Synthetic methods for dendrimer preparation

Abstract

Processes for preparing an alkylated phenolic ester and provided and comprise reacting an alkylating agent with a phenolic ester at an elevated temperature in the presence of a polar solvent and at least about 5 equivalents of a base. Compositions comprising the compounds described herein and a ribonucleic acid are also described, as are methods of delivering ribonucleic acids to cells in a patient using the compositions described herein.

Description

25-10889 | 103241.007490 SYNTHETIC METHODS FOR DENDRIMER PREPARATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the priority of U.S. Provisional Patent Application No.63 / 693,086, filed September 10, 2024 and U.S. Provisional Patent Application No.63 / 694,525, filed September 13, 2024, the disclosure of which are incorporated by reference herein. GOVERNMENT RIGHTS

[0002] This invention was made with Government support under Contract Nos. DMR-2104554, DMR-1720530, and CHE-1827457 awarded by the National Science Foundation. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The disclosure pertains to methods for preparing dendrimer and uses thereof. BACKGROUND

[0004] The field of nanomedicine relies on the targeted delivery of nucleic acids into cells and / or their nucleus by viral and nonviral vectors. Nonviral vectors have as their main advantage an infinite number of synthetic capabilities. The extraordinary success of Covid-19 vaccines promoted the four-component based lipid nanoparticles (LNPs) to the forefront of the mRNA delivery with synthetic rather than viral vectors.

[0005] One-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer delivery system for mRNA are known. They co-assemble with mRNA by simple injection of their ethanol solution into a pH = 4 acetate buffer containing the nucleic acid, into monodisperse dendrimersome nanoparticles (DNPs) with predictable dimensions. DNPs are competitive with 4-component lipid nanoparticles (LNPs) which are used in commercial COVID-19 vaccines, except that ionizable amphiphilic Janus dendrimers (IAJD)s are prepared in fewer reaction steps than each individual component of the LNPs. This simple methodology to the synthesis of IAJDs and of their co-assembly with mRNA into DNPs, together with the precise placement of their individual25-10889 | 103241.007490 components and indefinite stability at room temperature in air, make them attractive candidates for the development of nanomedicine based targeted mRNA delivery.

[0006] This system known for short as IAJD self-assemble vesicles, DNPs, is complementary to the 4-component based LNPs except that it contains the ionizable amine in a precise location of the IAJD rather than in a non-defined distribution of its 4- components. IAJDs were inspired from amphiphilic Janus dendrimers (JDs) to self- assemble by simple injection in water and in buffer into monodisperse vesicles of predictable dimensions known as dendrimersomes (DSs).

[0007] The number of steps to synthesize IAJDs compared to the 4-components of LNPs, as well as the methodology to assemble DNPs and LNPs is important. The assembly of the LNPs by microfluidic or T-tube technologies followed by fractionation and dialysis requires several days, while the preparation of the DNPs requires less than one minute.

[0008] Easily attainable and accelerated synthetic methods that do not demand a lot of expertise and complex instrumentation are needed in the synthesis of IAJDs such as IAJD97. SUMMARY

[0009] In some embodiments, the disclosure provides processes for preparing an alkylated phenolic ester, the process comprising reacting an alkylating agent with a phenolic ester at an elevated temperature in the presence of a polar solvent and at least about 5 equivalents of a base.

[0010] In other embodiments, the disclosure provides compounds prepared using the processes described herein.

[0011] In further embodiments, the disclosure provides compositions comprising the compounds described herein and a ribonucleic acid.

[0012] In yet other embodiments, the disclosure provides methods of delivering ribonucleic acids to cells in a patient.25-10889 | 103241.007490 BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The foregoing summary, as well as the following detailed description, will be better understood when read in conjunction with the appended drawings, which show exemplary embodiments for the purposes of illustration.

[0014] FIG.1 is the (a) TLC and (b) MALDI-TOF analysis as a function of time during the alkylation of propyl gallate.

[0015] FIG.2 are images of mice showing the in-vivo Luc activity of IAJD-97 as a function of the number of purifications performed for the first series of experiments.

[0016] FIG.3 are images of mice showing the in-vivo Luc activity of IAJD97 as a function of number of purifications performed for the second time. IAJD97* experiments are from Zhang, J. Am. Chem. Soc.2021, 143 (31), 12315–12327.

[0017] FIG.4 is the1H-NMR spectra of (a) freshly prepared IAJD97 and (b) IAJD97 after six month stored at 23.5oC in the chemistry laboratory air.

[0018] FIG.5 is the1H NMR spectrum of propyl 3,4,5-tris((2- ethylhexyl)oxy)benzoate.

[0019] FIG.6 is the1H NMR spectrum of (3,4,5-tris((2- ethylhexyl)oxy)phenyl)methanol.

[0020] FIG.7 is the1H NMR spectrum of 3,4,5-tris((2-ethylhexyl)oxy)benzyl 4- bromobutanoate.

[0021] FIG.8 is the1H NMR spectrum of IAJD-97.

[0022] FIG.9 is the13C NMR spectrum of propyl 3,4,5-tris((2- ethylhexyl)oxy)benzoate.

[0023] FIG.10 is the13C NMR spectrum of (3,4,5-tris((2- ethylhexyl)oxy)phenyl)methanol.

[0024] FIG.11 is the1H NMR spectrum of 3,4,5-tris((2-ethylhexyl)oxy)benzyl 4-bromobutanoate.

[0025] FIG.12 is the1H NMR spectrum of IAJD-97.

[0026] FIG.13 are HPLC traces (UV) of (a)-(c) intermediates and (d) final product of the synthesis of IAJD-97.

[0027] FIG.14 are HPLC traces (RI) of (a)-(c) intermediates and (d) final product of the synthesis of IAJD-97.25-10889 | 103241.007490

[0028] FIG.15 are MALDI-TOF spectra of (a)-(c) intermediates and (d) final product of the synthesis of IAJD-97. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0029] The disclosure may be more fully appreciated by reference to the following description, including the following definitions and examples. Certain features of the disclosed compositions and methods which are described herein in the context of separate aspects, may also be provided in combination in a single aspect. Alternatively, various features of the disclosed compositions and methods that are, for brevity, described in the context of a single aspect, may also be provided separately or in any sub combination. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the disclosure.

[0030] In the present disclosure the singular forms “a,” “an,” and “the” include the plural reference, and reference to a particular numerical value includes at least that particular value, unless the context clearly indicates otherwise. Thus, for example, a reference to “a compound” is a reference to one or more of such compounds and equivalents thereof known to those skilled in the art, and so forth. The term “plurality,” as used herein, means more than one.

[0031] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0032] When a range of values is expressed, another embodiment includes from the one particular and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it is understood that the particular value forms another embodiment. All ranges are inclusive and combinable.25-10889 | 103241.007490

[0033] The term “alkyl,” when used alone or as part of a substituent group, refers to a straight- or branched-chain alkyl group having from 1 to 12 carbon atoms (“C1-12”), preferably 1 to 6 carbons atoms (“C1-6”), in the chain. Examples of alkyl groups include methyl (Me, C1alkyl) ethyl (Et, C2alkyl), n-propyl (C3alkyl), isopropyl (C3alkyl), butyl (C4alkyl), isobutyl (C4alkyl), sec-butyl (C4alkyl), tert-butyl (C4alkyl), pentyl (C5alkyl), isopentyl (C5alkyl), tert-pentyl (C5alkyl), hexyl (C6alkyl), isohexyl (C6alkyl), and groups that in light of the ordinary skill in the art and the teachings provided herein would be considered equivalent to any one of the foregoing examples. An alkyl moiety is optionally substituted with one, two, or three substituents selected from halo (F, Cl, Br, or I, preferably F), -OH, -OC1-6alkyl, -CN, -NH2, -NH(C1-6alkyl), -NH(C1-6alkyl)2, C3-8cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0034] The term “C1-8alk” refers to “alkyl” defined herein, but bound through two carbons. Examples include –CH2-, –CH(CH3)-, -CH(CH3)-CH2-, and –C(CH3)2-.

[0035] The term “alkoxy,” when used alone or as part of a substituent group, refers to a straight- or branched-chain alkyl group having from 1 to 12 carbon atoms (“C1-12”), preferably 1 to 6 carbons atoms (“C1-6”), and one oxygen in the chain. Examples of alkoxy groups include methoxy (OMe, C1alkoxy) ethoxy (OEt, C2alkoxy), n-propoxy (C3alkoxy), isopropoxy (C3alkoxy), butoxy (C4alkoxy), isobutoxy (C4alkoxy), sec-butoxy (C4alkoxy), tert-butoxy (C-alkoxy), pentoxy (C5alkoxy), isopentoxy (C5alkoxy), tert- pentoxy (C5alkyl), hexoxy (C6alkoxy), isohexoxy (C6alkoxy), and groups that in light of the ordinary skill in the art and the teachings provided herein would be considered equivalent to any one of the foregoing examples. An alkoxy moiety is optionally substituted with one, two, or three substituents selected from halo (F, Cl, Br, or I, preferably F), -OH, -OC1-6alkyl, -CN, -NH2, -NH(C1-6alkyl), -NH(C1-6alkyl)2, C3-8cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0036] The term “heterocycloalkyl” when used alone or as part of a substituent group refers to any three to ten membered monocyclic or bicyclic, saturated ring structure containing at least one heteroatom selected from the group consisting of O, N and S. Heterocycloalkyl groups of the disclosure include monocyclic groups, as well as multicyclic groups such as bicyclic and tricyclic groups. In those embodiments having at least one multicyclic heterocycloalkyl group, the cyclic groups can share one common atom (i.e., spirocyclic). In other embodiments having at least one multicyclic25-10889 | 103241.007490 heterocycloalkyl group, the cyclic groups share two common atoms. The term -C3-C6heterocycloalkyl refers to a heterocycloalkyl group having between three and six carbon ring atoms. The term -C3-C10heterocycloalkyl refers to a heterocycloalkyl group having between three and 10 ring atoms. The heterocycloalkyl group may be attached at any heteroatom or carbon atom of the ring such that the result is a stable structure. Examples of suitable heterocycloalkyl groups include, but are not limited to, azepanyl, aziridinyl, azetidinyl, pyrrolidinyl, dioxolanyl, imidazolidinyl, pyrazolidinyl, piperazinyl, piperidinyl, dioxanyl, morpholinyl, dithianyl, thiomorpholinyl, oxazepanyl, oxiranyl, oxetanyl, quinuclidinyl, tetrahydrofuranyl, tetrahydropyranyl, piperazinyl, azepanyl, diazepanyl, oxepanyl, dioxepanyl, azocanyl diazocanyl, oxocanyl, dioxocanyl, azaspiro[2.2] pentanyl, oxaazaspiro[3.3]heptanyl, oxaspiro[3.3]heptanyl, dioxaspiro[3.3]heptanyl, and the like. A heterocycloalkyl is optionally substituted with one, two, or three substituents selected from halo (F, Cl, Br, or I, preferably F), C1-6alkyl, -OH, -OC1-6alkyl, C1-6haloalkyl, -CN, - NH2, -NHC(O)(C1-6alkyl), -NH(C1-6alkyl), -NH(C1-6alkyl)2, C3-8cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.

[0037] The term “-heterocycloalk-” refers to “heterocycloalkyl” defined herein but bound through two points on the heterocycloalkyl. In some embodiments, the “- heterocycloalk-” is bound through one carbon atom and one heteroatom. In other embodiments, the “-heterocycloalk-” is bound through two carbon atoms. In further embodiments, the “-heterocycloalk-” is bound through two heteroatoms.

