BRPI0819828B1 - MORPHOLINE COMPOUNDS AND PROCESSES FOR SYNTHESIZING A MORPHOLINE OLIGOMER - Google Patents

Abstract

MORPHOLINE OLIGOMER SYNTHESIS PROCESS. Morphyline compounds are provided having the structure (I): wherein R1 is selected from the group consisting of lower alkyl, di(lower alkyl)amino, and phenyl; R2 is selected from the group consisting of lower alkyl, monocyclic arylmethyl (arloxy)methyl; R3 is selected from the group consisting of triarylmethyl and hydrogen; and Y is selected from the group consisting of: a protected or unprotected hydroxyl or amino group; a chlorophosphoramidate group; and a phosphorodiamidate linkage to the ring nitrogen of an additional morpholino compound or a morpholino aligomer. Such compounds include doubly protected morpholino guanine (MoG) monomers. Its use in the synthesis of morpholino oligomers and other improved procedures for the synthesis of morpholino oligomers, referring to the deprotection of the nitrogen of the protected morpholino ring in each monomer coupling step, are also described.

Description

Field of Invention

[001]The invention relates to processes for synthesizing phosphorodiamidate-linked morpholino oligomers by coupling morpholino subunit monomers and in particular to improved procedures for deprotecting the protected morpholino ring nitrogen in each coupling step and to the use of morpholino guanine (MoG) subunits with protection at both the N2 and O6/N1 groups of the guanine base. Morpholino oligomers synthesized using these modifications are obtained with higher purity and yields compared to those synthesized using mono-protected guanine subunits and/or conventional ring nitrogen deprotection procedures.

References

[002] Albert, A., Physical Methods in Heterocyclic Chemistry, Vol. I, A. R. Katritzky, Ed., Academic Press, pp 44 (1963).

[003] Fisher, A., Galloway, W. J., and Vaughan, J., J. Chem. Soc. 3591 (1964).

[004]Garrison, A.W. and Boozer, C.E., J. Am. Chem. Soc. 90 (13): 34863494 (1968).

[005] Gough et al. (1979) Nucleic Acids Research 7:1955-1964.

[006]Hata et al. (1983) Tetrahedron Lett. 24:2775–2778.

[007]Jones et al. (1982A) Tetrahedron Lett. 23:2253-2256.

[008]Jones et al. (1982B) Tetrahedron Lett. 23:2257-2260.

[009] Mitsunobu, O. (1981) Synthesis 1: 1-28.

[010]Ravikumar, V. et al., U.S. Patent No. 5,510,476.

[011]Reese et al. (1981) Tetrahedron Lett. 22:4755–4758.

[012] Reese et al. (1984) J.Chem. Soc, Perkins Trans. 1 1263-1270.

[013] Rogne, O., J. Chem. Soc. 727 (1970).

[014]Summerton, J.E. and Weller, D.D. (1993) U.S. Patent No. 5,185,444.

[015]Summerton, J.E. and Weller, D.D., Antisense Nucl. Acid Drug Dev. 7(3): 187-195 (1997).

[016]Summerton, J.E. and Weller, D.D., U.S. Patent No. 5,185,444 (1993).

Basis

[017]Phosphorodiamidate-linked morpholino oligomers, or PMOs, are nucleic acid analogs that bind rigidly and sequence-specifically to complementary RNA and are useful in modulating protein synthesis and thus gene expression. These oligomers are composed of base-pairing recognition groups (heterocyclic bases) supported by a morpholino backbone system. Morpholino subunits for use in the synthesis of such oligomers can be prepared readily from the corresponding ribonucleosides, which are readily available and inexpensive precursors (see, for example, Summerton and Weller, 1993, 1997).

[018]During such a synthesis, as in conventional oligonucleotide synthesis, the functional groups on the heterocyclic bases are typically masked to avoid interference with the synthetic transformations. For example, activation of the N-tritylated morpholino monomer (1a-f; Figure 1) causes the 5'-hydroxyl to react with a suitable phosphoramido dihydrochloride to form the activated 2a-f subunit. On a large scale (50-100 gallon reactor), the crude activated subunit is often contaminated with a high level of byproducts. After chromatographic purification, the activated subunit is isolated in approximately 50% yield for A, C, I, T, U, and their protected forms, but only in approximately 5% yield for the singly activated protected G subunit, which is believed to be due to the presence of the unprotected O6 oxygen.

[019]The unprotected O6 subunit also gives rise to side reactions at the oligomer stage. For example, the O6 oxygen can react with the activated subunit during the coupling steps to form phosphorylated or derivative species, and during the final cleavage of the base protecting groups with ammonia, the ammonia can react at C6 to displace these species, affording a diaminopurine derivative. Such impurities are difficult to remove by chromatography and cause a large loss in yield. Several protection schemes have been proposed in the art to reduce side reactions of the unprotected O6 guanine positions in conventional oligonucleotide synthesis (see, for example, Gough et al. 1979; Reese et al., 1981, 1984; Jones et al. 1982A, 1982B). However, these protocols have been largely unsuccessful when applied to PMO synthesis. Consequently, improved methods are sought to increase yield and purity in PMO syntheses, particularly in the use of morpholino G subunits.

[020]The morpholino nitrogen of a morpholino subunit is also protected prior to use, typically with a trityl or substituted trityl species. During oligomer synthesis, this group needs to be removed during each cycle to allow incorporation of the next subunit. Failure to completely remove the protecting group leads to N-I elimination sequences that contaminate the desired oligomer product.

[021]Trityl groups are conventionally removed with acid, and the deprotection reagents used for PMO synthesis have traditionally been carboxylic acids (Summerton et al. 1993, 1997). However, phosphorodiamidate groups are also acid sensitive, and carboxylic acids useful for detritylation are also capable of promoting hydrolysis of phosphorodiamidate linkages to amidate species, as shown in Fig. 1, with the possibility of more extensive main chain degradation. For example, cyanoacetic acid in 20% acetonitrile/DCM is an effective deprotecting reagent, but it is found to cause substantial (5-10%) hydrolysis of the phosphorodiamidate linkages in the PMO product.

[022]Carboxylic acids also need to be completely removed from the support resin for synthesis prior to the coupling reaction; otherwise by-products consisting of truncated oligomers containing a 3'-acylated species are formed.

[023]For these reasons, improved reagents are needed for the deprotection of the morpholino nitrogen in PMO synthesis.

Summary

[024]In one aspect, the invention provides a morpholino compound comprising structure I: wherein R 1 is selected from the group consisting of lower alkyl, di(lower alkyl)amino, and phenyl; R 2 is selected from the group consisting of lower alkyl, monocyclic arylmethyl, and monocyclic (aryloxy)methyl; R 3 is selected from the group consisting of triarylmethyl and hydrogen; and in selected embodiments, Y is selected from the group consisting of a protected or unprotected hydroxyl group and a chlorophosphoramidate group, for example, a chlorophosphoramidate group of the form -OP(=O)-N(CH 3 ) 2 Cl. When Y is a protected hydroxyl group, it is preferably a protected trialkylsilyl group.

[025]The group R3 is preferably selected from trityl (triphenylmethyl), 4-methoxytrityl, 4-methyltrityl, 4,4'-dimethyltrityl and 4,4',4"-trimethyltrityl. The group R1 is preferably lower alkyl, especially C1-C4 alkyl and more particularly -C(CH3)3 (tert-butyl). The group R2 is preferably selected from benzyl and -CH(CH3)2 (isopropyl).

[026] In a related aspect, the invention provides an improved method of synthesizing a morpholino oligomer, the method comprising: (a) reacting a solid-phase supported morpholino subunit having an unprotected ring nitrogen with a base-protected morpholino subunit monomer having a protected triarylmethyl ring nitrogen and an activated phosphoramidate group on a 5'-exocyclic carbon, thereby forming a phosphoramidate bond between the 5'-exocyclic carbon and the deprotected ring nitrogen; (b) deprotecting the protected ring nitrogen to form a deprotected ring nitrogen; and (c) repeating steps (a) and (b) one or more times with other base-protected morpholino subunit monomers; wherein at least one of the base-protected morpholino subunit monomers is a doubly protected guanine morpholino compound having the structure I: wherein R 1 is selected from the group consisting of lower alkyl, di(lower alkyl)amino, and phenyl; R 2 is selected from the group consisting of lower alkyl, monocyclic arylmethyl, and monocyclic (aryloxy)methyl; R 3 is selected from the group consisting of triarylmethyl and hydrogen, and Y is a chlorophosphoramidate group. Selected embodiments of the variables represented in the above structure include those described above.

[027] In another aspect, the invention provides an improved method of synthesizing a morpholino oligomer, the method comprising: (a) reacting a solid-phase supported morpholino subunit having a deprotected ring nitrogen with a base-protected morpholino subunit monomer having a protected triarylmethyl ring nitrogen and an activated phosphoramidate group on a 5'-exocyclic carbon, thereby forming a phosphoramidate bond between the 5'-exocyclic carbon and the deprotected ring nitrogen; (b) deprotecting the protected ring nitrogen to form a deprotected ring nitrogen; and (c) repeating steps (a) and (b) one or more times with other base-protected morpholino subunit monomers; wherein said deprotection comprises exposing the triarylmethyl-protected ring nitrogen to a reagent solution comprising a heterocyclic amine salt in a solvent containing trifluoroethanol, the salt being a salt of a heterocyclic amine having a pKa in the range of 1-4 in its protonated form, with an acid selected from a sulfonic acid, trifluoroacetic acid, and hydrochloric acid.

