35 mM imidazole) and unsolubilized material was removed by ultracentrifugation. The solubilized fraction was then applied to Ni-NTA agarose resin (Qiagen) which was equilibrated with solubilization buffer. After 30 minutes incubation, the unbound material was let washed through and the column was washed with 15 CV of wash buffer (10 mM sodium phosphate (pH 8.0), 300 niM NaCl, 0.2% (vol/vol) Triton X- 100, 35 mM imidazole). After this washing step, the column is further washed with a second wash buffer which contains all the components as first wash buffer except for imidazole. Then the column matrix was incubated with TCO-4 in DMSO (2 mg/ml final concentration in column). After 45 minutes at room temperature the column was washed with the second wash buffer and 50 mM histidine buffer (wash buffer containing 50 mM Histidine). Finally, the protein was eluted with 235 mM histidine buffer (wash buffer containing 235 mM Histidine) and the fractions were analyzed for protein content by the Bradford assay. Protein Re constitution into Liposomes

Proteins were reconstituted into synthetic liposomes according to Kocer et al. (Kocer et al, 2007, Nat Prot). Briefly, lipid suspension (azolectin) was homogenized by extrusion 11 times through a 400-nm filter. Liposomes were destabilized by the addition of Triton X- 100. Protein and lipids were mixed at 1 :50 weight ratio and incubated for 30 min at 50 °C. Subsequently, the mixture was supplemented with 6 mg (wet weight) Biobeads (SM-2 Absorbents; Bio-Rad) per microliter of detergent (10% Triton X- 100) used in the sample and lipid preparation. For detergent removal, the sample was incubated overnight (□ 16 h) at 4 °C under mild agitation.

Before fluorescence assay, the mixture was applied to size exclusion column

(Sephadex G50 Pharmacia) in order to collect the liposomes.

Fluorescence Assay

Elution fractions were assayed in a Varian Cary Eclipse Fluorometer at an excitation wavelength wavelength of 495 nm and recording the emission at 515 nm. In a standard assay, 5 iL calcein-filled proteoliposomes were diluted into 2,200 μΕ efflux buffer (10 mM NaPi pH:8.0, 150 mM Na). At t = 1 min, tetrazine was added. The fluorescence was measured continuously, and the total fluorescence of the sample was determined by dissolving the proteoliposomes by the addition of 0.5% (vol/vol) Triton X- 100 at t = 100 min. As a control, empty liposomes were recorded in the presence of tetrazine. The datasets were normalized by using the initial fluorescence of each sample as 0% and the signal after the Triton X-100 addition as 100%.

Results The results are shown in Figure 2. In summary, tetrazine 8 afforded selective release of calcein from TCO-protein containing liposomes at pH 8. No release was observed for control liposomes without protein-TCO.

Claims

Claims:

1. A liposome, comprising a lipid bilayer enclosing a cavity, wherein the bilayer comprises a channel protein releasably linked to an eight-membered non-aromatic cyclic alkenylene group, preferably a cyclooctene group, and more preferably a trans -cyclooctene group.

2. A liposome according to claim 1, wherein eight-membered non-aromatic cyclic alkenylene group satisfies the following formula (la):

(la)

Wherein A and P each independently are CR¾ or CR^aX^D provided that at least one is CR^aX^O. χο i_s (0-C(0))_p-(L^D)_N-(DD), S-C(0)-(LD)„-(DD), O-C(S)- (L^D)_n-(D^D), S-C(S)-(L^D)_n-(D^D), 0-S(0)-(L^D)n-(DD), wherein p - 0 or 1.

Preferably, X^D is (0-C(0))_p-(L^D)_n-(D^D), where p = 0 or 1, preferably 1, and n = 0 or 1; Y,Z,X,Q each independently are selected from the group consisting of CR¾, C=CR¾ C=0, C=S, C=NR^b, S, SO, SO₂, O, NR^b, and SiR^C2, with at most three of Y, Z, X, and Q being selected from the group consisting of

wherein two R moieties together may form a ring, and with the proviso that no adjacent pairs of atoms are present selected from the group consisting of O-O, O-NR^b, S- NR^b, O-S, O-S(O), O-S(O)₂, and S-S, and such that Si is only adjacent to CR¾ or O.

