A Synthesis of 6-(2,5-Dimethoxy-4-(2-aminopropyl)phenyl)- hexylthiol. A Ligand for Conjugation with Fluorescent Cadmium Selenide/Zinc Sulfide Core/Shell Nanocrystals and Biological Imaging.

The synthesis of 6-(2,5-dimethoxy-4-(2-aminopropyl)phenyl)hexylthiol, an agonist with a very high affinity for the 5HT2A serotonin receptor subtype is reported. This agonist was designed to be attached to highly fluorescent cadmium selenide/zinc sulfide core/shells via a thiol at the end of a linker arm. This conjugate has applications in biological assays and biological imaging.


Introduction
When a narrow band gap semiconductor nanocrystal (or quantum dot) is coated with a shell of a wider band gap semiconductor, a new material, a core/shell nanocrystal, is produced. An example are CdSe/ZnS core/shell nanocrystals [1]. These core/shells have novel physical characteristics, one of the most important being size-tunable, narrow fluorescence emission bands. The fluorescence emission is a result of radiative recombination of the quantum confined electron-hole pair within the CdSe core. The core ranges in diameter from 18Å to 70Å and controls the emission wavelength. Small cores emit in the blue, while large cores emit in the red. The shell is several monolayers thick and serves both to passivate dangling bonds on the core surface (these act as traps for the electron and hole and reduce the fluorescence quantum yield [2]) and to confine the photoexcited electron-hole pair to the core. Fluorescent CdSe/ZnS core/shells offer many distinct advantages over conventional dye molecules. They exhibit greater photostability and are not easily photobleached. Their quantum yields are comparable to or greater than organic dyes, and their absorption spectra are continuous above the first excitation feature, enabling all sizes of nanocrystals, and hence all colors, to be excited with a single excitation source. We are involved in utilizing the optical properties of core/shell nanocrystals for both static and dynamic fluorescent imaging of biological systems. Several groups have demonstrated that proteins and antibodies may be attached to core/shells [3][4][5], and these nanoconjugates have been shown to be capable of labeling specific cellular components. The narrow emission spectra of core/shell nanocrystals enables multiplexing experiments, where different biomolecules or drugs conjugated to different sizes of quantum dots can target multiple cellular components, and their distribution and dynamics can be visually observed. There is considerable effort being invested in optimizing fluorescent quantum dots for biological labeling systems [6]. It is hoped that such systems may find applications in genomics, proteomics, and high-throughput screening [7] Our group is interested both in compounds that have biological activity in the central nervous system (CNS) and in synthesizing drug-core/shell conjugates that can be used to image receptor proteins, ion channels, and transporter proteins in neurons. This paper describes the synthesis and characterization of a ligand that has high affinity for the 5HT 2A and 5HT 2C serotonin receptors. [8] These receptors are G protein coupled receptors [9], and 5-HT 2A receptors have been linked to a wide range of behaviors and physiological functions including sleep, memory, hallucinations, anxiety, and aggression, as well as psychotic and affective diseases such as schizophrenia and unipolar depression. Our synthetic strategy is based upon attaching a flexible linker arm to the drug and attaching the other end to Zn atoms of the nanocrystal via a thiol group. We have already demonstrated that similar nanoconjugates are biologically active and can be used to image serotonin transporter proteins transfected in HEK cells [10]. 1-(2-Aminopropyl)-2,5-dimethoxybenzene ( Figure 1) has been shown to have a high affinity for the 5HT 2 receptors. Recent publications indicated that there is a region in the molecule where increasing steric bulk is tolerated, and substituents with a large steric bulk have little or no detrimental effect on its biological activity [8,11]. This position is para to the isopropylamine; consequently we decided to attach an alkyl chain to this position giving the ligand 6-(2,5-dimethoxy-4-(2-aminopropyl)phenyl)hexylthiol (14).

Conclusions
A synthetic route for the synthesis of 6-(2,5-dimethoxy-4-(2-aminopropyl)phenyl)hexylthiol (14) has been developed. This compound has been shown to be a full agonist with an EC 50 for the 5HT 2A receptor of 88 nM [13]. Conjugates of this compound with highly fluorescent cadmium selenide/zinc sulfide core/shells have been demonstrated to be biologically active and have been used in biological fluorescence studies [13].

Acknowledgements
We would like to thank Professor Ned Porter for a critical reading of this manuscript and Vanderbilt University for supporting this research in part by providing an intramural discovery grant. We would also like to acknowledge the National Institute of Health for provision of grant # 5R03191-161874-02.