[028]The heterocyclic amine is preferably selected from the group consisting of: a pyridine substituted with an electron-withdrawing group, thiazole, pyridazine, pyrazole, triazole, and substituted derivatives thereof substituted with an electron-withdrawing group. Such electron-withdrawing groups (EWG) include halogen, cyano, aldehyde, keto, carboxyester, and carboxamide.

[029] Preferably, the heterocyclic amine is a pyridine substituted with an electron-withdrawing group, such as a chloro- or cyano-substituted pyridine. The amine salt is preferably a salt of a sulfonic acid, such as an alkylsulfonate, (fluoroalkyl)sulfonate, or p-toluenesulfonate, or a trifluoroacetate. In selected embodiments, the salt is selected from 3-chloropyridinium methanesulfonate (CPM) and 4-cyanopyridinium trifluoroacetate (CYTFA). The TFE-containing solvent preferably comprises dichloromethane and trifluoroethanol in a volume ratio ranging from approximately 90:10 to 25:75, and more preferably in a volume ratio of approximately 80:20 DCM:TFE.

[030]The triarylmethyl protecting group is selected from the group consisting of trityl (triphenylmethyl), 4-methoxytrityl, 4-methyltrityl, 4,4'-dimethyltrityl, and 4,4',4"-trimethyltrityl.

[031]The modifications and improvements described herein may be combined such that steps (a)-(c) above are carried out wherein: (1) at least one of the base-protected morpholino subunit monomers is a doubly protected guanine morpholino compound having structure I as set forth above, and (ii) deprotecting the protected ring nitrogen comprises exposing the triarylmethyl-protected ring nitrogen to a solution of a reagent comprising a heterocyclic amine salt in a solvent containing trifluoroethanol, the salt being a salt of a heterocyclic amine having a pKa in the range of 1-4 in its protonated form, with an acid selected from a sulfonic acid, trifluoroacetic acid, and hydrochloric acid. Typically, the synthesis also comprises cleaving the morpholino oligomers from the solid phase and deprotecting the bases according to standard procedures.

[032]These and other objects and features of the invention will become more apparent when the following detailed description is read in conjunction with the accompanying drawings.

Brief Description of the Drawings

[033]Figure 1 illustrates the formation of an activated morpholino subunit. Figure 2 illustrates a formation pathway for a derivative of a doubly protected morpholino G subunit (DPG) in which the N2 position is phenylacetylated and the O6 position is protected with the 4-nitrophenethyl (NPE) group.

[034]Figure 3 illustrates an alternative formation pathway for a doubly protected morpholino G subunit (DPG) derivative in which the N2 position is phenylacetylated and the O6 position is protected with the 4-nitrophenethyl (NPE) group.

[035]Figure 4 illustrates the formation of a DPG derivative in which the N2 position is phenylacetylated and the O6 position is protected with the phenylsulfonylethyl (PSE) group or the methylsulfonylethyl (MSE) group.

[036]Figure 5 illustrates the formation of a DPG derivative in which the N2 position is phenylacetylated and the O6 position is protected with the trimethylsilylethyl (TMSE) group. Figure 6 illustrates the formation of a DPG derivative in which the N2 position is phenylacetylated and the O6 position is protected with a series of aryl derivatives.

[037]Figure 7 illustrates the formation of a DPG derivative in which the N2 position is phenylacetylated and the O6 position is protected with a series of carbamoyl derivatives. Figure 8 illustrates the formation of the DPG derivative in which the N2 position is phenylacetylated and the O6 position is protected with the 4-(pivaloyloxy)benzyloxy (POB) group.

[038]Figure 9 shows the conversion of the phosphorodiamidate (PDA) linkage to phosphoramidate (amidate) linkages in a side reaction that can occur by treating phosphoramidate-linked morpholino oligomers (PMO) with carboxylic acids. Figure 10 illustrates the preparation of a disulfide anchor for use in modifying a synthesis resin used for the stepwise preparation of a morpholino oligomer, which allows for facile release of oligomers by treatment with a thiol.

[039]Figure 11 illustrates the preparation of a triethylene glycol containing the ("Tip") group that increases the aqueous solubility of synthetic antisense oligomers. Figure 12 illustrates the preparation of resins useful for the solid-phase synthesis of morpholino oligomers.

Detailed Description of the Invention I. Definitions

[040]The following terms, as used herein, have the following meanings, unless otherwise indicated:

[041] A "morpholino oligomer" refers to a polymeric molecule having a backbone bearing bases capable of hydrogen bonding to typical polynucleotides, wherein the polymer lacks a pentose sugar backbone group and more specifically a phosphodiester-linked ribose backbone that is typical of nucleotides and nucleosides, but instead contains a ring nitrogen with coupling via the ring nitrogen. A preferred morpholino oligomer is composed of "morpholino subunit" structures, as shown below, which in the oligomers are preferably linked together by phosphoramidate (thio)linkages, associating the morpholino nitrogen of one subunit with the 5' exocyclic carbon of an adjacent subunit. Each subunit includes a purine or pyrimidine Pi base pairing group that is effective for linking, by base-specific hydrogen bonding, to a base in a polynucleotide.

[042]Morpholino oligomers are detailed, for example, in the same Assignee's U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185,444, 5,521,063, and 5,506,337, all of which are expressly incorporated herein by reference in this case.

[043] A "phosphoramidate" group comprises phosphorus having two attached oxygen atoms and two attached nitrogen atoms and herein may also refer to phosphorus having one attached oxygen atom and three attached nitrogen atoms. In the intersubunit linkages of the oligomers described herein, one nitrogen is typically pendant to the main chain link and the second nitrogen is the ring nitrogen in a morpholino ring structure, as shown in formula II below. Alternatively or in addition, a nitrogen may be present at the 5'-exocyclic carbon, as shown in formulas III and IV below.

[044]In a phosphoramidate thioligation, an oxygen atom, typically a main chain pendant oxygen in the oligomers described herein, is replaced with sulfur. A "solid-phase supported morpholino subunit" can be the first or any subsequent morpholino subunit monomer incorporated into morpholino oligomers by stepwise solid-phase synthesis as described herein. The subunit is attached to the solid support, or to an oligomer chain growing on the solid support, via its 5' exocyclic carbon. "Base-protected" refers to the protection of the base-pairing groups, e.g., purine or pyrimidine bases, on the morpholino subunits with suitable protecting groups to prevent reaction or interference of the base-pairing groups during stepwise oligomer synthesis.

[045]An "activated phosphoramidate group" is typically a chlorophosphoramidate group, which has substitution on the nitrogen which is desirable in the eventual phosphoramidate linkage in oligomers. An example is (dimethylamino)chlorophosphoramidate, i.e., -O-P(=O)(NMe2)Cl.

[046]The terms "charged", "uncharged", "cationic" and "anionic" as used herein refer to the predominant state of a chemical group at near-neutral pH, e.g., approximately 6 to 8. Preferably, the term refers to the predominant state of the chemical group at physiological pH, i.e., approximately 7.4.

[047]"Lower alkyl" refers to an alkyl radical of one to six carbon atoms, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl, isoamyl, n-pentyl, and isopentyl. In selected embodiments, a "lower alkyl" group has one to four carbon atoms or 1-2 carbon atoms; i.e., methyl or ethyl. Analogously, "lower alkenyl" refers to an alkenyl radical of two to six, preferably three or four, carbon atoms, as exemplified by allyl and butenyl. A "non-perturbing" substituent is a substituent that does not adversely affect the ability of an antisense oligomer as described herein to bind to its intended target. Such substituents include small and preferably non-polar groups such as methyl, ethyl, methoxy, ethoxy, hydroxy, or fluoro.

II. Protection Based on PMO Synthesis

[048]Due to the specific challenges of morpholino chemistry, a base protecting group must meet several requirements. The protecting group must be easily introduced onto the heterocyclic group and then be stable under the conditions of subunit activation, subunit purification, and solid-phase synthesis. The protecting group must be nonreactive with the amine group of the growing chain morpholino and must allow the activated morpholino subunit to couple cleanly with the growing oligomer chain. The protecting group must be cleaved, preferably by ammonia, without introducing new impurities. Finally, this must result in crystalline subunit derivatives, avoiding the need for chromatographic purification prior to activation.

[049]As described below and in the Comparative Examples, the protecting groups reported in the literature for doubly protected guanosines, as used for nucleic acid synthesis, did not adequately satisfy these criteria. Thus, a new protecting strategy for the morpholino G subunits was needed. As described below, it was found that the use of the 4-(pivaloyloxy)benzyloxy group in 06 satisfies all of the above criteria.