[0038] The term “aryl” refers to carbocyclic aromatic groups having from 6 to 10 carbon atoms (“C6-10”) such as phenyl, naphthyl, and the like. An aryl is optionally substituted with one, two, or three substituents selected from halo (F, Cl, Br, or I, preferably F), C1-6alkyl, -OH, -OC1-6alkyl, C1-6haloalkyl, -CN, -NH2, -NHC(O)(C1-6alkyl), -NH(C1-6alkyl), -NH(C1-6alkyl)2, C3-8cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0039] The term “-ar-” refers to “aryl” defined herein but bound through two points on the aryl. In some embodiments, the “-ar-” is bound through two carbon atoms.

[0040] “Heteroaryl” refers to a 5- to 18-membered aromatic radical, e.g., C5-18heteroaryl, that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur, and which may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system. Whenever it appears herein, a numerical range such as “5 to 18” refers to each integer in the given range, e.g., “5 to 18 ring atoms” means that the heteroaryl group may contain 525-10889 | 103241.007490 ring atoms, 6 ring atoms, etc., up to and including 18 ring atoms. An N-containing heteroaryl moiety refers to an aromatic group in which at least one of the skeletal atoms of the ring is a nitrogen atom. The polycyclic heteroaryl group may be fused or non-fused. The heteroatom(s) in the heteroaryl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl may be attached to the rest of the molecule through any atom of the ring(s). Examples of heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4- benzodioxany], benzonaphthofuranyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzofurazanyl, benzothiazolyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d|pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6- dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]|cinnolinyl, 6,7-dihydro- 5Hbenzo[6,7|cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo[3,2-c]pyridinyl, 5,6,7,8,9,10- hexahydrocycloocta[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, 5,8-methano- 5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2- oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1- phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d|pyrimidinyl, pyridinyl, pyrido[3,2- d]pyrimidinyl, pyrido[3,4-d|pyrimidiny], pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, 5,6,7,8- tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9- tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5- c]pyridazinyl, thiazolyl, thiadiazolyl, thiapyranyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d|pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl (i.e. thienyl). A heteroaryl is optionally substituted with one, two, or three substituents selected from halo (F, Cl, Br, or I, preferably F), -OH, C1-6alkyl, -O1-6alkyl, -CN, -NH2, - NH(C1-6alkyl), -NH(C1-6alkyl)2, C3-8cycloalkyl, heterocyclyl, aryl, or heteroaryl.25-10889 | 103241.007490

[0041] The term “-heteroar-” refers to “heteroaryl” defined herein but bound through two points on the heterocycloalkyl. In some embodiments, the “-heteroar-” is bound through one carbon atom and one heteroatom. In other embodiments, the “- heteroar-” is bound through two carbon atoms. In further embodiments, the “-heteroar-” is bound through two heteroatoms.

[0042] The terms “halogen” and “halo” represent chlorine, fluorine, bromine, or iodine. The term “halo” represents chloro, fluoro, bromo, or iodo. The Processes

[0043] Access to large scale synthesis of IAJDs without the need for sophisticated technologies, instrumentation and synthetic skills is expected to open numerous new opportunities world-wide in nanomedicine. This disclosure provides an accelerated ten-gram scale synthesis of IAJD97 from inexpensive food additives obtained from renewable plant phenolic acid starting materials by methodologies accessible to any laboratory. This accelerated synthesis can be accomplished in only four days.

[0044] The disclosure provides an accelerated 10 gram scale synthesis of the IAJD97 that delivers Luc-mRNA predominantly to spleen and lymph nodes. Propyl gallate, an inexpensive food additive derived from renewable plants phenolic acid, gallic acid, was employed to generate this synthesis in 4 days. Co-assembly of IAJD97 with Luc-mRNA is generated by simple injection of an ethanol solution of the synthetic vector IAJD97 into a pH = 4 acetate buffer containing Luc-mRNA. Total and targeted Luc activities in vivo were more than one order of magnitude higher than the art. IAJD97 is stable for at least 7 months stable in an organic chemistry laboratory at room temperature (23.5oC) in air, as compared to IAJD97, as prepared in the art, which is stable at best for several days .

[0045] The preparation the gallic acid-based simplified SS (sSS) IAJD97 starts from the inexpensive food additive propyl gallate which under suitable conditions should be alkylated with 100% conversion in order to construct the hydrophobic domain of the IAJD (Scheme 1).2Therefore, the rate determining step for the synthesis of both sSS and nonsymmetric simplified SS (nsSS) IAJDs, is the alkylation reaction employed to construct the hydrophobic region of IAJDs. For IAJD97, the propyl gallate precursor (1) has three phenol groups that must be quantitatively substituted with 2-ethylhexyl bromide (2). The pathway to complete trialkylation occurs through monoalkylation of the most25-10889 | 103241.007490 reactive 4-position followed by dialkylation in the 3- and / or 5- positions. All these intermediary compounds of IAJD97 can be monitored by a combination of thin layer chromatography (TLC), HPLC and MALDI-TOF (FIG.1).Scheme 1

[0046] According to the disclosure, processes for preparing an alkylated phenolic ester are provided. The processes comprise reacting an alkylating agent with a phenolic ester. The alkylated phenolic ester is of Formula (I):

[0047] In this structure, R1is C2-10alkyl. In some embodiments, R1is C2alkyl. In other embodiments, R1is C3alkyl. In further embodiments, R1is C4alkyl. In yet other embodiments, R1is C2alkyl. In still further embodiments, R1is C2alkyl. In other25-10889 | 103241.007490 embodiments, R1is C5alkyl. In further embodiments, R1is C6alkyl. In yet other embodiments, R1is C7alkyl. In still further embodiments, R1is C8alkyl. In other embodiments, R1is C9alkyl. In further embodiments, R1is C10alkyl.

[0048] In this structure, R2to R6are, independently, H, -OSO2CH3, -O-C1-24alkyl, optionally substituted aryl, or -O-C1-8alk-(optionally substituted aryl); provided that at least one of R2to R6is not H. For example R2to R6may be the phenyl substituents identified in Percec, “An Accelerated Modular-Orthogonal Ni-Catalyzed Methodology to Symmetric and Nonsymmetric Constitutional Isomeric AB2to AB9Dendrons Exhibiting Unprecedented Self-Organizing Principles,” J. Am. Chem. Soc.2021, 143, 17724-17743, which is incorporated by reference herein.

[0049] In some embodiments, R2is H. In other embodiments, R2is -O-C24alkyl, such as -O-C1alkyl, -O-C2alkyl, -O-C3alkyl, -O-C4alkyl, -O-C5alkyl, -O-C6alkyl, -O- C7alkyl, -O-C8alkyl, -O-C9alkyl, -O-C10alkyl, -O-C11alkyl, -O-C12alkyl, -O-C13alkyl, -O- C14alkyl, -O-C15alkyl, -O-C16alkyl, -O-C17lkyl, -O-C18alkyl, -O-C19alkyl, -O-C20alkyl, -O- C21alkyl, -O-C22alkyl, -O-C23alkyl, or -O-C24alkyl. In yet further embodiments, R2is - OSO2CH3. In further embodiments, R2is optionally substituted aryl such as:In yet other embodiments, R2is -O-C1-8alk-(optionally substituted aryl) such as:

[0050] In some embodiments, R3is H. In other embodiments, R3is -O-C24alkyl, such as -O-C1alkyl, -O-C2alkyl, -O-C3alkyl, -O-C4alkyl, -O-C5alkyl, -O-C6alkyl, -O- C7alkyl, -O-C8alkyl, -O-C9alkyl, -O-C10alkyl, -O-C11alkyl, -O-C12alkyl, -O-C13alkyl, -O- C14alkyl, -O-C15alkyl, -O-C16alkyl, -O-C17lkyl, -O-C18alkyl, -O-C19alkyl, -O-C20alkyl, -O- C21alkyl, -O-C22alkyl, -O-C23alkyl, or -O-C24alkyl. In yet further embodiments, R3is - OSO2CH3. In further embodiments, R3is optionally substituted aryl such as:25-10889 | 103241.007490In yet other embodiments, R3is -O-C1-8alk-(optionally substituted aryl) such as:

[0051] In some embodiments, R4is H. In other embodiments, R4is -O-C24alkyl, such as -O-C1alkyl, -O-C2alkyl, -O-C3alkyl, -O-C4alkyl, -O-C5alkyl, -O-C6alkyl, -O- C7alkyl, -O-C8alkyl, -O-C9alkyl, -O-C10alkyl, -O-C11alkyl, -O-C12alkyl, -O-C13alkyl, -O- C14alkyl, -O-C15alkyl, -O-C16alkyl, -O-C17lkyl, -O-C18alkyl, -O-C19alkyl, -O-C20alkyl, -O- C21alkyl, -O-C22alkyl, -O-C23alkyl, or -O-C24alkyl. In yet further embodiments, R4is - OSO2CH3. In further embodiments, R4is optionally substituted aryl such as:In yet other embodiments, R4is -O-C1-8alk-(optionally substituted aryl) such as:

[0052] In some embodiments, R5is H. In other embodiments, R5is -O-C24alkyl, such as -O-C1alkyl, -O-C2alkyl, -O-C3alkyl, -O-C4alkyl, -O-C5alkyl, -O-C6alkyl, -O- C7alkyl, -O-C8alkyl, -O-C9alkyl, -O-C10alkyl, -O-C11alkyl, -O-C12alkyl, -O-C13alkyl, -O- C14alkyl, -O-C15alkyl, -O-C16alkyl, -O-C17lkyl, -O-C18alkyl, -O-C19alkyl, -O-C20alkyl, -O- C21alkyl, -O-C22alkyl, -O-C23alkyl, or -O-C24alkyl. In yet further embodiments, R5is - OSO2CH3. In further embodiments, R5is optionally substituted aryl such as:25-10889 | 103241.007490In yet other embodiments, R5is -O-C1-8alk-(optionally substituted aryl) such as:

[0053] In some embodiments, R6is H. In other embodiments, R6is -O-C24alkyl, such as -O-C1alkyl, -O-C2alkyl, -O-C3alkyl, -O-C4alkyl, -O-C5alkyl, -O-C6alkyl, -O- C7alkyl, -O-C8alkyl, -O-C9alkyl, -O-C10alkyl, -O-C11alkyl, -O-C12alkyl, -O-C13alkyl, -O- C14alkyl, -O-C15alkyl, -O-C16alkyl, -O-C17lkyl, -O-C18alkyl, -O-C19alkyl, -O-C20alkyl, -O- C21alkyl, -O-C22alkyl, -O-C23alkyl, or -O-C24alkyl. In yet further embodiments, R6is - OSO2CH3. In further embodiments, R6is optionally substituted aryl such as:In yet other embodiments, R6is -O-C1-8alk-(optionally substituted aryl) such as:

[0054] According to the disclosure, the alkylated phenolic ester is of Formula (I- 1), (I-2), or (I-3):-10889 | 103241.007490In some embodiments, the alkylated phenolic ester is of Formula (I-1). In other embodiments the alkylated phenolic ester is of Formula (I-2). In further embodiments, the alkylated phenolic ester is of Formula (I-3). In further embodiments, the alkylated phenolic ester is of Formula (I-4):In yet other embodiments, the alkylated phenolic ester is:.

[0055] According to the disclosure, the phenolic ester is of Formula (II):25-10889 | 103241.007490

[0056] In this structure, R10to R14are, independently, H or -OH, provided that at least one of R10to R14is -OH.

[0057] In some embodiments, R10is H. In other embodiments, R10is -OH.

[0058] In some embodiments, R11is H. In other embodiments, R11is -OH.

[0059] In some embodiments, R12is H. In other embodiments, R12is -OH.

[0060] In some embodiments, R13is H. In other embodiments, R13is -OH.