3. A liposome according to claim 1, wherein eight-membered non-aromatic cyclic alkenylene group satisfies the following formula (la):

(la)

Wherein A and P each independently are CR¾ or CR^AX^D provided that at least one is CR^AX^D. X^D is (O-C(O))_P-(LD)_N-(DD), S-C(0)-(LD)„-(DD), O-C(S)- (L^D)n-(D^D), S-C(S)-(L^D)_n-(D^D), O-S(O)-(L^D)n-(D^D), wherein p = 0 or 1. Preferably, X^D is (0-C(0))_p-(L^D)_n-(D^D), where p = 0 or 1, preferably 1, and n = 0 or 1; wherein one of the bonds PQ, QX, XZ, ZY, YA is part of a fused ring or consists of CR^a=CR^a, such that two exocyclic bonds are fixed in the same plane, and provided that PQ and YA are not part of an aromatic 5-or 6-membered ring, of a conjugated 7-membered ring, or of CR^a=CR^a; when not part of a fused ring P and A are independently CR¾ or CR^aX^D provided that at least one is CR^aX^D ; when part of a fused ring P and A are independently CR^a or CX^D provided that at least one is CX^D; the remaining groups (Y,Z,X,Q) being independently from each other CR¾, C=CR¾ C=0, C=S, C=NR^b, S, SO, S0₂, O, NR^b, SiR¾ such that at most 1 group is

and no adjacent pairs of atoms are present selected from the group consisting of O-O, 0-NR^b, S-NR^b, O-S, O- S(O), 0-S(0)2, and S-S, and such that Si, if present, is adjacent to CR¾ or O, and the

bond, if present, is adjacent to CR¾ or

groups.

4. A liposome according to any one of the preceding claims, wherein the eight -membered non-aromatic cyclic alkenylene group is a iran-s-cyclooctene moiety that satisfies formula (lb):

(lb)

wherein, in addition to the optional presence of at most two exocyclic bonds fixed in the same plane, each R^a independently denotes H, or, in at most four instances, a substituent selected from the group consisting of alkyl, aryl, OR', SR', S(=0)R"', S(=0)₂R'", S(=0)₂NR'R", Si-R'", Si-O-R'", OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', F, CI, Br, I, N₃, S0₂H, S0₃H, S0₄H, PO3H, PO4H, NO, N0₂, CN, OCN, SCN, NCO, NCS, CF₃, CF2-R', NR'R", C(=0)R', C(=S)R', C(=0)0-R', C(=S)0-R', C(=0)S-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'C(=0)-R"\ NR'C(=S)-R"', NR'C(=0)0-R"', NR'C(=S)0-R"', NR'C(=0)S-R"', NR'C(=S)S-R'", OC(=0)NR'-R"',

SC(=0)NR'-R"', OC(=S)NR'-R"', SC(=S)NR'-R"', NR'C(=0)NR"-R",

NR'C(=S)NR"-R", CR'NR", with each R' and each R" independently being H, aryl or alkyl and R'" independently being aryl or alkyl;

Each R^e as above indicated is independently selected from the group consisting of H, alkyl, aryl, OR', SR', S(=0)R"'_; S(=0)₂R'", Si-R'", Si-0-R"'_; OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R'", F, CI, Br, I, N₃, S0₂H, S0₃H, P0₃H, NO, N0₂, CN, CF₃, CF₂-R', C(=0)R', C(=S)R', C(=0)0-R', C(=S)0-R', C(=0)S-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'C(=0)-R"', NR'C(=S)- R'", NR'C(=0)0-R"', NR'C(=S)0-R"', NR'C(=0)S-R'", NR'C(=S)S-R"', NR'C(=0)NR"-R", NR'C(=S)NR"-R", CR'NR", with each R' and each R" independently being H, aryl or alkyl and R"' independently being aryl or alkyl; wherein two R^{a e} moieties together may form a ring; and wherein T and F each independently denote H, or a substituent selected from the group consisting of alkyl, F, CI, Br, and I, and X^D is (0-C(0))_p-(LD)_n-(DD),

S-C(0)-(L^D)n-(D^D), 0-C(S)-(LD)„-(DD), S-C(S)-(LD)„-(D^D), 0-S(0)-(LD)_n-(D^D), wherein p = 0 or 1.