A. O6 Protecting Groups: Comparative Data A1. 4-nitrophenethyl ether (NPE)

[050]This derivative was prepared as shown in Figure 2 (Mitsunobu 1981) or Figure 3 (Jones et al. 1982B). Although the crude O6-protected subunit could be prepared in reasonable yield, the compound was not readily crystalline and could be adequately purified only by silica gel chromatography, which is undesirable for large-scale production. After testing a wide range of resuspension and/or recrystallization conditions, it was found that solvent combinations containing butoxyethanol could, with some difficulty, crystallize the material. However, excess butoxyethanol could not be removed from the final product because the compound likely crystallized as a solvate. The presence of excess alcoholic solvent would not be acceptable in the activation reaction.

[051]The NPE group is cleaved with strong base by a β-elimination mechanism. These conditions tend to generate the reactive byproduct 4-nitrostyrene, which can then react with the reactive sites on the oligomers. Although various expelling agents (e.g., thiols and 1,3-dicarbonyl compounds) were introduced into the deprotection mixture in an attempt to prevent byproduct entrapment by the oligomers, none were completely successful in eliminating this backflow problem. Even after purification, oligomers prepared with this subunit had a yellow tint.

A2. Phenylsulfonylethyl (PSE) and Methylsulfonylethyl (MSE).

[052]These groups were introduced via the corresponding 2-thioethanol derivatives (Jones et al. 1982A, 1982B), as shown in Figure 4. However, no successful crystallization procedure could be found for the resulting subunits. Like the NPE group above, these groups are cleaved by an e-elimination mechanism. After incorporation into an oligomer, these derivatives gave the same problems observed with the NPE group; i.e., internal return of the reactive alkene byproduct formed during deprotection.

A3. Trimethylsilylethyl ether

[053]As reported by Jones (Jones et al. 1982B), an O6-TMSE modified morpholino guanine subunit was prepared as shown in Figure 5, but it was not stable during oligomer synthesis. The oligomers obtained with this subunit showed a range of byproducts obtained from unprotected G subunits O6.

A4. Phenyl ether

[054]The α6-phenyl-substituted morpholino guanine subunits (Figure 6) were prepared according to the procedure of Reese et al. (1981, 1984). Derivatives included unsubstituted phenyl, 2,5-dichlorophenyl, pentafluorophenyl, and 3-fluorophenyl. Such subunits could be incorporated into PMO, but deprotection with the usual reagents, such as 2-nitrobenzaldehyde oxime and strong base, could not be carried to completion without degradation of the oligomers.

A5. Carbamate

[055]Several carbamate derivatives of O6 were synthesized according to the procedure of Hata et al. 1983 (Figure 7). Use of these derivatives in oligomer synthesis gave variable results depending on the derivative used. For the more labile species, such as the diphenylcarbamoyl analog, transfer of the protecting group to the 3'-nitrogen of the growing chain was observed during the coupling step of the solid-phase synthesis, resulting in truncated oligomers containing a 3'-diphenylcarbamoyl group. Furthermore, carbamates of O6 have two possible reaction sites with ammonia. Although the more reactive groups such as the diphenylcarbamoyl group provide a relatively selective attack on the carbonyl, the more stable dimethyl and pyrrolidinyl carbamates showed a significant competitive reaction of ammonia at the C6 position with conversion to diaminopurine.

B. 4-(Pivaloyloxy)benzyloxy protecting group

[056]The 4-(Pivaloyloxy)benzyloxy alcohol (4a, Figure 8) was introduced into the morpholino guanine subunit by an efficient, high-yield synthesis. The subunit before activation (compound If in Figures 1 and 8) can be synthesized and reproducibly isolated on a large scale without chromatographic purification, and it can be crystallized with a variety of solvents (e.g., THF/water, THF/heptane, acetonitrile, various ester/hydrocarbon mixtures).

[057]Ten batches of this subunit obtained on the 50-200 gallon scale (batch size: 8 - 27 kg of compound Ic) provided an average yield of 65% of product, which has a purity (by HPLC) of 97.6% to 99.2%.

[058]The subunit is converted to an activated subunit (i.e., conversion to the 5'-chlorophosphoramidate compound) much more cleanly than the mono-protected G and can be more easily purified by silica gel chromatography. At scale, the overall yield of compound If to compound 2f (Fig. 1) is approximately 50%.

[059]The POB protecting group may be employed with other combinations of protecting groups for the N2 and morpholino ring nitrogens. Suitable N2 protecting groups include phenylacetyl (as illustrated in Fig. 8) as well as acetyl, propionyl, isobutyryl, and phenoxyacetyl. Suitable trityl species for protecting the morpholino ring nitrogen between coupling steps include unsubstituted trityl, 4-methyl-, 4,4'-dimethyl-, and 4,4',4"-trimethyltrityl, and 4-methoxytrityl.

[060]Other acyl protecting groups may also be used in place of pivaloyl for the phenol group of the POB group. Suitable alternatives include N,N-dimethylcarbamoyl and benzoyl.

[061]During the synthesis of PMO, no products are observed in which the pivaloyl group became attached to the 3'-terminus of the smaller fragments of full-length PMO, a side reaction common to the O6 carbamates discussed above. The only notable side product detected was a PMO-containing phenolic residue resulting from the reaction with the deprotection byproduct quinone methide. However, this byproduct could be reduced to trace levels by sufficient dilution of the ammoniacal deprotection solution. Furthermore, it is easily removed due to the strong binding of the phenolic residue to the polymeric resins used for strong anion exchange chromatography. Overall, the overall yield of purified PMO is greatly increased, as seen in Table 1.

[062]The improvement in PMO production stimulated by the POB-protected guanine group is even more evident in the purification following solid-phase PMO synthesis, where difficulty in removing diaminopurine and related byproducts can lead to severe loss during strong anion exchange chromatography (SAX). For example, the crude purities for AVI-4126 prepared with CPM and MPG (monoprotected guanine subunit, 2c) are in the range of 68-73%, which calculates to approximately 58% crude yield of PMO. During the Trityl-On and Trityl-Off purifications, significant material is lost to obtain the pure product, and the overall chromatographic recovery is 52%. For AVI-4126 obtained using CYTFA and DPG (di-protected guanine subunit), the crude purities are 7075%, with comparable NI levels by mass spectrometry (indicating that the efficiencies of the CYTFA and CPM reagents are approximately equivalent) and crude yields of approximately 61%. However, application of the usual purification methods recovers 80% of the PMO from the crude mixture.Table 1.

[063]Syntheses were carried out according to the methods described in the same Applicant's patent application US 11/801,885 using the modifications indicated in the table; see Examples 3-6 below. All PMOs have a 5'-end "Final Product" and are unsubstituted at the 3'-terminus. CAA = 11% cyanoacetic acid (wt/wt) in a mixture of 20% acetonitrile/DCM (v/v); CPM = 2% 3-chloropyridinium methanesulfonate (wt/v) and 0.9% ethanol (v/v) in 20% trifluoroethanol/DCM (v/v); CYTFA = 2% 3-cyanopyridinium trifluoroacetate (wt/v) and 0.9% ethanol (v/v) in 20% trifluoroethanol/DCM (v/v).2 Scale is the starting resin weight in grams. Resin loading is 480-520 micromoles/g3 Combined yield of 4 x 12 g runs and 1 x 8 g runs.4 Combined yield of 2 x 12 g runs.5 Combined yield of 4 x 12 g runs.

[064]Addition of the final C subunit was accomplished with an activated morpholino C subunit with 4-methoxytrityl protection on the morpholino nitrogen.

[065]Thus, use of the doubly protected MoG monomer of the invention provides a method of synthesizing a morpholino oligomer in higher purified yield relative to prior art methods and particularly compared to purified yields observed when a mono-protected MoG monomer or another non-invention protected MoG monomer is employed. In particular, the method generally preferably provides a reduced level of diaminopurine species than would be obtained using a non-invention MoG monomer.

III. Doubly Protected Guanine Morpholino Subunits

[066]The doubly protected guanine (DPG) morpholino subunits of the invention have the structure wherein R 1 is selected from the group consisting of lower alkyl, di(lower alkyl)amino, and phenyl; R 2 is selected from the group consisting of lower alkyl, monocyclic arylmethyl, and monocyclic (aryloxy)methyl; R 3 is selected from the group consisting of triarylmethyl and hydrogen; and Y is selected from the group consisting of: a protected or unprotected hydroxyl or amino group; a chlorophosphoramidate group; and a phosphoramidate linkage to the ring nitrogen of another morpholino compound or a morpholino oligomer.

[067] In selected embodiments, Y is a protected or unprotected hydroxyl group (as in the pre-activated monomer) or a chlorophosphoramidate group (as in the activated monomer). Preferred protecting groups for the hydroxyl group include trialkylsilyl groups, such as tert-butyldimethylsilyl (TBDMS).

[068] Embodiments in which Y is a phosphoramidate linkage to the ring nitrogen of another morpholino compound or a phosphoramidate linkage to a morpholino oligomer refer to the species formed during the synthesis of a morpholino oligomer, prior to base deprotection.