[0061] In some embodiments, R14is H. In other embodiments, R14is -OH.

[0062] In some embodiments, the phenolic ester is of Formula (II-1), (II-2), or (II-3):In some embodiments, the phenolic ester is of formula (II-1). In other embodiments, the phenolic ester is of Formula (II-2). In further embodiments, the phenolic ester is of Formula (II-3). In yet other embodiments, the phenolic ester is:.

[0063] According to the disclosure, the alkylated phenolic ester may be prepared at an elevated temperature in the presence of a polar solvent and at least about 5 equivalents of a base.

[0064] In some embodiments, the polar solvent has a boiling point of about 50 to about 220°C. In some embodiments, the polar solvent has a boiling point of about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210 or about25-10889 | 103241.007490 220°C. In other embodiments, the polar solvent has a boiling point of about 80°C. In further embodiments, the polar solvent has a boiling point of about 120°C. In still other embodiments, the polar solvent has a boiling point of about 150°C. In yet further embodiments, the polar solvent has a boiling point of about 165°C. In other embodiments, the polar solvent has a boiling point of about 190°C. In further embodiments, the polar solvent has a boiling point of about 200°C.

[0065] In some embodiments, the polar solvent is a dipolar, aprotic solvent. Examples of dipolar, aprotic solvents include dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMAC), or N-methylpyrrolidone (NMP), or a combination thereof. In some embodiments, the dipolar, aprotic solvent is DMF. In other embodiments, the dipolar, aprotic solvent is DMSO. In further embodiments, the dipolar, aprotic solvent is DMAC. In yet other embodiments, the dipolar, aprotic solvent is NMP.

[0066] In some embodiments, the polar solvent is acetonitrile (MeCN or ACN), cyclohexanol (CyO), or methyl isobutyl ketone (MIBK), or a combination thereof. In some embodiments, the polar solvent is MeCN. In other embodiments, the polar solvent cyclohexanol (CyO). In further embodiments, the polar solvent is MIBK.

[0067] In some embodiments, the base is potassium carbonate, potassium bicarbonate, sodium carbonate, cesium carbonate, cesium bicarbonate, sodium bicarbonate, sodium hydroxide, or potassium hydroxide, or a combination thereof. In other embodiments, the base is potassium carbonate. In further embodiments, the base is sodium carbonate. In yet other embodiments, the base is cesium carbonate. In still further embodiments, the base is cesium bicarbonate. In other embodiments, the base is sodium bicarbonate. In further embodiments, the base is sodium hydroxide. In still other embodiments, the base is potassium hydroxide.

[0068] In certain embodiments, about 6 to about 10 molar equivalents of the base are used. In some embodiments, about 6 equivalents of the base are used. In other embodiments about 7 equivalents of the base are used. In further embodiments, about 8 equivalents of the base are used.

[0069] In some embodiments, the alkylating agent is an alkyl halide such as an alkyl bromide, alkyl iodide, or alkyl chloride. In other embodiments, the alkylating agent is an alkyl bromide. In further embodiments, the alkylating agent is an alkyl iodide. In25-10889 | 103241.007490 yet other embodiments, the alkylating agent is an alkyl chloride. In some embodiments, the alkylating agent is an alkyl sulfonate such as an alkyl tosylate, alkyl mesylate, or alkyl triflate. In other embodiments, the alkylating agent is an alkyl tosylate. In further embodiments, the alkylating agent is an alkyl mesylate. In yet other embodiments, the alkylating agent is an alkyl triflate.

[0070] According to the disclosure, the alkylating agent comprises 1 to 24 carbon atoms. In some embodiments, the alkylating agent comprises 1, 2, 3, 4, 5, 6, 7, 8,910, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbon atoms. In other embodiments, the alkyl portion of the alkylating agent is branched. In further embodiments, the alkyl portion of the alkylating agent is linear.

[0071] According to the disclosure, about 4 equivalents to about 8 equivalents of the alkylating agent are used. In some embodiments, about 4 equivalents of the alkylating agent are used. In other embodiments, about 5 equivalents of the alkylating agent are used. In further embodiments, about 6 equivalents of the alkylating agent are used.

[0072] According to the disclosure, the process is performed for at least about 15 minutes. In some embodiments, the process is performed for about 15 minutes to about 24 hours. In other embodiments, the process is performed for about 20 minutes. In further embodiments, the process is performed for about 40 minutes. In yet other embodiments, the process is performed for about 50 minutes. In still further embodiments, the process is performed for about 55 minutes. In other embodiments, the process is performed for about 70 minutes. In further embodiments, the process is performed for about 2 hours. In still other embodiments, the process is performed for about 2.5 hours. In yet further embodiments, the process is performed for about 3 hours. In other embodiments, the process is performed for about 4 hours. In further embodiments, the process is performed for about 5 hours. In still other embodiments, the process is performed for about 16 hours. In yet further embodiments, the process is performed for about 17 hours. In other embodiments, the process is performed for about 20 hours.

[0073] In some embodiments, the temperature is at least about 40°C. In further embodiments, the temperature is about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, or 180°C. In other embodiments, the temperature is about 80 to about 180°C. In further embodiments, temperature is about 80°C. In yet other embodiments, temperature25-10889 | 103241.007490 is about 90°C. In still further embodiments, temperature is about 100°C. In other embodiments, temperature is about 110°C. In further embodiments, temperature is about 120°C. In still other embodiments, temperature is about 130°C. In yet further embodiments, temperature is about 135°C. In other embodiments, temperature is about 140°C. In further embodiments, temperature is about 150°C.

[0074] In certain embodiments, the alkylated phenolic ester is prepared using at least about 4 equivalents of alkylating agent, about 1 equivalent of the phenolic ester, and at least about 6 equivalents of the base.

[0075] According to the disclosure, the processes may further comprise reducing the alkylated phenolic ester to form a reduced phenol. In some embodiments, the reduced phenol is of Formula (III), wherein R2-R6are defined herein:In some embodiments, the reduced phenol is of Formula (III-1), (III-2), or (III-3):In other embodiments, the reduced phenol is of Formula (III-1). In further embodiments, the reduced phenol is of Formula (III-2). In yet other embodiments, the reduced phenol is of Formula (III-3). In still further embodiments, the reduced phenol is:25-10889 | 103241.007490.

[0076] According to the disclosure, the reducing is performed using a reducing agent that reduces an ester to an alcohol. Thus, the reduction may be performed using reducing agents known in the art. In some embodiments, the reducing agent is a strong reducing agent such as lithium aluminum hydride, diisobutylaluminum hydride (DIBAL- H), lithium borohydride (LiBH4), borane-dimethyl sulfide (BH3-SMe2), sodium borohydride (NaBH4).

[0077] According to the disclosure, the reducing is performed at reduced temperature. In some embodiments, the reduced temperature is about 0 to about 24°C. In other embodiments, the reduced temperature is about 0, about 5, about 10, about 15, about 20, or about 24°C. In further embodiments, the reduced temperature is about 0 to about 20, about 0 to about 15, about 0 to about 10, about 5 to about 24, about 5 to about 20, about 5 to about 15, about 10 to about 24, about 10 to about 20, or about 15 to about 24°C.

[0078] According to the disclosure, the reduced phenol is esterified to form an esterified phenol. In some embodiments, the esterified phenol is of Formula (IV):wherein, R2-R6is defined herein.

[0079] According to the disclosure, X1is Cl, Br, or F. In some embodiments, X1is Cl. In other embodiments, X1is Br. In further embodiments, X1is F. In some embodiments, the –(C2-8alk)- group is –(C2alk)-. In other embodiments, the –(C2-8alk)- group is –(C3alk)-. In further embodiments, the –(C2-8alk)- group is –(C4alk)-. In yet other embodiments, the –(C2-8alk)- group is –(C5alk)-. In still further embodiments, the –25-10889 | 103241.007490 (C2-8alk)- group is –(C6alk)-. In other embodiments, the –(C2-8alk)- group is –(C7alk)-. In further embodiments, the –(C2-8alk)- group is –(C8alk)-.

[0080] In some embodiments, the esterified phenol is of Formula (IV-1), (IV-2), or (IV-3):In other embodiments, the esterified phenol is of Formula (IV-1). In further embodiments, the esterified phenol is of Formula (IV-2). In yet other embodiments, the esterified phenol is of Formula (IV-3). In still further embodiments, the esterified phenol is:. In other embodiments, the esterified phenol is:25-10889 | 103241.007490.

[0081] According to the disclosure, the esterification is performed using X1-C1-8alk-C(O)-X2, wherein X1and X2are, independently, Cl, Br, or F. In some embodiments, X1is Cl. In other embodiments, X1is Br. In further embodiments, X1is F. In further embodiments, X2is Cl. In other embodiments, X2is Br. In further embodiments, X2is F. In yet other embodiments, X1and X2are the same. In still further embodiments, X1and X2differ.

[0082] The esterification may be performed at reduced temperature, such as about 0 to about 23°C. In other embodiments, the reduced temperature is about 0, about 5, about 10, about 15, about 20, or about 23°C. In further embodiments, the reduced temperature is about 0 to about 20, about 0 to about 15, about 0 to about 10, about 5 to about 23, about 5 to about 20, about 5 to about 15, about 10 to about 23, about 10 to about 20, or about 15 to about 23°C.

[0083] According to the disclosure, the ester group of the esterified phenol may be coupled with any group. In some embodiments, the ester group is coupled with HY1- C1-8alk-OH, wherein Y1is a chemical moiety comprising two nitrogen atoms. In some embodiments, the coupling forms a compound of Formula (V) or (V-A):wherein, Y1is -optionally substituted C3-8cycloalk-, -optionally substituted heterocycloalk- , -optionally substituted heteroar-, or -optionally substituted ar-; and Y2is OH, NH2, - NH(C1-6alkyl), -NH(C1-6alkyl)2, optionally substituted C1-6alkyl, optionally substituted C1-25-10889 | 103241.0074906alkoxy, substituted C3-8cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl.

[0084] In some embodiments, Y1is -optionally substituted C3-8cycloalk-. In other embodiments, Y1is -optionally substituted heterocycloalk- such as optionally substituted piperazinyl. In further embodiments, Y1is -optionally substituted heteroar-. In yet other embodiments, Y1is -optionally substituted ar-. In some embodiments, Y1is a 5-8-membered heterocycloalkyl comprising two nitrogen atoms. In other embodiments, Y1is piperazinyl.

[0085] In some embodiments, Y2is OH. In other embodiments, Y2is NH2. In further embodiments, Y2is -NH(C1-6alkyl). In yet other embodiments, Y2is -NH(C1-6alkyl)2. In still further embodiments, Y2is optionally substituted C1-6alkyl. In other embodiments, Y2is optionally substituted C1-6alkoxy. In further embodiments, Y2is optionally substituted C3-8cycloalkyl. In still other embodiments, Y2is optionally substituted heterocycloalkyl. In yet further embodiments, Y2is optionally substituted aryl. In other embodiments, Y2is optionally substituted heteroaryl.

[0086] In other embodiments, the coupling forms a compound of Formula (V-1), (V-2), or (V-3): O(C2- -Y1 C1- OHIn further embodiments, the coupling forms a compound of Formula (V-1). In yet other embodiments, the coupling forms a compound of Formula (V-2). In still further25-10889 | 103241.007490 embodiments, the coupling forms a compound of Formula (V-3). In other embodiments, the coupling forms:. In further embodiments, the coupling forms:.

[0087] The disclosure also provides compounds prepared according to the process described herein.