5. A liposome according to any one of the preceding claims, wherein the irans-cyclooctene moiety satisfies any one of the following formulae:

^■~>~ = rest of attached D^D L^D-D^D

— = rest of attached D^D, L^D-D^D

126

6. A liposome according to any one of the claims 1-4, wherein the irc -cyclooctene moiety satisfies any one of the following formulae:

= rest of attached D^D, L^D

= rest of attached D^D or L^D-D^C

7. A liposome according to any one of the claims 1-4, wherein the trans -cyclooctene moiety is comprised in a conjugate with the channel protein the conjugate satisfying the formula:

8. A liposome according to any one of the claims 1-4, wherein the trans -cyclooctene moiety comprises the structure obtained from coupling a compound of the formula::

9. A liposome according to any one of the claims 1-4, wherein the trans -cyclooctene moiety comprises any one of the following structures:

10. A liposome according to any one of the preceding claims, wherein the eight-membered non-aromatic cyclic alkenylene group is linked to a cysteine in the channel protein.

11. A liposome according to any one of the preceding claims, wherein the channel protein is a cysteine mutant of a wild-type channel protein.

12. A liposome according to any one of the preceding claims, wherein the channel protein is a mechanosensitive channel of large conductance (MscL).

13. A liposome according to claim 12, wherein the MscL is E coli MscL mutant comprising a Cys at a position selected from positions 15 - 45.

14. A liposome according to claim 12, wherein the MscL is E coli MscL comprising a Gly22Cys mutation

15. A liposome according to any one of the preceding claims, wherein the liposome formulation is DOPC:cholesterol:DSPE-PEG2000 (70:20: 10 mol%),

wherein the abbreviations have the following meanings:

DOPC: l,2-Dioleoyl-sn-glycero-3-phosphocholine

DSPE: l,2-Distearoyl-sn-glycero-3-phosphoethanolamine

PEG: polyethylene glycol.

16. A kit for the administration and activation of an activatable liposome, the kit comprising a liposome, the hposomal membrane of which comprises a channel protein linked to a Trigger, and an Activator for the Trigger, wherein the Trigger comprises an eight-membered non-aromatic cyclic alkenylene group as defined in any one of the preceding claims, and the Activator comprises a diene.

17. A kit according to claim 16, wherein the Trigger (T^R) is linked to the channel protein accordin to any one of the following formulae.

wherein X^s is oxygen, sulfur, nitrogen, carbonyl, aromate, alkene, alkyn, ester, ether, thioester, thioether, amide, amine, imine, alkane, carbamate, carbonate, carboxylic acid ester, sulfate, sulfonate, aminooxy, disulfide, (oligo)dimethylsiloxane, sulfoxide, phosphate and phosphite and Z^s is a thiol reactive group.

18. A kit according to claim 16 or 17, wherein the diene satisfies any one of the following formulae (2) to (4):

(2)

wherein R¹ is selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', OR', SR', C(=0)R', C(=S)R', C(=0)0-R\ C(=0)S-R\ C(=S)0-R', C(=S)S-R", C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=0)OR", NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR",

NR'C(=0)NR"R", and NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl; A and B each independently are selected from the group consisting of alkyl-substituted carbon, aryl substituted carbon, nitrogen, N⁺O", N⁺R with R being alkyl, with the proviso that A and B are not both carbon; X is selected from the group consisting of O, N-alkyl, and C=0, and Y is CR with R being selected from the group consisting of H, alkyl, aryl, C(=0)OR', C(=0)SR', C(=S)OR', C(=S)SR', C(=0)NR'R" with R' and R" each independently being H, aryl or alkyl;

(3)

wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', N0₂, OR', SR', C(=0)R', C(=S)R', OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R'", S(=0)R', S(=0)₂R"',

S(=0)₂NR'R", C(=0)0-R', C(=0)S-R', C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=0)OR",

NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR", OC(=0)NR'R", SC(=0)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=0)NR"R", and NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl; A is selected from the group consisting of N-alkyl, N-aryl, C=0, and CN-alkyl; B is O or S; X is selected from the group consisting of N, CH, C-alkyl, C-aryl, CC(=0)R', CC(=S)R', CS(=0)R', CS(=0)₂R"', CC(=0)0-R', CC(=0)S-R', CC(=S)0-R', CC(=S)S-R',