[069]As discussed below, the substituents on the chlorophosphoramidate group (in the activated monomer) can vary depending on the specific phosphoramidate linkage desired.

[070]The invention also correspondingly provides a process of synthesizing a morpholino oligomer, the process comprising: (a) reacting a solid-phase supported morpholino subunit having a deprotected ring nitrogen with a base-protected morpholino subunit monomer having a triarylmethyl-protected ring nitrogen and an activated phosphoramidate group on a 5'-exocyclic carbon, thereby forming a phosphoramidate bond between said 5'-exocyclic carbon and said deprotected ring nitrogen; (b) deprotecting said protected ring nitrogen to form a deprotected ring nitrogen; and (c) repeating steps (a) and (b) one or more times with other base-protected morpholino subunit monomers; wherein at least one of said base-protected morpholino subunit monomers is a doubly protected guanine morpholino compound having the structure: wherein R 1 is selected from the group consisting of lower alkyl, di(lower alkyl)amino, and phenyl; R 2 is selected from the group consisting of lower alkyl, monocyclic arylmethyl, and monocyclic (aryloxy)methyl; R 3 is selected from the group consisting of triarylmethyl and hydrogen, and Y is a chlorophosphoramidate group. Preferred triarylmethyl protecting groups for the nitrogen of the morpholino ring (R 3 ) include trityl (triphenylmethyl), 4-methoxytrityl, 4-methyltrityl, 4,4'-dimethyltrityl, and 4,4',4"-trimethyltrityl.

[071]The R1 substituent on the O6 protecting group is preferably C1 to C4 alkyl, especially -C(CH3)3 (tert-butyl), as in the 4-(pivaloyloxy)benzyloxy (POB) group. However, R1 may also be di(lower alkyl)amino, such as dimethylamino or phenyl.

[072]As noted above, the substitution of the chlorophosphoramidate group Y in “activated” monomers varies depending on the structure of the desired phosphoramidate bond. For the preparation of the uncharged PMO “standardized” bond 5'-O-P(=O)(-N(CH3)2)-3' (as shown in Formula II above when R is methyl), the chlorophosphoramidate group Y is 5'-O-P(=O)Cl-NR2 (see, e.g., compound 2f, Figure 8).

[073] As described in the same Applicant's patent application having USSN 11/801,885, filed on May 10, 2007, which is incorporated herein by reference, advantageous properties can be obtained by preparing PMOs having cationic as well as neutral intersubunit linkages. In such oligomers, at least one intersubunit linkage between two consecutive morpholino ring structures contains a pendant cationic group. The pendant group contains a distal nitrogen atom that may bear a positive charge at neutral or near-neutral (e.g., physiological) pH.

[074]For the preparation of such linkages, the chlorophosphoramidate group Y in the monomers of the subunit of the invention may have one of the following structures: where R is lower alkyl, such as methyl or ethyl; X = -R4-NHC(=O)Rf, where R4 is divalent alkyl or oligo PEG and Rf is fully or partially fluorinated methyl, ethyl, or isopropyl and Z = X as defined above or lower alkyl. Note that the A-containing group results in a 5'-amine-containing linkage.

[075]The term "oligo PEG" refers to a group such as -(CH2-CH2-O)n-CH2- CH2- where n is typically 1 to 3 and "bivalent alkyl" is typically C2 to C8 alkyl.

[076]After preparing oligomers using monomers having activated chlorophosphoramidate groups, the C(=O)Rf protecting groups are removed from the terminal nitrogen atoms, which may be further modified, for example, to form terminal guanidinyl groups, as described in the same Applicant's patent application USSN 11/801,885.

IV. Improved Conditions for Morpholino Ring Nitrogen Deprotection in PMO Synthesis

[077]As noted above, deprotection of the nitrogen group of the morpholino ring, which is typically protected by a triarylmethyl group such as trityl, in the PMO synthesis needs to be sufficiently complete at each step to minimize N-I elimination species. However, studies in support of the invention have demonstrated that reagents used in the prior art for this purpose caused an undesirable amount of main chain hydrolysis (see Fig. 1) and degradation. Therefore, efficient deprotection reagents that at the same time minimize such hydrolysis were sought.

[078]A simple assay was used to test the efficiency of various reagents in deprotecting (typically detritylating) N-protected morpholino subunits. A model compound, tritylated moCBz (i.e., the benzoyl-protected morpholino cytosine subunit) shown below, is dissolved in the detritylation solution to be investigated. At various time points (e.g., 1, 2, 4 minutes), an aliquot was quenched and analyzed by TLC or HPLC for complete deprotection of the morpholino nitrogen. Generally, to predict efficient detritylation during solid-phase synthesis of PMO, this model reaction should be complete within approximately 2 minutes at room temperature.

[079]Using this assay and further experimentation, it was determined that various pyridinium salts of strong acids in mixtures of trifluoroethanol (TFE) and dichloromethane (DCM) are excellent catalysts for removing the triarylmethyl protecting group, e.g., a trityl group, from the morpholino nitrogen during solid-phase PMO synthesis.

[080]A minimal amount of TFE (~10% v/v or greater) is preferred for reasonable reaction rates and solubilization of the pyridinium salts. Since TFE alone does not swell bare polystyrene, mixtures with DCM (dichloromethane) are preferred, especially in the earlier PMO synthesis cycles. Preferred solvent compositions include 10 to 75% TFE.

[081]The use of the solvent TFE is believed to improve the selectivity of the detritylation reaction with respect to amidate formation (hydrolysis) and phosphoramidate (PDA) cleavage, described above, by controlling different mechanisms of PDA cleavage and detritylation. TFE is a potent hydrogen-bonding solvent and decreases the reactivity of nucleophiles in solution; therefore, it is believed to retard the attack on phosphorus required for P-N bond cleavage. TFE also promotes SN1-type solvolysis reactions. The solvolytic character of amine detritylation reactions with TFE is evidenced by the yellow color of the detritylation reaction mixtures and the orange color of the demethoxytritylation reaction mixtures. Therefore, increasing the concentration of TFE is believed to both suppress nucleophilic attack on the PDA bond and promote detritylation.

[082]Unsubstituted pyridinium salts are not sufficiently acidic for optimal deprotection, but the use of pyridinium species containing electron-withdrawing groups (EWG) (e.g., halogen, carbonyl, cyano) allows rapid cleavage of the protecting group. Generally, at least 2% (w/v) of such a salt in the TFE:DCM solvent is sufficient for rapid detritylation. Preferred levels of pyridinium salts are 2 to 10% (w/v).

[083] Acids useful in the formation of pyridinium salts include sulfonic acids such as methanesulfonic, trifluoromethanesulfonic, and p-toluenesulfonic acids, trifluoroacetic acid, and hydrochloric acid. Although a carboxylic acid, trifluoroacetic acid does not cap the growing PMO chain if present during the coupling reaction, and its carboxylate is not sufficiently nucleophilic to promote amidate formation. Particularly preferred are trifluoroacetic acid and especially methanesulfonic acid.

[084]Pyridines useful in the formation of pyridinium salts include halogen-substituted pyridines, especially the less costly chloropyridines, of which 4-cyanopyridine is preferred. 3- and 4-cyanopyridines are inexpensive, readily available bulk chemicals. In general, the efficiency of salts correlates inversely with the pKa of the pyridinium species. Pyridines with electron-withdrawing groups are in the pKa range of approximately 1 to 4 (Fisher et al. 1964, Rogne 1970).

[085]Also useful are nicotinic acid derivatives (i.e., esters such as ethyl nicotinate and nicotinamide), as well as their ketone and aldehyde congeners. Generally, however, these are less potent reagents than the cyanopyridinium salts.

[086] It will be appreciated that salts of heterocycles other than pyridines may function as selective detritylation reagents under the conditions described, provided that the pKa of the protonated form is similar to that of the substituted pyridines of the invention. Examples can be found in the many pKa tables for heterocycles found in the literature (e.g., Albert 1963). Examples include thiazole (pKa 2.53), pyridazine (pKa 2.33), pyrazole (pKa 2.47), triazole (pKa 2.30), and substituted derivatives thereof, especially EWG-substituted derivatives as described above.

[087] Two particularly preferred salts are 3-chloropyridinium methanesulfonate (CPM) and 4-cyanopyridinium trifluoroacetate (CYTFA), and particularly preferred embodiments of detritylation reagents include solutions of 2% (w/v) CPM or CYTFA in 20% trifluoroethanol/DCM (v/v) containing 0.9% ethanol (v/v). As shown in Table 1 above, the use of these reagents resulted in a significant increase in yield over conventional cyanoacetic acid reagents.

[088] It has been found that the more acidic CYTFA is slightly more efficient than the CPM and CYTFA reagents in the Table can be attributed to the use of doubly protected guanine (DPG) monomer in which the 06-position is protected with a 4-(pivaloyloxy)benzyloxy group, as disclosed in the same Applicant's concurrently filed provisional patent application entitled "Improved Synthesis of Oligomers of Morpholinos using Doubly Protected Guanine Subunits of Morpholino". In general, the use of DPG monomer reduces the amount of diaminopurine-containing side products, although the improved detritylation reagents reduce the amount of hydrolyzed or truncated main chain products.