[0088] According to the disclosure, compositions comprising the compounds described herein and a ribonucleic acid (RNA) are provided. One of skill in the art would be able to select suitable ribonucleic acids for delivery. In some embodiments, the RNA is messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotide (ASO), or aptamers thereof. In other embodiments, the RNA is mRNA. In further embodiments, the RNA is siRNA. In yet other embodiments, the RNA25-10889 | 103241.007490 is miRNA. In still further embodiments, the RNA is ASO. In other embodiments, the RNA is Luc-mRNA.

[0089] The RNA may be delivered to one or more cells of a patient, by administered one or more compositions described herein to the patient. See, International Patent Publication No. WO-2022 / 251191, which is incorporated herein by reference. In some embodiments, the cell is a tissue. In other embodiments, the cell includes lung tissue, liver tissue, spleen tissue, or a lymph node. Aspects I

[0090] Aspect I-1. A process for preparing a compound of Formula (I):wherein, R1is C2-8alkyl; the process comprising reacting at least about 4 equivalents of ethylhexyl halide with about 1 equivalent of a compound of Formula (II) at an elevated temperature in the presence of at least about 6 equivalents of potassium carbonate and a polar solvent:

[0091] Aspect I-2. The process of Aspect I-1, wherein R1is C2alkyl, C3alkyl, C4alkyl, C5alkyl, C6alkyl, C7alkyl, or C8alkyl, preferably C3alkyl.

[0092] Aspect I-3. The process of Aspect I- 1 or 2, wherein the temperature is at least about 80°C.

[0093] Aspect I-4. The process of any one of Aspects I-1 to I-3, wherein the temperature is about 80 to about 180°C, or such as about 80°C, or such as about 90°C, or25-10889 | 103241.007490 such as about 100°C, or such as about 120°C, or such as about 130°C, or such as about 135°C, or such as about 140°C, or such as about 150°C.

[0094] Aspect I-5. The process of any one of the preceding Aspects I, wherein the polar solvent has a boiling point of about 50 to about 220°C, or such as about 80°C, or such as about 120°C, or such as about 150°C, or such as about 165°C, or such as about 190°C, or such as about 200°C,.

[0095] Aspect I-6. The process of any one of the preceding Aspects I, wherein the polar solvent is a dipolar, aprotic solvent.

[0096] Aspect I-7. The process of Aspect I-6, wherein the dipolar, aprotic solvent is dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMAC), or N-methylpyrrolidone (NMP), or a combination thereof.

[0097] Aspect I-8. The process of any one of Aspects I-1 to I-5, wherein the polar solvent is acetonitrile (MeCN or ACN), CyO, or methyl isobutyl ketone (MIBK), or a combination thereof.

[0098] Aspect I-9. The process of any one of the preceding Aspects I, comprising about 6 to about 10 molar equivalents of potassium carbonate, or such as about 6 equivalents, or such as about 7 equivalents, or such as about 8 equivalents.

[0099] Aspect I-10. The process of any one of the preceding Aspects I, comprising about 4 equivalents to about 8 equivalents of ethylhexyl bromide, or such as about 4 equivalents, or such as about 5 equivalents, or such as about 6 equivalents.

[0100] Aspect I-11. The process of any one of the preceding Aspects I, that is performed for at least about 15 minutes.

[0101] Aspect I-12. The process of any one of the preceding Aspects I, that is performed for about 15 minutes to about 24 hours, or such as about 20 minutes, about 40 minutes, about 50 minutes, about 55 minutes, about 70 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, about 5 hours, about 16 hours, about 17 hours, or about 20 hours.

[0102] Aspect I-13. The process of any one of the preceding Aspects I, wherein the compound of Formula (I) is:25-10889 | 103241.007490.

[0103] Aspect I-14. The process of any one of the preceding Aspects I, wherein the compound of Formula (II) is:.

[0104] Aspect I-15. The process of any one of the preceding Aspects I, further comprising reducing the compound of Formula (I) to form compound III:

[0105] Aspect I-16. The process of Aspect I-15, wherein the reducing is performed using lithium aluminum hydride.

[0106] Aspect I-17. The process of Aspect I-15 or Aspect I-16, that is performed at reduced temperature, such as about 0 to about 24°C.

[0107] Aspect I-18. The process of any one of Aspects I-15 to I-17, wherein the compound III is esterified to form a compound of Formula (IV):25-10889 | 103241.007490wherein, X1is Cl, Br, or F.

[0108] Aspect I-19. The process of Aspect I-18, wherein the esterification is performed using X1-C1-8alk-C(O)-X2, wherein X1and X2are, independently, Cl, Br, or F.

[0109] Aspect I-20. The process of Aspect I-18 or Aspect I-19, that is performed at reduced temperature, such as about 0 to about 23°C.

[0110] Aspect I-21. The process of any one of Aspects I-18 to I-20, wherein the compound of Formula (IV) is:.

[0111] Aspect I-22. The process of any one of Aspects I-18 to I-21, further comprising reacting the compound of Formula (IV) with HY1-C1-8alk-OH to form a compound of Formula (V):25-10889 | 103241.007490wherein Y1is a chemical moiety comprising two nitrogen atoms.

[0112] Aspect I-23. The process of Aspect I-22, wherein Y1is a 5-8-membered heterocycloalkyl comprising two nitrogen atoms.

[0113] Aspect I-24. The process of Aspect I-22 or Aspect I-23, wherein Y1is piperazinyl.

[0114] Aspect I-25. The process of any one Aspects I-22 to I-24, wherein the compound of Formula (V) is:.

[0115] Aspect I-26. A compound prepared according to the process of any one of Aspects I-1 to I-25.

[0116] Aspect I-27. A composition comprising the compound of Aspect I-26 and a nucleic acid.25-10889 | 103241.007490

[0117] Aspect I-28. The composition of Aspect I-27, wherein the nucleic acid is RNA, such as mRNA, or such as Luc-mRNA.

[0118] Aspect I-29. A method for delivering a nucleic acid to a cell in a patient, comprising delivering the compound of Aspect I-26 or composition of Aspect I-27 or Aspect I-28 to the patient.

[0119] Aspect I-30. The method of Aspect I-29, wherein the cell is a tissue, such as lung tissue, liver tissue, spleen tissue, or a lymph node.

[0120] Aspect I-31. The method of Aspect I-29 or Aspect I-30, wherein the nucleic acid is RNA, such as mRNA, or such as Luc-mRNA. Aspects II

[0121] Aspect II-1. A process for preparing an alkylated phenolic ester, the process comprising reacting an alkylating agent with a phenolic ester at an elevated temperature in the presence of a polar solvent and at least about 5 equivalents of a base.

[0122] Aspect II-2. The process of Aspect II-1, wherein the alkylated phenolic ester is of Formula (I):wherein: R1is C2-10alkyl; and R2to R6are, independently, H, -O-C1-24alkyl, optionally substituted aryl, or -O-C1-8alk-(optionally substituted aryl); provided that at least one of R2to R6is not H.

[0123] Aspect II-3. The process of Aspect II-1, wherein R1is C2alkyl, C3alkyl, C4alkyl, C5alkyl, C6alkyl, C7alkyl, or C8alkyl, preferably C3alkyl.

[0124] Aspect II-4. The process of Aspect II-1 or Aspect II-2, wherein R2is H.

[0125] Aspect II-5. The process of Aspect II-1 or Aspect II-2, wherein R2is -O- C1-24alkyl.

[0126] Aspect II-6. The process of any one of the preceding Aspects II, wherein R2is H.25-10889 | 103241.007490

[0127] Aspect II-7. The process of any one of Aspects II-1 to II-5, wherein R2is -O-C1-24alkyl.

[0128] Aspect II-8. The process of any one of the preceding Aspects II, wherein R3is H.

[0129] Aspect II-9. The process of any one of Aspects II-1 to II-7, wherein R3is -O-C1-24alkyl.

[0130] Aspect II-10. The process of any one of the preceding Aspects II, wherein R4is H.

[0131] Aspect II-11. The process of any one of Aspects II-1 to II-9, wherein R4is -O-C1-24alkyl.

[0132] Aspect II-12. The process of any one of the preceding Aspects II, wherein R5is H.

[0133] Aspect II-13. The process of any one of Aspects II-1 to II-11, wherein R5is -O-C1-24alkyl.

[0134] Aspect II-14. The process of any one of the preceding Aspects II, wherein the alkylated phenolic ester is of Formula (I-1), (I-2), or (I-3):

[0135] Aspect II-15. The process of any one of the preceding Aspects II, wherein the alkylated phenolic ester is of Formula (I-4):25-10889 | 103241.007490

[0136] Aspect II-16. The process of any one of the preceding Aspects II, wherein the alkylated phenolic ester is:.

[0137] Aspect II-17. The process of any one of the preceding Aspects II, wherein the phenolic ester is of Formula (II):wherein, R10to R14are, independently, H or -OH, provided that at least one of R10to R14is -OH.

[0138] Aspect II-18. The process of Aspect II-17, wherein R10is H.

[0139] Aspect II-19. The process of Aspect II-17, wherein R10is -OH.

[0140] Aspect II-20. The process of any one of Aspects II-17 to II-19, wherein R11is H.

[0141] Aspect II-21. The process of any one of Aspects II-17 to II-19, wherein R11is -OH.25-10889 | 103241.007490

[0142] Aspect II-22. The process of any one of Aspects II-17 to II-21, wherein R12is H.

[0143] Aspect II-23. The process of any one of Aspects II-17 to II-21, wherein R12is -OH.

[0144] Aspect II-24. The process of any one of Aspects II-17 to II-23, wherein R13is H.

[0145] Aspect II-25. The process of any one of Aspects II-17 to II-23, wherein R13is -OH.

[0146] Aspect II-26. The process of any one of Aspects II-17 to II-25, wherein R14is H.

[0147] Aspect II-27. The process of any one of Aspects II-17 to II-25, wherein R14is -OH.

[0148] Aspect II-28. The process of any one of the preceding Aspects II, wherein the phenolic ester is of Formula (II-1), (II-2), or (II-3):

[0149] Aspect II-29. The process of any one of the preceding Aspects II, wherein the phenolic ester is:.

[0150] Aspect II-30. The process of any one of the preceding Aspects II, wherein the polar solvent has a boiling point of about 50 to about 220°C, or such as about 80°C, or such as about 120°C, or such as about 150°C, or such as about 165°C, or such as about 190°C, or such as about 200°C,.

[0151] Aspect II-31. The process of any one of the preceding Aspects II, wherein the polar solvent is a dipolar, aprotic solvent.25-10889 | 103241.007490

[0152] Aspect II-32. The process of Aspect II-31, wherein the dipolar, aprotic solvent is dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMAC), or N-methylpyrrolidone (NMP), or a combination thereof, or preferably DMF.

[0153] Aspect II-33. The process of any one of Aspects II-1 to II-30, wherein the polar solvent is acetonitrile (MeCN or ACN), cyclohexanol (CyO), or methyl isobutyl ketone (MIBK), or a combination thereof.

[0154] Aspect II-43. The process of any one of the preceding Aspects II, wherein the base is potassium carbonate, potassium bicarbonate, sodium carbonate, cesium carbonate, cesium bicarbonate, sodium bicarbonate, sodium hydroxide, or potassium hydroxide, or a combination thereof.

[0155] Aspect II-35. The process of any one of the preceding Aspects II, comprising about 6 to about 10 equivalents of the base.

[0156] Aspect II-36. The process of any one of the preceding Aspects II, wherein the temperature is at least about 40°C.

[0157] Aspect II-37. The process of any one of the preceding Aspects II, wherein the temperature is about 80 to about 180°C, or such as about 80°C, or such as about 90°C, or such as about 100°C, or such as about 120°C, or such as about 130°C, or such as about 135°C, or such as about 140°C, or such as about 150°C.