CC(=0)NR'R", and CC(=S)NR'R", R' and R" each independently being H, aryl or alkyl and R'" independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and

(4) wherein R¹ and R² each independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R\ NO, NO₂, OR', SR', CN, C(=0)R', C(=S)R', OC(=0)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', S(=0)R', S(=O)₂R"', S(=0)₂OR', P0₃R'R", S(=0)₂NR'R", C(=0)0-R', C(=0)S-R', C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=O)OR", NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR", OC(=0)NR'R", SC(=O)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=0)NR"R", and

NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R" independently being aryl or alkyl; A is selected from the group consisting of N, C-alkyl, C-aryl, and N⁺O; B is N; X is selected from the group consisting of N, CH, C-alkyl, C-aryl, CC(=0)R', CC(=S)R', CS(=0)R', CS(=0)₂R"\ CC(=0)0-R', CC(=0)S-R', CC(=S)0-R', CC(=S)S-R', CC(=0)NR'R", CC(=S)NR'R", R' and R" each independently being H, aryl or alkyl and R'" independently being aryl or alkyl; Y is selected from the group consisting of CH, C-alkyl, C-aryl, N, and

19. A kit according to claim 16, wherein the diene satisfies formula (7) as defined in the description.

20. A kit according to claim 16, wherein the diene satisfies formula (8 a) or (8b):

wherein each R¹ and each R² independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R\ N0₂, OR', SR', C(=O)R', C(=S)R', OC(=O)R"', SC(=0)R"', OC(=S)R"', SC(=S)R"', S(=0)R', S(=O)₂R"', S(=O)₂NR'R", C(=O)O-R', C(=O)S-R', C(=S)O-R', C(=S)S-R', C(=O)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=0)OR", NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR", OC(=0)NR'R", SC(=0)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=0)NR"R", and NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl.

21. A kit according to claim 16, wherein the diene is satisfies a formula selected from the group consisting of (8c), (8d), (8e), (8f), and (8g):

wherein each R¹ and each R² independently are selected from the group consisting of H, alkyl, aryl, CF₃, CF₂-R', N0₂, OR', SR', C(=0)R', C(=S)R', OC(=0)R"', SC(=0)R"\ OC(=S)R"', SC(=S)R"', S(=0)R', S(=0)₂R"', S(=0)₂NR'R", C(=0)0-R', C(=0)S-R', C(=S)0-R', C(=S)S-R', C(=0)NR'R", C(=S)NR'R", NR'R", NR'C(=0)R", NR'C(=S)R", NR'C(=0)OR",

NR'C(=S)OR", NR'C(=0)SR", NR'C(=S)SR", OC(=0)NR'R", SC(=0)NR'R", OC(=S)NR'R", SC(=S)NR'R", NR'C(=0)NR"R", and NR'C(=S)NR"R" with each R' and each R" independently being H, aryl or alkyl, and R'" independently being aryl or alkyl.

22. A kit according to claim 16, wherein the diene satisfies any one of the formulae:

23. A kit according to claim 16, wherein the diene satisfies any one of the formulae:

24. A kit according to claim 16, wherein the diene satisfies any one of the formulae:

136

25. A kit according to claim 16, wherein the diene satisfies the formul

26. A kit according to claim 16, wherein the diene satisfies the formul

27. A liposomal composition, comprising:

(a) a Construct of a liposome comprising a lipid bilayer enclosing a cavity, wherein the bilayer comprises a channel protein and, optionally, a Targeting Moiety;

(b) an eight-membered non-aromatic cyclic alkenylene group,

preferably a cyclooctene group, and more preferably a trans- cyclooctene group, linked to the Construct,

wherein the liposome is as defined in any one of the claims 1 to 15.

28. The use of an eight-membered non-aromatic cyclic alkenylene group as a dienophile, preferably a cyclooctene group, and more preferably a ircms-cyclooctene group, as a Trigger on an activatable liposome, wherein the liposome comprises a channel protein linked to the Trigger, and wherein reaction of the dienophile with a diene results in activation of the liposome, wherein the liposome is as defined in any one of the claims 1 to 15 and the diene is as defined in any one of the claims 16 to 26.