[089] Thus, this modification provides a process for synthesizing a morpholino oligomer with reduced hydrolysis of the phosphoramidate bond in the backbone, and preferably a reduced or equivalent level of N-1 elimination species, relative to prior art methods. In another aspect, the invention provides a method of deprotecting a nitrogen in the triarylmethyl-protected morpholino ring during the synthesis of a morpholino oligomer ring, with reduced hydrolysis of the phosphoramidate bond in the backbone of the morpholino oligomers relative to that observed when cyanoacetic acid is used as the deprotection reagent. Preferably, the method also provides a reduced or equivalent level of N-1 elimination species than would be observed when cyanoacetic acid is used as the deprotection reagent. Another useful modification is the use of a trityl trapping agent, such as a thiol, to shift the reaction equilibrium toward the products. The use of trityl trapping agents has been employed for nucleic acid synthesis (Ravikumar et al., U.S. Patent No. 5,510,476). Mercaptoethanol is a readily available, inexpensive agent useful for this purpose. The presence of the hydroxyl group is not critical for trapping, because simple thiols such as benzylmercaptan work equally well. Alcohols, such as ethanol and butanol, and even water also serve as trapping agents for the trityl cation.

Examples Example 1: Synthesis of N2-PhAc, O6-POB Doubly Protected Morpholino G (DPG) Subunit (See Fig. 8)

[090]Preparation of 3 (Starting from 35 kg of Ic): A 100 G reactor is charged with Ic (35 kg; 1.0 eq), imidazole (5.0 kg; 1.3 eq), and dichloromethane (279 kg). The batch is cooled to 3° C. A 50 G reactor is cooled to 3° C and charged with t-butylchlorodimethylsilane (10.1 kg; 1.2 eq) and dichloromethane (93 kg). The solution in the 50 G reactor is transferred to the 100 G reactor, and the batch is adjusted to 20°C. After completion of the reaction (1–3 hours), methanol (1.8 kg; 1.0 eq) is charged to the 100 G reactor. After 30 minutes, the solution in the 100 G reactor is charged to a 200 G reactor containing pH 3 citrate buffer (376 kg of 1 M citric acid adjusted to pH 3 with solid NaOH). The batch is stirred for 30 minutes, and the layers are separated. The lower organic layer is washed once more with pH 3 citrate buffer and once with brine solution (287 kg of 2.5% (wt:wt) NaCl/water). The resulting organic solution is distilled at <35°C until the Karl Fischer analysis of the batch shows <0.05% water. This solution is cooled to 3°C in the 100 G reactor and is used directly in the preparation of compound 4.

[091]Preparation of 4: The 100 G reactor containing the solution of compound 3 is charged with triethylamine (6.8 kg; 1.2 eq), 4-dimethylaminopyridine (0.68 kg; 0.1 eq), and triisopropylbenzenesulfonyl chloride (18.6 kg; 1.1 eq). The batch is heated to 20°C. After completion of the reaction (3-9 hours), the solution is charged to a 200 G reactor containing pH 4.5 phosphate buffer (228 kg of 1 M KH2PO4). The batch is stirred for 30 minutes and the layers are separated. The lower organic layer is washed with brine (212 kg of 2.5% NaCl/water (wt:wt)). The resulting organic solution is distilled at <35°C until Karl Fischer analysis of the batch shows <0.01% water. This solution is cooled to 3°C in the 100 G reactor and is used directly in the preparation of compound 5.

[092]Preparation of 4a (Starting from 60 kg of 4-hydroxybenzaldehyde): A 750 G reactor is charged with 4-hydroxybenzaldehyde (60 kg; 1.0 eq), toluene (260 kg), and 1-methyl imidazole (8.1 kg; 0.2 eq). To this solution is charged a solution of potassium bicarbonate (100 kg; 2.0 eq) in water (400 kg), followed by trimethylacetyl chloride (83 kg; 1.4 eq). This two-phase mixture is stirred at 20°C. After completion of the reaction (1-5 hours), methanol (15.7 kg; 1.0 eq) is charged to the batch. The batch is stirred at 20°C for 1 hour. The layers are separated. To the upper organic layer is charged water (200 kg). The batch is stirred for 30 min and the layers are separated. To the upper organic layer is charged pH 4.5 phosphate buffer (16.5 kg of KH2PO4 in 242 kg of water). The batch is stirred for 30 min and the layers are separated. To the upper organic layer is charged water (200 kg). The batch is stirred for 30 min and the layers are separated. The upper organic layer is vacuum distilled at <30 °C to achieve a batch volume of 200 L. THF (70 kg) is charged to the batch and the batch is transferred to a 500 G reactor containing Pd/C (9.6 kg; 0.004 eq; 5% Pd/C, 50% Johnson Matthey Type A405028-5 or A570129-5 wet). The reactor is initially pressurized to 34.5 kPa (5 psi) H2 with stirring set at 50 rpm. Both the pressure and stirring rate are slowly increased as the reaction proceeds, up to a maximum of 172 kPa (25 psi) H2 and 90 rpm. After completion of the reaction (8–48 hours), the batch is filtered through a Celite pad followed by a 0.1 micron in-line filter. The Celite pad is rinsed with toluene (20 kg). To the batch is charged phosphate buffer solution at pH 6.5 (2.7 kg of KH2PO4 and 2.3 kg of potassium phosphate, dibasic, trihydrate in 200 kg of water). The batch is stirred for 30 minutes, and the layers are separated. The upper organic layer is vacuum distilled at <30°C to achieve a batch volume of 140 L. Toluene (126 kg) is charged to the batch, and the batch is vacuum distilled at <30°C to achieve a batch volume of 140 L. The batch is adjusted to 20°C and transferred to a 500 G reactor containing n-heptane (821 kg) and seed crystals of compound 4a (100 grams) maintained at 0°C. The batch is maintained at 0°C for 1-2 hours. A second portion of seed crystals (100 grams) is added, and the batch is maintained at 0°C for 1-2 hours. Compound 4a is isolated by filtration. Yield = 70-80% with 4-hydroxybenzaldehyde.

[093]The derivative in which the phenol group is protected as its N,N-dimethylcarbamate instead of the pivalate ester is obtained under conditions similar to 4a. To force completion of the reaction between 4-hydroxybenzaldehyde and dimethylcarbamoyl chloride, the reaction is carried out in dichloromethane under reflux in the presence of N-methylimidazole as base and 0.2 eq of DMAP as catalyst.

[094]Preparation of 5: A 100 G reactor containing the solution of compound 4 is charged with N-methylpyrrolidine (9.5 kg; 2.0 eq dissolved in 23 kg of dichloromethane). After 10 minutes, compound 4a (14.0 kg; 1.2 eq) is added, followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (10.2 kg; 1.2 eq in 23 kg of dichloromethane). The batch is heated to 20° C. After completion of the reaction (1-9 hours), the solution is diluted with 327 kg of dichloromethane and charged to a 200 G reactor containing phosphate buffer at pH 4.5 (334 kg of 1 KH2PO4M). The batch is stirred for 30 minutes and the layers are separated. The lower organic layer is washed once more with a phosphate buffer at pH 4.5 (111 kg of 1 M KH2PO4), then once with brine (212 kg of 2.5% NaCl/water (wt:wt)). The resulting organic solution is distilled at <35°C until Karl Fischer analysis of the batch shows <0.05% water. This solution is used directly in the preparation of compound If.

[095]Preparation of If: A 100 G reactor containing compound 5 solution is charged with triethylamine trihydrofluoride (18.0 kg; 2.0 eq). The batch is stirred at 20°C. After completion of the reaction (4-20 hours), the batch is charged to a 200 G reactor. The 200 G reactor is charged with NaHCO3 solution (230 kg of a 5% (wt:wt) solution). The batch is stirred for 30 minutes and the layers are separated. The lower organic layer is washed once more with NaHCO3 solution (230 kg of a 5% (wt:wt) solution), then once with phosphonate buffer at a pH of 6.5 (9.3 kg of KH2PO4 and 14.0 kg of K2HPO4 in 215 kg of water). The resulting organic solution is solvent exchanged into THF (to achieve <1% DCM by weight in the batch). The solution is diluted with THF (124 kg) and heated to 60°C. Water (8 kg per kg of compound If in solution based on LOE analysis; preheated to 60°C) is slowly charged to the THF solution. The solution is slowly cooled to 3°C and held for >4 hours. Crude compound If is isolated by filtration. The crude material is redissolved in THF (342 kg) and heated to 60°C. Water (315 kg; preheated to 60°C) is slowly charged to the THF solution. The solution is slowly cooled to 3°C and held for >4 hours. Compound If is isolated by filtration. A second recrystallization can be performed to further purify compound If if desired. Yield = 53–73% starting from Ic.