General
Adipic acid, N-bromosuccinimide, potassium thioacetate, N,N-phthalimido-2-(5-hydroxy-1Hindole-3-yl)ethylamine, potassium thioacetate and triphenyl phosphine were purchased from Aldrich. 1,4-Dimethoxybenzene, aluminum chloride, and lithium aluminum hydride were purchased from Lancaster Synthesis. Thionyl chloride was purchased from ACROS. Reagents were used as they were received. Thin-layer chromatography was carried out on pre-coated plates, and the products were visualized using UV light. Column chromatography on silica refers to silica gel obtained from Scientific Adsorbents, Inc., catalogue number 02826-25. All NMR data was obtained using a Brucker 300 MHz machine with CDCl 3 as a solvent unless otherwise stated. Chemical shifts were measured in ppm relative to TMS and coupling constants are measured in Hz. GC-MS was performed on a Hewlett Packard 5890 series II gas chromatogram coupled to a Hewlett Packard 5971 mass spectroscope using electron impact as the ionization method. Low resolution mass spectra (MS) were obtained using a Finnegan Thermoquest TSQ 7000 triple quadrupole LC-MS equipped with an API-1 electrospray ionization source (ES). Samples for elemental analysis were routinely dried at ca. 10mmHg. Elemental analysis was performed by Atlantic Microlabs, Georgia, and the analysis is corrected for hydrates when necessary. (1) Adipoyl chloride (50 mL) and aluminum chloride (10g, 7.4 mmols) were dissolved in nitrobenzene (50 mL) and cooled to 0 o C. 1,4-Dimethoxybenzene (10g, 7.2 mmols) dissolved in nitrobenzene (50 mL) was added dropwise over a 3 hour period, during which the temperature was maintained below 5 o C. The resulting mixture was stirred for a further 2 hours at 0 o C, then crushed ice was added. The reaction mixture was allowed to warm to room temperature over an 18 hour period and the solution was filtered. The organic solution was separated and extracted into 3M sodium hydroxide solution (3 x 100 mL) and the aqueous solution was acidified to pH 1 using 4M hydrochloric acid. After extracting this solution with diethyl ether (3 x 200 mL) the ethereal extracts were combined and dried over magnesium sulfate. The solution was filtered and evaporated giving the crude product as a brown solid. It was purified by recrystallization from a mixture of ethyl acetate and hexane. This gave the product 11.4g (60%) as a colorless solid Rf = 0.38 (silica, 9:1 dichloromethane-methanol); m.p.  (2) 6-(2,5-Dimethoxyphenyl)-6-oxohexanoic acid (1, 4.2g, 160 mmols) was added to a mixture of methanol (100 mL) and concentrated sulfuric acid (2 drops). The solution was heated at reflux over a period of 18 hours with stirring. After cooling to room temperature the solution was evaporated and the crude product was dissolved in diethyl ether (100 mL). This was washed with saturated sodium bicarbonate (50 mL) and water (50 mL). It was dried over magnesium sulfate, filtered and evaporated. The product was purified using column chromatography on silica gel (dichloromethane elution). This Powdered zinc (22.5g) was added to a solution of mercuric chloride (940 mg) in concentrated hydrochloric acid (0.93 mL) and water (23.1 mL). This suspension was shaken for 5 minutes and the liquid was decanted. The amalgamated zinc was placed in a 500ml 3 necked flask and concentrated hydrochloric acid (12 mL) was added. The flask was heated to cause a gentle reflux and a solution of methyl-6-(2,5-dimethoxyphenyl)-6-oxohexanoate (2, 4.2g, 15 mmols) in methanol (7 mL) and concentrated hydrochloric acid (23 mL) was added dropwise [15]. The mixture was heated at reflux for 3 hours following the addition of compound 2, then filtered. The aqueous solution was extracted with diethyl ether (4 x 100 mL) and the combined ethereal extracts were washed with saturated sodium bicarbonate (50 mL) and water (50 mL). After drying over magnesium sulfate the solution was filtered and evaporated. The product was purified by column chromatography on silica gel (98:2 dichloromethane-methanol). This gave 1.35g (33%) of the product as a pale yellow oil; R f = 0.67 (silica, 98:2 dichloromethane-methanol); 1

Methyl 6-(2,5-dimethoxy-4-formylphenyl)hexanoate (4).
A mixture of phosphorus oxychloride (1mL) and N-methylformanilide (1.81 g) were allowed to incubate at room temperature for 30 minutes. Methyl-6-(2,5-dimethoxyphenyl)hexanoate (3, 1 g, 4 mmols) was added and the mixture was heated for 2 hours. After cooling to room temperature water (50 mL) was added and the mixture was left standing at room temperature for 18 hours. Then the solution was extracted with dichloromethane (2 x 100mL) dried over magnesium sulfate, filtered and evaporated. The resulting oil was leached with boiling hexanes (4 x 100mL) and the combined solutions were evaporated under reduced pressure [16]. Purification of the product was accomplished by column chromatography on silica gel (98:2 dichloromethane-methanol). This gave 0.4g (35%) of the product as a colorless solid;