[0158] Aspect II-38. The process of any one of the preceding Aspects II, that is performed for at least about 15 minutes.

[0159] Aspect II-39. The process of any one of the preceding Aspects II, that is performed for about 15 minutes to about 24 hours, or such as about 20 minutes, about 40 minutes, about 50 minutes, about 55 minutes, about 70 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, about 5 hours, about 16 hours, about 17 hours, or about 20 hours.

[0160] Aspect II-40. The process of any one of the preceding Aspects II, comprising about 4 equivalents to about 8 equivalents of the alkylating agent, or such as about 4 equivalents, or such as about 5 equivalents, or such as about 6 equivalents.

[0161] Aspect II-41. The process of any one of the preceding Aspects II, wherein the alkylating agent is an alkyl halide such as an alkyl bromide, alkyl chloride, or alkyl iodide.25-10889 | 103241.007490

[0162] Aspect II-42. The process of any one of the preceding Aspects II, wherein the alkylating agent is an alkyl sulfonate such as an alkyl tosylate, alkyl mesylate, or alkyl triflate.

[0163] Aspect II-43. The process of any one of the preceding Aspects II, wherein the alkylating agent comprises 1 to 24 carbon atoms.

[0164] Aspect II-44. The process of any one of the preceding Aspects II, wherein the alkylating agent is branched.

[0165] Aspect II-45. The process of any one of Aspects II-1 to II-42, wherein the alkylating agent is linear.

[0166] Aspect II-46. The process of preceding Aspect II-1, comprising at least about 4 equivalents of the alkylating agent, about 1 equivalent of the phenolic ester, and at least about 6 equivalents of the base.

[0167] Aspect II-47. The process of any one of the preceding Aspects II, further comprising reducing the alkylated phenolic ester to form a reduced phenol.

[0168] Aspect II-48. The process of Aspect II-46, wherein the reduced phenol is of Formula (III):

[0169] Aspect II-49. The process of Aspect II-46 or II-47, wherein the reduced phenol is of Formula (III-1), (III-2), or (III-3):25-10889 | 103241.007490

[0170] Aspect II-50. The process of any one of Aspects II-46 to II-48, wherein the reduced phenol is:.

[0171] Aspect II-51. The process of Aspect II-49, wherein the reducing is performed using a reducing agent that reduces an ester to an alcohol, such as lithium aluminum hydride.

[0172] Aspect II-52. The process of Aspect II-49 or II-50, wherein the reducing is performed at reduced temperature, such as about 0 to about 24°C.

[0173] Aspect II-53. The process of any one of Aspects II-15 to II-17, wherein the reduced phenol is esterified to form an esterified phenol.

[0174] Aspect II-54. The process of Aspect II-52, wherein the esterified phenol is of Formula (IV):wherein, X1is Cl, Br, or F.

[0175] Aspect II-55. The process of Aspect II-52 or II-53, wherein the esterified phenol is of Formula (IV-1), (IV-2), or (IV-3):25-10889 | 103241.007490

[0176] Aspect II-56. The process of any one of Aspects II-52 to II-54, wherein the esterified phenol is:.

[0177] Aspect II-57. The process of any one of Aspects II-52 to II-55, wherein the esterified phenol is:.25-10889 | 103241.007490

[0178] Aspect II-58. The process of any one of Aspects II-52 to II-56, wherein the esterification is performed using X1-C1-8alk-C(O)-X2, wherein X1and X2are, independently, Cl, Br, or F.

[0179] Aspect II-59. The process of any one of Aspects II-52 to II-57, wherein the esterification is performed at reduced temperature, such as about 0 to about 23°C.

[0180] Aspect II-60. The process of any one of Aspects II-52 to II-58, further comprising coupling the ester group of the esterified phenol with HY1-C1-8alk-OH, wherein Y1is a chemical moiety comprising two nitrogen atoms.

[0181] Aspect II-61. The process of Aspect II-59, wherein the coupling forms a compound of Formula (V) or (V-A):wherein: Y1is -optionally substituted C3-8cycloalk-, -optionally substituted heterocycloalk-, -optionally substituted heteroar-, or -optionally substituted ar-; and Y2is OH, NH2, -NH(C1-6alkyl), -NH(C1-6alkyl)2, optionally substituted C1-6alkyl, optionally substituted C1-6alkoxy, optionally substituted C3-8cycloalkyl, substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0182] Aspect II-62. The process of Aspect II-59 or II-60, wherein the coupling forms a compound of Formula (V-1), (V-2), or (V-3):25-10889 | 103241.007490(V-3).

[0183] Aspect II-63. The process of any one of Aspects II-59 to II-61, wherein the coupling forms the following compound:.

[0184] Aspect II-64. The process of any one of Aspects II-59 to II-62, wherein the coupling forms the following compound:.25-10889 | 103241.007490

[0185] Aspect II-65. The process of any one of Aspects II-59 to II-62, wherein Y1is a 5-8-membered heterocycloalkyl comprising two nitrogen atoms.

[0186] Aspect II-66. The process of any one of Aspects II-59 to II-64, wherein Y1is piperazinyl.

[0187] Aspect II-67. A compound prepared according to the process of any one of Aspects II-1 to II-65.

[0188] Aspect II-68. A composition comprising the compound of Aspect II-26 and a ribonucleic acid.

[0189] Aspect II-69. The composition of Aspect II-67, wherein the nucleic acid is mRNA, or such as Luc-mRNA.

[0190] Aspect II-70. A method for delivering a nucleic acid to a cell in a patient, comprising administering the composition of Aspect II-67 or II-68 to the patient.

[0191] Aspect II-71. The method of Aspect II-69, wherein the cell is a tissue, such as lung tissue, liver tissue, spleen tissue, or a lymph node.

[0192] The following examples are exemplary, only and should not be construed as limiting the scope of the invention.

[0193] Example 1: Materials and General Methods

[0194] (i) Materials

[0195] Propyl gallate (Acros Organics, 98%), 2-ethylhexyl bromide (TCI, > 97%), lithium aluminum hydride (LiAlH4) (Oakwood Chemical), 4-bromobutyric acid (Acros Organics, 98%), thionyl chloride (SOCl2) (Thermo Scientific, 99%), triethylamine (NEt3) (TCI, > 99%), 4-dimethylaminopyridine (DMAP) (TCI, > 99%), N-(2- hydroxyethyl)piperazine (98.5%, Acros Organics), anhydrous potassium carbonate (K2CO3) (Fisher Chemical), sodium sulfate (Na2SO4) (Fisher Chemical), sodium bicarbonate (NaHCO3) (Fisher Chemical, Certified ACS), and anhydrous magnesium sulfate (MgSO4) (Fisher Chemical) were used as received. Dimethylformamide (DMF), ethyl acetate (EtOAc), hexane (Hex), tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile (MeCN), and methanol (MeOH) were all ACS Grade solvents from Fisher. DCM and NEt3were dried over CaH2and distilled before use. THF was dried over sodium / benzophenone and distilled before use.

[0196] Acetate buffer (10mM, pH = 4.00) was prepared by dissolving sodium acetate (2.3 mM) and acetic acid (7.7 mM) in ultra-pure water. The final buffer pH was25-10889 | 103241.007490 adjusted with either 0.1 M HCl or 0.1 M NaOH solutions. Nucleoside-modified mRNA encoding the luciferase enzyme (Luc-mRNA) was produced as discussed in Pardi, In Vitro Transcription of Long RNA Containing Modified Nucleosides. Methods Mol. Biol.2013, 969, 29-42. DPBS (Corning), OptiMEM (Gibco), UltraPure DNase / RNase-Free Distilled Water (Invitrogen), Trypsin-EDTA (0.25%, Gibco), Trypan Blue (Sigma-Aldrich), Cell Culture Lysis 5X Reagent (Promega), Luciferase Assay System (Promega) and D-luciferin sodium salt (Regis Technologies) were used as received.

[0197] (ii) General Methods

[0198] 1H spectra were recorded at 400 MHz and13C NMR spectra at 101 MHz on a Bruker NEO NMR spectrometer. All NMR characterizations were carried out at 23.5°C in CDCl3with 0.05 % TMS. NMR spectra were analyzed using MNova 14. Chemical shifts (δ) are reported in ppm. The resonance multiplicities for the1H NMR spectra are labeled as “s” for singlet, “d” for doublet, “t” for triplet, “m” for multiplet, and “br” for broad resonance. Residual protonic solvent, CHCl3(1H, δ 7.26 ppm;13C, δ 77.16 ppm) and tetramethyl silane (TMS, δ 0 ppm) were used as internal reference in all NMR spectra. TLC was used to monitor reactions and evaluate compound purity using silica gel 60 F254precoated plates (E. Merck). Individual aromatic compounds were visualized by UV light (λ = 254 nm). Purification by column chromatography on SiO2was performed using silica gel from Silicycle (60 Å, 40-63 μm). High pressure liquid chromatography (HPLC) analysis was performed using a Shimadzu LC-20AD high-performance liquid chromatograph pump, a PE Nelson Analytical 900 Series integration data station, a Shimadzu SPD-10A VP (UV-vis, λ = 254 nm), Shimadzu RID-10A refractive index (RI) detector, and three AM gel columns (guard column, two 500 Å, 10 μm columns). THF with 5% triethylamine was used as the solvent for all HPLC characterizations which were carried out at 23.5°C. Molar Mass of all compounds were determined by MALDI-TOF mass spectrometry using a PerSeptive Biosystem-Voyager-DE (Framingham, MA) mass spectrometer equipped with a nitrogen laser (337 nm) operating in linear mode. Angiotensin II and Bombesin were used as standards for calibration. To prepare the sample solution, the compound for characterization was first dissolved in THF (5-10 mg / mL). The matrix (2,5-dihydroxybenzoic acid, 99%, Thermo Scientific) was also dissolved in THF 10 mg / mL and the two solutions were mixed in a 1 / 5 (v / v, compound solution / matrix solution) ratio. One drop of solution was dried on the MALDI-TOF plate25-10889 | 103241.007490 at 23.5°C, the plate was inserted into the vacuum chamber for analysis, and laser intensity / voltages applied were adjusted for the molar mass and nature of each compound.

[0199] (iii) Synthesis

[0200] A modular-orthogonal methodology was employed. Structural analysis of all compounds was performed by a combination of1H and13C-NMR spectroscopy, TLC, HPLC, and MALDI-TOF.1H-NMR spectra are shown in FIGs.5-8,13C-NMR spectra in FIGs.9-12, HPLC traces are in FIGs.13 and 140 and MALDI-TOF spectra are in FIG.15.

[0201] (iv) Dynamic Light Scattering (DLS).

[0202] DLS was performed to determine the sizes and polydispersities of DNPs using a Malvern Instruments particle sizer (Zetasizer Nano S, Malvern Instruments, UK) equipped with a 4 mW He-Ne laser (633 nm), an avalanche photodiode positioned at 175° to the beam, and a temperature-controlled cuvette holder. Parameters and measurement times were set automatically.0.4 mL of sample solution was placed in a semi-micro cuvette (1.6 mL, polystyrene, 10 × 10 × 45 mm, Greiner Bio-One) and the experiments were performed at 23°C. See, Lu, “Targeted and Equally Distributed Delivery of mRNA of mRNA to Organs with Pentaerythritol-Based One-Component Ionizable Amphiphilic Janus Dendrimers,” J. Am. Chem. Soc.2023, 145, 18760-18766; Zhang, “Targeted Delivery of mRNA with One-Component Ionizable Amphiphilic Janus Dendrimers,” J. Am. Chem. Soc.2021, 143, 17975-17982; Zhang, “The Unexpected Importance of the Primary Structure of the Hydrophobic Part of One-Component Ionizable Amphiphilic Janus Dendrimers in Targeted mRNA Delivery Activity,” J. Am. Chem. Soc.2022, 144, 4746-4753; Zhang, “One-Component Multifunctional Sequence-Defined Ionizable Amphiphilic Janus Dendrimer Delivery Systems for mRNA,” J. Am. Chem. Soc.2021, 143, 12315-12327; and Lu, “Screening Libraries to Discover Molecular Design Principles for the Targeted Delivery of mRNA with One-Component Ionizable Amphiphilic Janus Dendrimers Derived from Plant Phenolic Acids,” Pharmaceutics 2023, 15, 1572.