[096]Preparation of 2f (Starting from 12 kg of If): A 50 G reactor is charged with compound If (12 kg; 1.0 eq), dichloromethane (159 kg), 2,6-lutidine (2.5 kg; 1.6 eq), and 1-methylimidazole (0.36 kg; 0.3 eq). This solution is distilled to a batch volume of 69 L and cooled to 5° C. N,N-Dimethylphosphoramido dihydrochloride (3.8 kg; 1.6 eq) is charged in batch. The batch is adjusted to 20° C. After completion of the reaction (6-16 hours), toluene (78 kg) is charged in batch. The resulting mixture is distilled at 25°C to obtain a batch volume of 126 L (GC analysis of the batch should show 30–45% DCM by weight) and transferred to a 100 G reactor containing citrate buffer at pH 3 (15.4 kg citric acid monohydrate, 1.4 kg NaOH, 80 kg water). The batch is stirred for 10 minutes, and the layers are separated. The lower aqueous layer is sent for disposal. The upper organic layer is transferred to the 50 G reactor containing sodium sulfate (8.0 kg). The batch is stirred for 30 minutes, and the sodium sulfate waste cake is removed by filtration. The sodium sulfate cake is rinsed with dichloromethane (16 kg). The resulting product solution is distilled in the 50 G reactor to achieve a batch volume of 53 L (GC analysis of the batch should show 11-15% DCM by weight). The 100 G reactor is charged with heptane (238 kg). The batch in the 50 G reactor is transferred to the 100 G reactor for 2 hours. After the transfer, the batch is maintained at 20°C for 4-16 hours. Crude compound 6 is collected by filtration. The crude material is charged to the 100 G reactor. A solution of toluene (16 kg) and heptane (50 kg) is added to the crude solids. This mixture is stirred for 3 hours and filtered. The resuspension is repeated one or more times. Yield of crude 2f = 80% starting from If.

[097]Purification of Compound 2f by Silica Gel Chromatography (Starting from ~6.5 kg of compound 2f): crude: The "concentration" of crude compound 2f is calculated by correcting the weight of the crude material for purity and volatiles by HPLC. For this purification step, 5.75 kg of material (corrected for concentration) is used for injection onto a 50 cm chromatography column. The 50 cm chromatography column is packed with a heptane/silica gel slurry (51.8 kg of silica gel). The crude material is loaded onto the column as a solution in dichloromethane/2,6-lutidine (15 kg of dichloromethane, 0.16 kg of 2,6-lutidine). The product is eluted with a two-step gradient of 4-methyl-2-pentanone (MIBK)/heptane/2,6-lutidine (first step is 827 L of 39:61 (wt:wt) MIBK:heptane with 0.06% 2,6-lutidine (wt:wt); second step is 1343 L of 73:27 MIBK:heptane (wt:wt) with 0.06% 2,6-lutidine (wt:wt)). The passed fraction pool is concentrated by thin film evaporation to a concentration of 150 g/L. This concentrated pool is precipitated over 6 volumes of heptane. Purified 2f is isolated by filtration. Yield of purified 2f = 50% starting from If; 65% starting from crude 2f.

Example 2. Preparation of CYTFA Detritylation Pyridinium Salt Solution

[098]To a solution of 4-cyanopyridine (10.1 g; 1.055 eq) in dichloromethane (790 mL) is added trifluoroacetic acid (10.5 g; 1.0 eq) followed by 2,2,2-trifluoroethanol (198 mL) and ethanol (10 mL) and the solution is stirred for 10-30 minutes.

Example 3: Preparation of Disulfide Anchor (See Fig. 10)

[099]Preparation of N-trityl piperazine, succinate salt (NTP): To a cooled solution of piperazine (10 eq) in toluene/methanol-(toluene/methanol 5:1 (volume:volume); 5 mL/g piperazine) was slowly added a solution of triphenylmethyl (trityl) chloride (1.0 eq) in toluene (5 mL/g trityl chloride). After completion of the reaction (1 - 2 hours), this solution was washed four times with water. To the resulting organic solution was added an aqueous solution of succinic acid (1.1 eq; 13 mL water/g succinic acid). This mixture was stirred for 90 minutes and the solid product was collected by filtration. Crude NTP was purified by two resuspensions in acetone. Yield = 70%.

[0100] Preparation of 7:1 Symmetrical disulfide, 1'-carbonyldiimidazole (CDI) (12.402 g; 2.2 eq.) was suspended in dichloromethane (5.25 mL/g) and cooled in an ice bath. Hydroxyethyl disulfide 6 (5.36 g; 1 eq.) was dissolved in dichloromethane (10 mL/g) and tetrahydrofuran (1 mL/g). The diol solution was added to CDI slowly such that the temperature of the mixture remained below 4 °C throughout the reaction. After completion of the reaction (once the addition was complete), deionized water (93.8 μL, 0.15 eq.) was added to quench the reaction. Independently, N-trityl piperazine succinate salt (NTP) (32.59 g; 2.1 eq.) was dissolved in toluene (8 mL/g NTP), dichloromethane (2 mL/g NTP), and methanol (2 mL/g NTP). K2CO3 (22.09 g; 4.6 eq.) was dissolved in deionized water (10 mL/g). The K2CO3 solution was added to the NTP solution; the mixture was stirred and then separated into two layers. The cloudy organic layer was distilled to remove 90 grams; the resulting water droplets were separated, and acetone (8 mL/g NTP) was added to the organic layer. The CDI-activated disulfide solution was added to the free base solution and concentrated to 225 mL. Acetone (10 mL/g NTP) was added, and the mixture was concentrated to 225 mL. The mixture was heated to reflux and the solid began to crystallize out of solution. After completion, the reaction mixture was cooled and the solid (7) was isolated by filtration. Yield: 27.92 g; 93.1% (based on weight assay).

[0101] Preparation of Alcohol Disulfide 8:7 (36.00 g; 32.1 mmol; 1 eq.) was suspended in acetone (2.8 mL/g of 7). Hydroxyethyl disulfide (78.51 mL; 20 eq.) was added followed by acetone (1.7 mL/g of 7). 5% Na-OH/methanol (2.85 mL; 0.1 eq.) was added; the pH of the mixture was found to be 10 by pH paper. Triphenylphosphine (8.42 g; 1 eq.) was added followed by acetone (1.1 mL/g of 7). All of the solids went into solution and then began to crystallize out. After sixteen hours, the reaction mixture was neutralized with acetic acid (2.4 g; 0.2 eq.). The crude product was isolated by filtration. The crude product 8 was resuspended twice at reflux with acetone (5 mL/g of 7). After filtration, the crude product was suspended in dichloromethane (7.25 mL/g of 7).

[0102]The mixture was heated until a clear solution formed (35° C). The solution was extracted five times with an equal volume of deionized water, and the final organic layer was concentrated to 155 mL. Dichloromethane (4.3 mL/g of 7) was added, and the solution was again concentrated to 155 mL. CDI (9.17 g; 1.1 eq.) was added, and the mixture was stirred at room temperature. After completion of the reaction (~20 min), the reaction mixture was washed twice with an equal volume of deionized water, then ethylbenzene (2.1 mL/g of 7) was added. The solution was concentrated to 65.2 g, reducing the dichloromethane in the solution to 0.17% and stirred over an ice bath to crystallize the product. Product 9 was isolated by filtration. Yield: 44%.

Example 4: Triethylene Glycol Final Product (See Fig. 11)

[0103] Preparation of trityl piperazine phenyl carbamate 10: To a cooled suspension of NTP in dichloromethane (6 mL/g NTP) was added a solution of potassium carbonate (3.2 eq) in water (4 mL/g potassium carbonate). To this two-phase mixture was slowly added a solution of phenyl chloroformate (1.03 eq) in dichloromethane (2 g/g phenyl chloroformate). The reaction mixture was heated to 20°C. After completion of the reaction (1-2 hours), the layers were separated. The organic layer was washed with water and dried over anhydrous potassium carbonate. Product 10 was isolated by crystallization with acetonitrile. Yield = 80%

[0104] Preparation of carbamate alcohol 11: Sodium hydride (1.2 eq) was suspended in 1-methyl-2-pyrrolidinone (32 mL/g sodium hydride). To this suspension were added triethylene glycol (10.0 eq) and compound 10 (1.0 eq). The resulting slurry was heated to 95°C. After completion of the reaction (1-2 hours), the mixture was cooled to 20°C. To this mixture were added 30% dichloromethane/methyl tert-butyl ether (v:v) and water. The organic layer containing the product was washed successively with aqueous NaOH, aqueous succinic acid, and saturated aqueous sodium chloride. Product 11 was isolated by crystallization with dichloromethane/methyl tert-butyl ether/heptane. Yield = 90%.

[0105] Acid preparation of final product 12: To a solution of compound 11 in tetrahydrofuran (7 mL/g of 11) was added succinic anhydride (2.0 eq) and DMAP (0.5 eq). The mixture was heated to 50°C. After completion of the reaction (5 hours), the mixture was cooled to 20°C and adjusted to pH 8.5 with aqueous NaHCO3. Methyl tert-butyl ether was added and the product was extracted into the aqueous layer. Dichloromethane was added and the mixture was adjusted to pH 3 with aqueous citric acid. The organic layer containing the product was washed with a mixture of pH = 3 citrate buffer and saturated aqueous sodium chloride solution. This DCM solution of 12 was used without isolation in the preparation of compound 13.

[0106] Preparation of 13: To the solution of compound 12 was added N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB) (1.02 eq), 4-dimethylaminopyridine (DMAP) (0.34 eq) and then 1-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (1.1 eq). The mixture was heated to 55° C. After completion of the reaction (4-5 hours), the mixture was cooled to 20° C and washed successively with 0.2 M citric acid/brine 1:1 and brine. The dichloromethane solution was solvent exchanged to acetone and then to N,N-dimethylformamide, and the product was isolated by acetone/N,N-dimethylformamide precipitation in saturated aqueous sodium chloride. The crude product was reslurried several times in water to remove residual N,N-dimethylformamide and salts. Yield = 70% of 13 starting from compound 11. The introduction of the activated "Final Product" onto the disulfide anchor resin was carried out in NMP by the procedure used for subunit incorporation during solid-phase synthesis.

Example 5: Preparation of Solid Support for the Synthesis of Morpholino Oligomers Example 5a: Preparation of aminomethylpolystyrene disulfide resin

[0107]This procedure was performed in a silanized, jacketed peptide vessel (ordered from ChemGlass, NJ, USA) with a coarse porosity (40-60 μm) glass frit, overhead stirrer, and 3-way Teflon stopper to allow N2 to bubble through the frit or a vacuum stripper. Temperature control was achieved in the reactor by a circulating water bath.

[0108]The resin treatment/washing steps in the following procedure consist of two basic operations: resin fluidization and solvent/solution extraction. For resin fluidization, the stopper was positioned to allow N2 to flow upward through the frit, and the resin specified in the treatment/washing was added to the reactor and allowed to permeate and completely wet the resin. Mixing and suspension of the mixed resin was then initiated for the specified period of time. For solvent/solution extraction, mixing and N2 flow were stopped, and the vacuum pump was started, and then the stopper was positioned to allow evacuation of the treatment/washing resin to the tailings. All resin treatment/washing volumes were 15 mL/g resin unless otherwise noted.

[0109]To aminomethylpolystyrene resin (100-200 mesh; ~1.0 mmol/g N2 substitution; 75 g, 1 eq, Polymer Labs, UK, part #1464-X799) in a jacketed, silanized peptide vessel was added 1-methyl-2-pyrrolidinone (NMP; 20 ml/g resin) and the resin was allowed to swell with mixing for 1-2 hours. After evacuation of the solvent to swell, the resin was washed with dichloromethane (2 x 1-2 minutes), 5% diisopropylethylamine in 25% isopropanol/dichloromethane (2 x 3-4 minutes), and dichloromethane (2 x 1-2 minutes). After evacuation of the final wash, the resin was fluidized with a solution of disulfide anchor 9 in 1-methyl-2-pyrrolidinone (0.17 M; \5 mL/g resin, ∼2.5 eq) and the resin/reagent mixture was heated at 45°C for 60 hours. Upon completion of the ratio, heating was stopped and the anchor solution was evacuated and the resin washed with 1-methyl-2-pyrrolidinone (4 x 3-4 minutes) and dichloromethane (6 x 1-2 minutes). The resin was treated with a 10% (v/v) solution of diethyl dicarbonate in dichloromethane (16 mL/g; 2 x 5-6 minutes) and then washed with dichloromethane (6 x 1-2 minutes). Resin 14 was dried under a stream of N2 for 1-3 hours and then under vacuum to constant weight (±2%). Yield: 110 - 150% of the original weight of the resin.

Example 5b: Determination of Aminomethylpolystyrene disulfide Resin Loadings

[0110]Resin loading (number of potentially available reactive sites) is determined by a spectrometric assay for the number of triphenylmethyl (trityl) groups per gram of resin.

[0111]A known weight of dry resin (25 ± 3 mg) is added to a 25 mL silanized volumetric flask and ~5 mL of 2% (v/v) trifluoroacetic acid in dichloromethane. The contents are mixed by gentle shaking and then allowed to stand for 30 minutes. The volume is made up to 25 mL with an additional 2% (v/v) trifluoroacetic acid in dichloromethane and the contents mixed thoroughly. Using a positive displacement pipette, an aliquot of the trityl-containing solution (500 μL) is transferred to a 10 mL volumetric flask and the volume made up to 10 mL with methanesulfonic acid.

[0112]The trityl cation content in the final solution is measured by UV absorbance at 431.7 nm and the resin loading calculated in trityl groups per gram of resin (μmol/g) using the appropriate volumes, dilutions, extinction coefficient (ε: 41 μmol-1cm-1) and resin weight. The assay is performed in triplicate and a loading calculated.

[0113]The resin loading procedure in this example will provide resin with a loading of approximately 500 μmol/g. A loading of 300-400 μmol/g was obtained if the disulfide anchor incorporation step was carried out for 24 hours at room temperature.

Example 5c: Loading the final product (See Fig. 12)

[0114]Using the same setup and volumes as for the preparation of aminomethylpolystyrene disulfide resin, the final product can be introduced into the molecule. For the coupling step, a solution of 13 (0.2 M) in NMP containing 4-ethylmorpholine (NEM, 0.4 M) was used instead of the disulfide anchor solution. After 2 hours at 45°C, resin 15 was washed twice with 5% diisopropylethylamine in 25% isopropanol/dichloromethane and once with DCM. To the resin was added a solution of benzoic anhydride (0.4 M) and NEM (0.4 M). After 25 minutes, the reactor jacket was cooled to room temperature and the resin washed twice with diisopropylethylamine in 25% isopropanol/dichloromethane and eight times with DCM. Resin 15 was filtered and dried under high vacuum. The loading for resin 15 is defined as the loading of the original aminomethylpoistyrene disulfide resin 14 used in the loading of the Final Product.

Example 6: Synthesis of Morpholino Oligomers Example 6a: Solid Phase Synthesis

[0115]Capped oligomers were prepared manually by solid-phase oligomer synthesis on aminomethylpolystyrene-disulfide resin (~500 μmol/g loading) at the 10 g scale (original resin weight). The solutions used were as follows:Detritylation solutions: CAA = 11% cyanoacetic acid (w/w) in a mixture of 20% acetonitrile/DCM (v/v);CPM = 2% 3-chloropyridinium methanesulfonate (w/v) and 0.9% ethanol (v/v) in 20% trifluoroethanol/DCM (v/v);CYTFA = 2% 4-cyanopyridinium trifluoroacetate (w/v) and 0.9% ethanol (v/v) in 20% trifluoroethanol/DCM (v/v).Neutralization solution: 5% diisopropylethylamine in 25% isopropanol/dichloromethane;Coupling solutions: 0.165 M (for 2f (DPG), 2c and 2d or other T subunits) or 0.18 M (for 2a and 2b or other A/C subunits) Activated morpholino subunit and 0.4 M N-ethylmorpholine in 1, 3-dimethylimidazolidinone (DMI).

[0116]Activated MPG (2c) was prepared as in Summerton et al. (1993).

[0117]After transferring the resin to the synthesis reactor and before starting the synthesis cycles, 1-methyl-2-pyrrolidinone (NMP, 20 mL/g resin) was added and allowed to stand for 1-2 hours. After washing twice with dichloromethane (10 mL/g resin), the following synthesis cycle was used with addition of the activated morpholino subunit coupling solution of the desired base and the desired linkage type in each cycle to provide the appropriate sequence.

[0118]After incorporation of the final subunit, a final cycle (methoxytritylation) was performed with 0.32 M 4-methoxytriphenylmethyl chloride and 0.4 M N-ethylmorpholine in DMI. After methoxytritylation, the resin was washed 8 times with NMP and then treated with cleavage solution consisting of 0.1 M 1,4-dithiothreitol (DTT) and 0.73 M triethylamine in NMP (27 mL/g starting resin) for 30 minutes. After collection of the protected oligomer solution, the resin (significantly reduced in volume) was washed with two additional parts of cleavage solution (13 mL/g original resin for 15 minutes each) and the washes were combined with the bulk solution. To the protected oligomer solution in an appropriately sized pressure flask with a Teflon stopper (Ace Glass, NJ, USA) was added concentrated aqueous ammonia (106 mL/g starting resin, previously cooled to -20°C), the flask sealed, and the contents mixed by shaking. The flask was placed in an oven at 45°C for 16–20 h to remove backbone and backbone protecting groups.

[0119]After ammonolysis, the crude oligomer solution is cooled to room temperature and then diafiltered against 0.28% aqueous ammonia using a 3kd PLBC Regenerated Cellulose membrane (Millipore) to remove solvents and small molecules prior to ion exchange chromatography.

Example 6b: Purification of Morpholino Oligomers by Anion Exchange Chromatography

[0120]The crude oligomer solution obtained by diafiltration is adjusted to pH 11-11.5 and loaded onto a ToyoPearl Super-Q 650S anion exchange resin column (Tosoh Bioscience). The methoxytritylated oligomer is diluted with a 5-35% B gradient over 17 column volumes (Buffer A: 10 mM sodium hydroxide; Buffer B: 1 M sodium chloride in 10 mM sodium hydroxide) and fractions of acceptable purity (anion exchange HPLC (ion exchange and mass spec.)) are pooled.