[0203] (v) Production of Nucleoside-Modified Luc-mRNA.

[0204] mRNA was produced as previously discussed in Pardi using T7 RNA polymerase on linearized DNA encoding codon-optimized Firefly Luciferase and a 101nt poly(A) tail.1-Methylpseudouridine-5’-triphosphate was used instead of UTP. A trinucleotide cap1 analog was added co-transcriptionally. Purification was performed as25-10889 | 103241.007490 discussed in Baiersdörfer, Method for the Removal of DsRNA Contaminant from In Vitro-Transcribed mRNA. Mol. Ther. Nucleic Acids 2019, 15, 26-35. mRNA was analyzed for RNase, dsRNA, endotoxin, and other forms of contamination before storing frozen at -20°C.

[0205] (vi) Formulation of DNPs Co-Assembled from IAJDs and Luc-mRNA

[0206] Luc-mRNA was dissolved in UltraPure DNase / RNase-free distilled water with an initial concentration of 4.0 mg / mL. IAJDs were dissolved in ethanol at an initial concentration of 80 mg / mL. Luc-mRNA solution (12.5 μL) was placed into a clean RNase free Eppendorf (1.5 mL) and 463 μL of acetate buffer (10mM, pH 4.0) was added. Subsequently, the IAJD stock solution in ethanol (25 μL) was rapidly injected on the surface of the Luc-mRNA solution in acetate buffer with a syringe, followed by vortexing for 5 seconds. The pH of the assembly mixture increases after the addition of the IAJD solution from 4.0 when it only contains mRNA to about 4.8-5.0. See, the two Lu and three Zhang cited above in section (iv).

[0207] (vii) In Vivo mRNA Delivery in Mice with DNPs

[0208] All mice used were in accordance with the guidelines and approval from the Pennsylvania University Institution of Animal Care and Use Committee. Female or male BALB / c mice (6-8 weeks old, from Charles River Laboratories) were anesthetized with isoflurane (Piramal Healthcare Limited) and injected via retro-orbital sinus with 100 μL of DNP solution containing 10 μg of Luc-mRNA. After 4 hours post injection, mice underwent luminescence characterization. See, the two Lu and three Zhang cited above in section (iv).

[0209] (viii) Luminescence Characterization for In Vivo Transfection Experiments

[0210] Bioluminescence imaging was performed with an IVIS Spectrum imaging system (PerkinElmer, Waltham, MA). Mice were anesthetized with 3% isoflurane (Piramal Healthcare Limited) and intraperitoneally (i.p.) administered with D-luciferin (Regis Technologies) at a dose of 150 mg / kg of body weight. Ten minutes post administration of D-luciferin, mice were placed on the imaging platform with continued isoflurane via nose cone and imaged using variable exposure time (15, 30, or 60 seconds) and medium binning (binning = 8) to ensure that the signal was within operative detection range. Mice were sacrificed and heart, lungs, liver, and spleen were immediately collected25-10889 | 103241.007490 for IVIS imaging of organs. Bioluminescence values were quantified measuring photon flux (photons / second, p / s) using Living Image Software (Perkin Elmer) in the region of interest (ROI) on mice where bioluminescence signal emanated. To quantify luminescent flux, an oval ROI was put over each organ of interest and analyzed. See, the two Lu and three Zhang cited above in section (iv).

[0211] Example 2: General Procedure for the Two-gram Scale Synthesis of Propyl 3,4,5-Tris((2-ethylhexyl)oxy)benzoate (3).

[0212] Compound 3 was obtained by the alkylation of propyl gallate (1) with 2- ethylhexyl bromide (2). Propyl gallate (1, 2 g, 9.4 mmol) and K2CO3(9.1 g, 65 mmol) were stirred in 25 mL of DMF.2-Ethylhexyl bromide (8.15 g, 42 mmol) was added into the reaction mixture under stirring. The reaction mixture was bubbled with nitrogen for at least 30 min. The flask equipped with a reflux condenser was then introduced into an oil bath preheated to 130°C under stirring and the extent of the reaction was monitored by TLC. After 30 min, TLC analysis indicated complete conversion showing only the product 3 at Rf= 0.80. At this time, the reaction mixture was quenched in ice-water and extracted three times with 50 mL EtOAc. The organic layer was isolated, dried over anhydrous MgSO4and vacuum filtered. The filtrate was concentrated on a rotary evaporator and subsequently was purified by column chromatography using 95 / 5:Hex / EtOAc as eluent to yield 3 as a colorless oil (4.80 g, 93.1%).1H NMR (400 MHz, CDCl3) δ 7.26 (s, 2H, PhH), 4.26 (t, 2H, H×PhCOOCH2), 3.93-3.86 (m, 6H, 3×PhOCH2-), 1.87-1.78 (m, 3H, 3×PhOCH2CH(CH2CH3)(CH2)3CH32), 1.64-1.30 (m, 26H, 3×PhOCH2CH(CH2CH3)(CH2)3CH3,-OCH2CH2CH3), 1.05 (t, 3H, -OCH2CH2CH3), 0.98–25-10889 | 103241.007490 0.91 (m, 18H, 3×PhOCH2CH(CH2CH3)(CH2)3CH3).13C NMR (101 MHz, CDCl3) δ 166.76, 153.13, 142.44, 125.07, 107.58, 76.10, 71.40, 66.67, 40.78, 39.73, 30.69, 30.59, 29.44, 29.27, 23.99, 23.81, 23.28, 23.23, 22.33, 14.27, 14.23, 11.34, 11.32, 11.30, 11.27, 10.64. Purity by HPLC: 99+%. MALDI-TOF MS m / z of [M + Na]+ calculated for C34H60NaO5+: 571.43; Found: 571.05.

[0213] Example 3: General Procedure for the Ten-gram Scale Synthesis of Propyl 3,4,5-Tris((2-ethylhexyl)oxy)benzoate (3)

[0214] Compound 3 was obtained by the alkylation of propyl gallate (1) with 2- ethylhexyl bromide (2). Propyl gallate (1, 15.0 g, 70.7 mmol) and K2CO3(58.0 g, 424 mmol) were stirred in 200 mL of DMF.2-Ethylhexyl bromide (61.1 g, 318 mmol) was added into the reaction mixture under stirring. The solution as bubbled with nitrogen for at least 30 min. The flask equipped with a reflux condenser was then introduced into an oil bath preheated to 130°C under stirring for 55 min at which time TLC analysis indicated complete conversion showing only the product 3 at Rf= 0.80. The reaction mixture was allowed to cool to room temperature and DMF was removed in a rotary evaporator. The residue was mixed with 100 mL water and extracted 5 times with 100 mL EtOAc. The organic layer was isolated, dried over anhydrous MgSO4and vacuum filtered. The filtrate was concentrated on a rotary evaporator and further purified by column chromatography using 95 / 5:Hex / EtOAc as eluent to yield the title compound as a colorless oil (36.3 g, 95.3%). TLC (95 / 5:Hex / EtOAc): Rf= 0.8.1H NMR (400 MHz, CDCl3) δ 7.26 (s, 2H, PhH), 4.26 (t, 2H, H×PhCOOCH2), 3.93-3.86 (m, 6H, 3×PhOCH2-), 1.87-1.78 (m, 3H, 3×PhOCH2CH(CH2CH3)(CH2)3CH32), 1.64-1.30 (m, 26H,25-10889 | 103241.007490 3×PhOCH2CH(CH2CH3)(CH2)3CH3,-OCH2CH2CH3), 1.05 (t, 3H, -OCH2CH2CH3), 0.98– 0.91166.76, 153.13, 142.44, 125.07, 107.58, 76.10, 71.40, 66.67, 40.78, 39.73, 30.69, 30.59, 29.44, 29.27, 23.99, 23.81, 23.28, 23.23, 22.33, 14.27, 14.23, 11.34, 11.32, 11.30, 11.27, 10.64. Purity by HPLC: 99+%. MALDI-TOF MS m / z of [M + Na]+ calculated for C34H60NaO5+: 571.43; Found: 571.05.

[0215] Example 4: General Procedure for the Synthesis of (3,4,5-Tris((2- ethylhexyl)oxy)phenyl)methanol (4).

[0216] Compound 4 was obtained by the reduction of propyl 3,4,5-tris((2- ethylhexyl)oxy)benzoate (3) with LiAlH4. Propyl 3,4,5-tris((2-ethylhexyl)oxy)benzoate (3, 12 g, 21.8 mmol) was dissolved in 100 mL of anhydrous THF. LiAlH4(1.08 g, 20.1 mmol) was added slowly to the reaction mixture maintained at 0°C with an ice bath. The ice bath was subsequently removed, and the reaction was allowed to proceed at 23.5°C for 1.5 h at which time TLC analysis indicated complete conversion of compound 3 into compound 4. The reaction was quenched by adding dropwise a saturated aqueous solution of Na2SO4until bubbling stopped. The reaction mixture was filtered, the solid and the flask were rinsed with THF and DCM. The filtrate was dried over anhydrous MgSO4, vacuum filtered, and the filtrate was concentrated in a rotary evaporator to yield compound 4 as a colorless oil (10.4 g, 88.6%). TLC (50 / 50:Hex / DCM): Rf= 0.39.1H NMR (400 MHz, CDCl3) δ 6.58 (s, 2H, PhH), 4.62 (d, 2 H, PhCH2OH), 3.91-3.81 (m, 6H, 3×PhOCH2-), 1.80-1.69 (m, 3H, 3×PhOCH2CH(CH2CH3)(CH2)3CH3), 1.60–1.29 (m, 24H, 3×PhOCH2CH(CH2CH3)(CH2)3CH3), 0.98–0.90 (m, 18H,25-10889 | 103241.007490 3×PhOCH2CH(CH2CH3)(CH2)3CH3).13C NMR (101 MHz, CDCl3) δ 153.68, 137.54, 136.08, 104.82, 76.12, 71.34, 65.92, 40.74, 39.78, 30.66, 29.48, 29.26, 23.96, 23.87, 23.31, 23.24, 14.29, 14.25, 11.36, 11.34, 11.32, 11.24. Purity by HPLC: 99+%. MALDI- TOF MS m / z of [M + H]+ calculated for C31H57O4+: 492.4; Found: 492.78.

[0217] Example 5: General Procedure for the Synthesis of 3,4,5-Tris((2- ethylhexyl)oxy)benzyl 4-bromobutanoate (6).

[0218] Compound 6 was obtained by the esterification of (3,4,5-tris((2- ethylhexyl)oxy)phenyl)methanol (4) with 4-bromobutanoyl chloride freshly prepared just before use.4-Bromobutyric acid was dissolved in a mixture containing 6 mL anhydrous DCM and approximately 10 mL SOCl2(16.89 g, 142.1 mmol). A drop of DMF was added and the reaction was stirred at 23.5°C for 1 h to form 4-bromobutanoyl chloride. DCM and excess SOCl2were distilled in a rotary evaporator under reduced pressure.3,4,5-Tris((2- ethylhexyl)oxy)phenyl)methanol (4, 10 g, 20.3 mmol) was dissolved in 20 mL of dry DCM to which dry NEt3(2.45 g, 24.24 mmol) and a catalytic amount of DMAP (0.46 g, 4.06 mmol) were added. A small amount of anhydrous DCM was used to dissolve the 4- bromobutanoyl chloride, and the solution was added dropwise to an ice-water bath of 3,4,5-tris((2-ethylhexyl)oxy)phenyl)methanol (4) solution. The ice bath was subsequently removed, and the reaction was allowed to proceed under stirring at 23.5°C for 2 h at which25-10889 | 103241.007490 time TLC analysis indicated complete conversion. The reaction was quenched by adding the reaction mixture to 50 mL of water. This mixture was extracted 3 times with 50 mL of DCM. The organic layer was dried over anhydrous MgSO4, and vacuum filtered. The filtrate was concentrated on a rotary evaporator and purified by column chromatography using 50 / 50:Hex / DCM as eluent to yield compound 6 as a colorless oil (11.8 g, 90.5%). TLC (50 / 50:Hex / DCM): Rf= 0.76.1H NMR (400 MHz, CDCl3) δ 6.53 (s, 2 H, PhH), 5.03 (s, 2 H, -CH2Ph), 3.87-3.78 (m, 6 H, 3×PhOCH2-), 3.47 (t, 2 H, -CH2Br), 2.56 (t, 2 H, BrCH2CH2CH2COO-), 2.24-2.16 (m, 2 H, BrCH2CH2CH2COO-), 1.78–1.65 (m, 3 H, 3×PhOCH2CH(CH2CH3)-), 1.59–1.29 0.95–0.85 (m, 18 H, 3×PhOCH2CH172.50, 153.57, 153.53, 138.13, 130.69, 106.37, 76.05, 71.31, 67.02, 44.13, 40.69, 39.75, 32.75, 32.63, 31.38, 30.63, 29.46, 29.44, 29.42, 29.24, 27.88, 27.77, 23.93, 23.83, 23.28, 23.22, 14.27, 14.23, 11.33, 11.31, 11.29, 11.25, 8.70. Purity by HPLC: 99+%. MALDI- TOF MS m / z of [M + Na]+ calculated for C35H61BrNaO5+: 663.36; Found: 664.55.

[0219] Example 6: General Procedure for the Synthesis of IAJD-97

[0220] IAJD97 (8) was obtained by reacting N-(2-hydroxyethyl)piperazine with 3,4,5-tris((2-ethylhexyl)oxy)benzyl 4-bromobutanoate (6).3,4,5-Tris((2- ethylhexyl)oxy)benzyl 4-bromobutanoate (6, 11 g, 17.2 mmol), K2CO3(2.9 g, 21.125-10889 | 103241.007490 mmol), and N-(2-hydroxyethyl)piperazine (7, 2.74 g, 21.1 mmol) were stirred in 100 mL of MeCN at 95°C for 3 h at which time TLC analysis indicated complete conversion. The reaction was quenched by mixing the reaction mixture with 50 mL of water. This mixture was extracted 3 times with 20 mL of DCM. The organic layer was dried over anhydrous MgSO4, and vacuum filtered. The filtrate was concentrated in a rotary evaporator and purified by column chromatography using 40 / 1:DCM / MeOH and 50 / 1: DCM / MeOH as eluent to yield IAJD97 (8) as a colorless oil (10.2 g, 86.8%). The resulting oil was dissolved in DCM (25 mL) and washed with NaHCO3(2%, 25 mL). The aqueous phase was extracted by DCM (25 mL) for two additional times. The organic phase was combined and dried over anhydrous MgSO4. Filtration, evaporation of the solvent, and vacuum drying yielded IAJD97, 8, as a colorless oil (10.2 g, 86.8%). TLC (95 / 5:DCM / MeOH): Rf= 0.32.1H NMR (400 MHz, CDCl3) δ 6.52 (s, 2 H, PhH), 5.00 (s, 2 H, -CH2Ph), 3.84-3.78 (m, 6 H, 3×PhOCH2-), 3.59 (t, 2 H, -CH2CH2OH), 2.83 (br, 1 H, -CH2CH2OH), 2.62-2.29 (m, 14 H, N(CH2CH2)2, -OCOCH2CH2CH2- and -CH2CH2OH), 1.88-1.77 (m, 2 H, -OCOCH2CH2CH2-), 1.77-1.64 (m, 3 H, 3×PhOCH2CH(CH2CH3)-), 1.62-1.25 (m, 24 H, 3×PhOCH2CH(CH2CH3)(CH2)3CH3), 0.95-0.86 (m, 18 H, 3×PhOCH2CH(CH2CH3)(CH2)3CH3).13C NMR (101 MHz, CDCl3) δ 173.5, 153.5, 138.1, 130.9, 106.5, 76.1, 71.3, 66.8, 59.3, 57.8, 57.7, 53.3, 53.0, 40.7, 39.8, 32.4, 30.6, 30.6, 29.5, 29.4, 29.4, 29.2, 23.9, 23.8, 23.3, 23.2, 22.3, 14.3, 14.2, 11.3, 11.3, 11.3, 11.3. Purityby HPLC: 99+%. MALDI-TOF MS m / z of [M + H]+calculated for C41H75N2O6+: 691.6;Found: 691.1 and [M + Na]+calculated for C41H74N2NaO6+: 713.5; Found: 713.3.

[0221] Example 7: Results

[0222] A. Rates of Reaction

[0223] Attempts to increase the rate of reaction under closely related reaction conditions began with an increased reaction temperature (Table 1).25-10889 | 103241.00749025-10889 | 103241.007490

[0224] The low boiling point of the alkylating reagent 2-ethylhexyl bromide (bp = 75-77oC) limits the reaction temperature of the alkylation to 70 or 80oC. However, 2-ethylhexyl bromide makes an azeotropic mixture with the dipolar aprotic solvent DMF. Therefore, during the experiments in Table 1 the reaction temperature of the alkylation step was increased up to 150oC (entries 1-10, Table 1).

[0225] At 120 to 130oC the reaction time to complete conversion determined by TLC was 30 min, which is a substantial increase in rate compared to the 5 h required for less than 100% conversion and low isolated yields at lower temperatures (entries 1 and 2, Table 1). The number of equivalents of the alkylating agent and K2CO3base play a very important role in maintaining a low reaction time. For example, decreasing the number of equivalents of 2-ethylhexyl bromide from 4.5 to 4 (entry 11, Table 1) increased the reaction time from 30 min to 16 h with only incomplete conversion by TLC, and to 17 h with complete conversion by TLC (entry 12, Table 1). Therefore, an optimum number of 2-ethylhexyl bromide equivalents of 4.5 was maintained in all other experiments.

[0226] Decreasing the equivalents of K2CO3base from 7 to 6 (entry 13, Table 1) does not affect the reaction time. However, only 5 equivalents of base increased the reaction time from 30 min to 16 h with incomplete conversion by TLC (entry 14, Table 1), while a reaction time of 17 h provided complete conversion by TLC (entry 15, Table 1). Therefore, for 2 g or one equivalent of propyl gallate, the shorter reaction time of 30 min to complete conversion requires 4.5 equivalents of 2-ethylhexyl bromide and 6 equivalents of base in 25 mL DMF at 130oC (entry 13, Table 1) to yield 94.2 % of colorless trialkylated product after column chromatography purification. This product was also distilled in a Kulgelrohr apparatus at 200oC and remains colorless after storage at rt (23.5oC) in air for as long as required.25-10889 | 103241.007490

[0227] This reaction was then screened using several additional dipolar aprotic solvents (entries 16-18, Table 1) as well as other polar solvents (entries 19-21, Table 1). Longer reaction times to complete conversion by TLC were required for the dipolar aprotic solvents DMSO, DMAC and NMP. Only incomplete conversions were obtained in acetonitrile, cyclohexanone and methyl isobutyl ketone. The polar reaction solvents, which did not provide complete conversion, maintained a clear reaction mixture (entries 19-21, Table 1). Without complete conversion, after column chromatography, the final product turns yellow from a clear reaction mixture, while in solvents like DMF exhibiting a darkly colored reaction mixture, but yields a clear colorless product after purification. The optimized reaction conditions mentioned above were scaled to 15 grams of propyl gallate to provide in 36.3 g of entirely colorless in 55 minutes product after column chromatography and Kulgelrohr distillation.

[0228] B. Synthesis of IAJD97

[0229] The next step of this reaction involves the reduction of propyl 3,4,5- tris((2-ethylhexyl)oxy)benzoate (3) with LiAlH4in dry THF at 0 to 23.5oC. This classic reduction proceeds to 100 % conversion in 1.5 h when 12 g of compound 3 was used in the reduction reaction. The resulting benzyl alcohol 4 (10.4 g, 88.6%) was esterified with 4-bromobutanoyl chloride (5) in dry methylene chloride at 0 to 23.5oC in the presence of dry NEt3and a catalytic amount of the supernucleophilic catalyst DMAP. Compound 5 was freshly prepared from the corresponding acid in dry methylene chloride with thionyl chloride in the presence of one drop of DMF as catalyst followed by the distillation of the excess thionyl chloride and of the methylene chloride to yield 83.7% of compound 6 after column chromatography. Compound 6 was reacted with N-(2-hydroxyethyl)piperazine (7) in the presence of K2CO3as base in MeCN at 95oC to produce compound 8 IAJD97 in 86.8% isolated yield after 3 h and purification by column chromatography, washing with dilute NaHCO3followed by drying on MgSO4, evaporation of the solvent and vacuum drying.

[0230] C. Potential Side Reactions of IAJD97

[0231] The IAJD97 synthesized by alkylation of the gallic acid derivative 80oC under the conditions of Zhang, “Targeted Delivery of mRNA with One-Component Ionizable Amphiphilic Janus Dendrimers.” J. Am. Chem. Soc.2021, 143 (43), 17975– 17982 was unstable at room temperature. Originally, it was believed that this was a25-10889 | 103241.007490 property of the pure IAJD97 and therefore, investigated the structure of the degradation product and of its potential mechanism of degradation quite extensively. However, this was a side reaction mediated by a concentration of free phenol that cannot be detected by the combination of NMR, TLC, HPLC and MALDI-TOF instrumentation used in the current experiments. This undetectable free phenol is generated during the first step of this synthesis. Quantitative alkylation during the first step, eliminated the instability of IAJD97. Since this side-reaction influences the total and the targeted activity in vivo, a method was established to detect this side reaction by targeted delivery experiments mediated by IAJD97. These experiments are more sensitive to sample purity than any of the analytical methods employed during the synthesis process.

[0232] D. Estimating the Purity of IAJD97 by Targeted Delivery of Luc- mRNA with its DNPs

[0233] Targeted delivery experiments of firefly luciferase (Luc)-mRNA mediated by IAJD97 as a function of the number of purification experiments were performed as described in Zhang, J. Am. Chem. Soc.2021, 143 (43), 17975–17982. The accuracy of the IAJD purification depends on the expertise of the experimentalist. The inventors found that the change in activity in vivo (comparing the synthesis routes in the art and the synthesis route described herein) can be only due to the purification and not due to the in vivo delivery. The results from FIG.2 show the dependence of total activity as well as of targeted activities as a function of the purity. pKa of all IAJD97 is constant and is shown in the upper part of FIG.2. DNPs assembled from IAJD97 and Luc-mRNA were generated by simple injection in acetate buffer at pH = 4. Under neutral or slightly basic conditions IAJD97 does not self-assemble into vesicles. However, at pH = 4 in the absence of Luc-mRNA it self-assembles into empty vesicles. These vesicles can be considered as being supramolecular polymers with narrow polydispersity. In the presence of Luc-mRNA vesicles containing encapsulated Luc-mRNA known as DNPs are co- assembled. These DNPs also can be considered as supramolecular polymers encapsulating a monodisperse Luc-mRNA natural polymer. The dimensions in nm and the polydispersity (PDI) of the DNPs are shown in the third line from the top of FIG.2. The fourth line reports the total flux in p / s. The images of the whole mouse bodies are shown in line five of FIG.2. Lines six and seven report the activities for the delivery to lymph nodes while lines eight, nine and ten show the lung, liver and spleen with their luminescence. Lines25-10889 | 103241.007490 eleven, twelve and thirteen provide the values for the delivery to lung, live and spleen, all in p / s. Each purification was analyzed in vivo, with three mice per condition.

[0234] As seen in FIG.2, after two purifications, both the total activity (1.15x107, 6.78x106, 5.84x106p / s) and the activity for the delivery to spleen (1.00x107, 1.30x107, 1.10x107p / s) show similar values (107p / s) with the results for IAJD97 in Zhang, J. Am. Chem. Soc.2021, 143 (43), 17975–17982. After three purifications the total activity increases to 1.23x107, 1.31x107, 6.47x106while the activity to spleen and lymph nodes are minimally affected. Four purifications provide an increase in the total activity to 1.29x108, 1.28x108, 1.32x108with a corresponding activity for delivery to the spleen to 5.10x107, 4.90x107and 7.00x107. Unexpectedly the activity to liver and lung increases after four purifications from 105to 107while the activity to lymph nodes increases from 105-106and 106-107. Five purifications provided a remarkable result. Total activity is 2.67×108, 2.38×108and 3.22×108, activity to spleen is 4.32×108, 3.33×108and 5.33×108, activity to liver is 4.75×107, 5.06×107and 3.86×107, activity to lung is 5.66×107, 4.59×107and 3.06×107while activity to lymph nodes is 1.16×107, 1.03×107, 1.19×107, 1.09×107, 1.96×107and 1.17×107. These results demonstrate that although by conventional analytical methods in all cases the purity of IAJD97 was higher than 99% after two purifications, in vivo activities continue to increase as the number of purifications by column chromatography is higher. The in vivo activity as a function of number of purifications of the IAJD was then duplicated and the remarkable progress was demonstrated by the experiments shown in FIG.3. Previously published data on IAJD97, including in vivo targeted delivery data, are shown in the first column marked with a star.

[0235] Indeed, after only two purifications all activities are equal or higher than after the five purifications illustrated in FIG.2 and are with one order of magnitude higher both as total activity and as targeted activity to spleen than the data for alternate synthesis methods for preparing alternate synthesis methods for IAJD97 in Zhang, J. Am. Chem. Soc.2021, 143 (43), 17975–17982. Column chromatography experiments on silica gel were performed with no more than 2.5 % methanol in DCM which is widely accepted that does not dissolve silica gel. These experiments also demonstrated for the first time that impurities that affect the in vivo activity are not detectable by conventional analytical methods and that repeated purifications of IAJDs combined with determination of activity in vivo establish their maximum activity.25-10889 | 103241.007490

[0236] E. Stability of IAJD97 Investigated as a Function of Time by1H-NMR Spectroscopy

[0237] 1H-NMR analysis as a function of time combined with TLC, HPLC and MALDI-TOF demonstrated the stability of the IAJD97 at the room temperature of the laboratory (23.5oC) and in air for more than six months. The1H-NMR spectra demonstrating this stability are shown in FIG.4 while HPLC and MALDI-TOF data are shown in FIGs.13-15.

[0238] Nothing in this specification should be considered as limiting the scope of this disclosure. All examples presented are representative and non-limiting. The above- described embodiments can be modified or varied, as appreciated by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the embodiments disclosed herein can be practiced otherwise than as specifically described.

Claims

1. 25-10889 | 103241.007490 What is claimed is:

1. A process for preparing an alkylated phenolic ester, the process comprising reacting an alkylating agent with a phenolic ester at an elevated temperature in the presence of a polar solvent and at least about 5 equivalents of a base.

2. The process of claim 1, wherein the alkylated phenolic ester is of Formula (I):wherein: R1is C2-10alkyl; and R2to R6are, independently, H, -O-C1-24alkyl, optionally substituted aryl, or -O-C1-8alk-(optionally substituted aryl); provided that at least one of R2to R6is not H.

3. The process of claim 1, wherein R1is C2alkyl, C3alkyl, C4alkyl, C5alkyl, C6alkyl, C7alkyl, or C8alkyl, preferably C3alkyl.

4. The process of claim 1, wherein R2is H.

5. The process of claim 1, wherein R2is -O-C1-24alkyl.

6. The process of claim 1, wherein R2is H.

7. The process of claim 1, wherein R2is -O-C1-24alkyl.

8. The process of claim 1, wherein R3is H.

9. The process of claim 1, wherein R3is -O-C1-24alkyl.

10. The process of claim 1, wherein R4is H.

11. The process of claim 1, wherein R4is -O-C1-24alkyl.

12. The process of claim 1, wherein R5is H.-10889 | 103241.007490 13. The process of claim 1, wherein R5is -O-C1-24alkyl.

14. The process of claim 1, wherein the alkylated phenolic ester is of Formula (I-1), (I-15. The process of claim 1, wherein the alkylated phenolic ester is of Formula (I-4):

16. The process of claim 1, wherein the alkylated phenolic ester is:.

17. The process of claim 1, wherein the phenolic ester is of Formula (II):-10889 | 103241.007490wherein, R10to R14are, independently, H or -OH, provided that at least one of R10to R14is -OH.

18. The process of claim 17, wherein R10is H.

19. The process of claim 17, wherein R10is -OH.

20. The process of claim 17, wherein R11is H.

21. The process of claim 17, wherein R11is -OH.

22. The process of claim 17, wherein R12is H.

23. The process of claim 17, wherein R12is -OH.

24. The process of claim 17, wherein R13is H.

25. The process of claim 17, wherein R13is -OH.

26. The process of claim 17, wherein R14is H.

27. The process of claim 17, wherein R14is -OH.

28. The process of claim 1, wherein the phenolic ester is of Formula (II-1), (II-2), or-10889 | 103241.007490 29. The process of claim 1, wherein the phenolic ester is:.

30. The process of claim 1, wherein the polar solvent has a boiling point of about 50 to about 220°C, or such as about 80°C, or such as about 120°C, or such as about 150°C, or such as about 165°C, or such as about 190°C, or such as about 200°C,.

31. The process of claim 1, wherein the polar solvent is a dipolar, aprotic solvent.

32. The process of claim 31, wherein the dipolar, aprotic solvent is dimethylformamide (DMF), dimethylsulfoxide (DMSO), dimethylacetamide (DMAC), or N- methylpyrrolidone (NMP), or a combination thereof, or preferably DMF.

33. The process of claim 1, wherein the polar solvent is acetonitrile (MeCN or ACN), cyclohexanol (CyO), or methyl isobutyl ketone (MIBK), or a combination thereof.

34. The process of claim 1, wherein the base is potassium carbonate, potassium bicarbonate, sodium carbonate, cesium carbonate, cesium bicarbonate, sodium bicarbonate, sodium hydroxide, or potassium hydroxide, or a combination thereof.

35. The process of claim 1, comprising about 6 to about 10 equivalents of the base.

36. The process of claim 1, wherein the temperature is at least about 40°C.

37. The process of claim 1, wherein the temperature is about 80 to about 180°C, or such as about 80°C, or such as about 90°C, or such as about 100°C, or such as about 120°C, or such as about 130°C, or such as about 135°C, or such as about 140°C, or such as about 150°C.

38. The process of claim 1, that is performed for at least about 15 minutes.

39. The process of claim 1, that is performed for about 15 minutes to about 24 hours, or such as about 20 minutes, about 40 minutes, about 50 minutes, about 55-10889 | 103241.007490 minutes, about 70 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, about 5 hours, about 16 hours, about 17 hours, or about 20 hours.

40. The process of claim 1, comprising about 4 equivalents to about 8 equivalents of the alkylating agent, or such as about 4 equivalents, or such as about 5 equivalents, or such as about 6 equivalents.

41. The process of claim 1, wherein the alkylating agent is an alkyl halide such as an alkyl bromide, alkyl chloride, or alkyl iodide.

42. The process of claim 1, wherein the alkylating agent is an alkyl sulfonate such as an alkyl tosylate, alkyl mesylate, or alkyl triflate.

43. The process of claim 1, wherein the alkylating agent comprises 1 to 24 carbon atoms.

44. The process of claim 1, wherein the alkylating agent is branched.

45. The process of claim 1, wherein the alkylating agent is linear.

46. The process of claim 1, comprising at least about 4 equivalents of the alkylating agent, about 1 equivalent of the phenolic ester, and at least about 6 equivalents of the base.

47. The process of claim 1, further comprising reducing the alkylated phenolic ester to form a reduced phenol.

48. The process of claim 46, wherein the reduced phenol is of Formula (III):

49. The process of claim 46, wherein the reduced phenol is of Formula (III-1), (III-2), or (III-3):-10889 | 103241.00749050. The process of claim 46, wherein the reduced phenol is:.

51. The process of claim 49, wherein the reducing is performed using a reducing agent that reduces an ester to an alcohol, such as lithium aluminum hydride.

52. The process of claim 49 or 50, wherein the reducing is performed at reduced temperature, such as about 0 to about 24°C.

53. The process of claim 15, wherein the reduced phenol is esterified to form an esterified phenol.

54. The process of claim 52, wherein the esterified phenol is of Formula (IV):-10889 | 103241.007490wherein, X1is Cl, Br, or F.

55. The process of claim 52, wherein the esterified phenol is of Formula (IV-1), (IV- 2), or (IV-3):

56. The process of claim 52, wherein the esterified phenol is:.

57. The process of claim 52, wherein the esterified phenol is:-10889 | 103241.007490.

58. The process of claim 52, wherein the esterification is performed using X1-C1-8alk- C(O)-X2, wherein X1and X2are, independently, Cl, Br, or F.

59. The process of claim 52, wherein the esterification is performed at reduced temperature, such as about 0 to about 23°C.

60. The process of claim 52, further comprising coupling the ester group of the esterified phenol with HY1-C1-8alk-OH, wherein Y1is a chemical moiety comprising two nitrogen atoms.

61. The process of claim 59, wherein the coupling forms a compound of Formula (V) or (V-A):wherein: Y1is -optionally substituted C3-8cycloalk-, -optionally substituted heterocycloalk-, -optionally substituted heteroar-, or -optionally substituted ar-; and Y2is OH, NH2, -NH(C1-6alkyl), -NH(C1-6alkyl)2, optionally substituted C1-6alkyl, optionally substituted C1-6alkoxy, optionally substituted C3-8cycloalkyl,-10889 | 103241.007490 substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

62. The process of claim 59, wherein the coupling forms a compound of Formula (V- 1), (V-2), or (V-3):

63. The process of claim 59, wherein the coupling forms the following compound:.

64. The process of claim 59, wherein the coupling forms the following compound:-10889 | 103241.007490.

65. The process of claim 59, wherein Y1is a 5-8-membered heterocycloalkyl comprising two nitrogen atoms.

66. The process of claim 59, wherein Y1is piperazinyl.

67. A compound prepared according to the process of claim 1.

68. A composition comprising the compound of claim 26 and a ribonucleic acid.

69. The composition of claim 67, wherein the nucleic acid is mRNA, or such as Luc- mRNA.

70. A method for delivering a nucleic acid to a cell in a patient, comprising administering the composition of claim 67 to the patient.

71. The method of claim 69, wherein the cell is a tissue, such as lung tissue, liver tissue, spleen tissue, or a lymph node.

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