Example 6c: Demethoxytritlation of morpholino oligomers

[0121]To the pooled fractions from anion exchange chromatography is added acetonitrile (10% by volume) followed by H3PO4 to adjust the pH to 3. The solution is mixed for 45 minutes and then neutralized with concentrated aqueous ammonia to pH 7. The oligomer solution is diafiltered against 20 mM sodium acetate using a 3kd PLBC Regenerated Cellulose Membrane (Millipore) to exchange buffers prior to cation exchange chromatography.

Example 6d: Purification of Morpholino Oligomers by Cation Exchange Chromatography

[0122]The oligomer solution is adjusted to pH 4.5 with acetic acid and loaded onto a Source 3OS cation exchange resin column (GE Healthcare). The oligomer is eluted with a 0-35% B gradient over 17 column volumes (Buffer A: 20 mM sodium acetate, 25% acetonitrile, pH 4.5; Buffer B: 0.5 M sodium chloride, 20 mM sodium acetate, 25% acetonitrile, pH 4.5) and fractions of acceptable purity (cation exchange HPLC and mass spec.) pooled.

Claims (30)

1. Morpholino compound CHARACTERIZED by the fact that it has the following structure (I): wherein:R1 is C1-C6 alkyl, di(C1-C6 alkyl)amino, or phenyl;R2 is C1-C6 alkyl, monocyclic arylmethyl, or monocyclic (aryloxy)methyl;R3 is triarylmethyl or hydrogen; andY is a protected or unprotected hydroxyl group, a protected or unprotected amino group, a chlorophosphoramidate group; a phosphorodiamidate bond to the nitrogen on the morpholino ring of a morpholino subunit monomer; a phosphorodiamidate bond to the nitrogen on the morpholino ring of a morpholino subunit in a morpholino oligomer; or a bond to a solid support. 2. The compound of claim 1, wherein Y is a protected or unprotected hydroxyl group or a chlorophosphoramidate group. 3. The compound of claim 2, wherein Y is a trialkylsilyl-protected hydroxyl group or an unprotected hydroxyl group. 4. Compound, according to claim 2, CHARACTERIZED by the fact that Y is a chlorophosphoramidate group having the following structure: 5. The compound of claim 1 wherein R3 is trityl, 4-methoxytrityl, 4-methyltrityl, 4,4'-dimethyltrityl, or 4,4',4"-trimethyltrityl. 6. Compound, according to claim 1, CHARACTERIZED by the fact that R1 is C1-C6 alkyl. 7. The compound of claim 6, wherein R1 is -C(CH3)3. 8. The compound of claim 1, wherein R2 is benzyl or -CH(CH3)2. 9. A process for synthesizing a morpholino oligomer, the process comprising: (a) reacting a solid-phase supported morpholino subunit monomer comprising a nitrogen in the deprotected morpholino ring with a first morpholino subunit monomer comprising a nitrogen in the triarylmethyl-protected morpholino ring and an activated phosphoramidate group at a 5'-exocyclic carbon, thereby forming a phosphorodiamidate bond between the 5'-exocyclic carbon of the first morpholino subunit monomer and the nitrogen in the deprotected morpholino ring of the solid-phase supported morpholino subunit monomer; (b) deprotecting the nitrogen in the triarylmethyl-protected morpholino ring to form a product comprising a nitrogen in the deprotected morpholino ring; (c) optionally reacting the product from step (b) with a first morpholino subunit monomer of an additional morpholino comprising a nitrogen on the triarylmethyl-protected morpholino ring and an activated phosphoramidate group on a 5'-exocyclic carbon, thereby forming a phosphorodiamidate bond between the 5'-exocyclic carbon of the additional morpholino subunit monomer and the nitrogen on the deprotected morpholino ring of the product of step (b); and (d) optionally repeating steps (b) and (c) one or more times; wherein at least one of the first morpholino subunit monomer, the additional morpholino subunit monomer, or the solid-phase supported morpholino subunit monomer is a doubly protected guanine morpholino compound having the following structure (I): where:R1 is C1-C6 alkyl, di(C1-C6 alkyl)amino, or phenyl;R2 is C1-C6 alkyl, monocyclic arylmethyl, or monocyclic (aryloxy)methyl;R3 is triarylmethyl or hydrogen; andY is a chlorophosphoramidate group or a bond to a solid support. 10. Process, according to claim 9, CHARACTERIZED by the fact that Y is a chlorophosphoramidate group that has the following structure: 11. The process of claim 9 wherein R3 is selected from trityl, 4-methoxytrityl, 4-methyltrityl, 4,4'-dimethyltrityl or 4,4',4"-trimethyltrityl. 12. The process of claim 9, wherein R1 is C1-C6 alkyl. 13. The process of claim 12, wherein R1 is -C(CH3)3. 14. The process of claim 9 wherein R2 is benzyl or -CH(CH3)2. 15. The process of claim 9 wherein the deprotection of step (b) comprises exposing the triarylmethyl-protected ring nitrogen to a reagent solution comprising a heterocyclic amine salt in a solvent containing trifluoroethanol, the salt being a salt of a heterocyclic amine with sulfonic acid, trifluoroacetic acid, or hydrochloric acid and having a pKa in the range of 1 to 4 in its protonated form. 16. The process of claim 15 wherein the salt is 3-chloropyridinium methanesulfonate (CPM) or 4-cyanopyridinium trifluoroacetate (CYTFA). 17. Process, according to claim 15, CHARACTERIZED by the fact that the solvent comprises dichloromethane and trifluoroethanol in a volume ratio ranging from 90:10 to 25:75. 18. The process of claim 17, wherein the volume ratio is 80:20. 19. A process for synthesizing a morpholino oligomer, the process comprising: (a) reacting a solid-phase supported morpholino subunit comprising a nitrogen in the deprotected morpholino ring with a first morpholino subunit monomer comprising a nitrogen in the triarylmethyl-protected morpholino ring and an activated phosphoramidate group at a 5'-exocyclic carbon, thereby forming a phosphorodiamidate bond between the 5'-exocyclic carbon of the first morpholino subunit monomer and the nitrogen in the deprotected morpholino ring of the solid-phase supported morpholino subunit monomer; (b) deprotecting the nitrogen in the triarylmethyl-protected morpholino ring to form a product comprising nitrogen in the deprotected morpholino ring; (c) optionally reacting the product from step (b) with a first morpholino subunit monomer; additional morpholino comprising a nitrogen on the triarylmethyl-protected morpholino ring and an activated phosphoramidate group on a 5'-exocyclic carbon, thereby forming a phosphorodiamidate bond between the 5'-exocyclic carbon of the additional morpholino subunit monomer and the nitrogen on the deprotected morpholino ring of the product of step (b); and (d) optionally repeating steps (a) and (b) one or more times; wherein the deprotection comprises exposing the nitrogen of the triarylmethyl-protected ring to a solution of a reagent comprising a heterocyclic amine salt in a solvent containing trifluoroethanol, the salt being a salt of a heterocyclic amine with sulfonic acid, trifluoroacetic acid, or hydrochloric acid and having a pKa in the range of 1 to 4 in its protonated form. 20. The process of claim 19 wherein the heterocyclic amine is pyridine, thiazole, pyridazine, pyrazole, or triazole, wherein the pyridine, thiazole, pyridazine, pyrazole, or triazole is substituted with one or more electron-withdrawing groups. 21. The process of claim 20, wherein the heterocyclic amine is a pyridine substituted with one or more electron-withdrawing groups. 22. The process of claim 20 wherein the electron-withdrawing group is a halogen, cyano, aldehyde, keto, carboxyester, or carboxamide group. 23. The process of claim 21 wherein the heterocyclic amine is pyridine substituted with chlorine or cyano. 24. The process of claim 19, wherein the salt is an alkylsulfonate salt, a (fluoroalkyl)sulfonate, a p-toluenesulfonate, or a trifluoroacetate. 25. The process of claim 24 wherein the salt is 3-chloropyridinium methanesulfonate (CPM) or 4-cyanopyridinium trifluoroacetate (CYTFA). 26. Process, according to claim 19, CHARACTERIZED by the fact that the solvent comprises dichloromethane and trifluoroethanol in a volume ratio ranging from 90:10 to 25:75. 27. The process of claim 26 wherein the volume ratio is 80:20. 28. The process of claim 19 wherein the triarylmethyl is trityl, 4-methoxytrityl, 4-methyltrityl, 4,4'-dimethyltrityl, or 4,4',4"-trimethyltrityl. 29. The compound of claim 1, wherein the compound has the following structure: where Y is a bond to a solid support or a chlorophosphoramidate group having the following structure: 30. The process of claim 9 or 19 wherein at least one of the first morpholino subunit monomer, the additional morpholino subunit monomer, or the solid-phase supported morpholino subunit monomer has the following structure: where Y is a bond to a solid support or a chlorophosphoramidate group having the following structure: