Key words steroids - PET - fluorination - cholesterol - adrenal
The scintiscanning agent 6-(iodomethyl)-19-norcholest-5(10)-en-3-ol (NP-59) was first
reported in 1975 as part of an effort to develop a cholesterol analogue for imaging
diseases associated with the adrenal glands such as Cushing’s syndrome, aldosteronism,
and identification of adrenal remnants following adrenalectomy procedures.[1 ] NP-59 was identified as an impurity in the preparation of 19-iodocholesterol.[2 ] It was discovered that 19-iodocholesterol, upon heating as part of the isotopic
exchange reaction to incorporate iodine-131, would rearrange to give NP-59. Adrenal
uptake of NP-59 was greater with a better tissue to background ratio compared to 19-iodocholesterol,
and NP-59 showed improved stability to deiodination.
Interest in utilizing NP-59 for cortical adrenal imaging has continued with efforts
made to improve the agent by using alternate iodine isotopes to prepare NP-59 for
use in single-photon emission computed tomography (SPECT) imaging (123 I, 125 I)[3 ] or in positron emission tomography (PET) imaging (124 I).[4 ] [131 I]NP-59 is limited to scintigraphy and SPECT methods that have lower spatial resolution
than PET, which limits the diagnostic utility of the agent. The PET imaging agent
has the benefit of coincidence detection for better resolution but is limited by the
low positron output of iodine-124 (124 I decays by β+ 26% vs. 18 F, 97%) leading to noise that lowers image quality, and requires undesirably high
radiation dosimetry to the patient. A fluorine-18 analogue will improve the imaging
characteristics, by providing a PET imaging cholesterol analogue with better spatial
resolution.
Additionally, NP-59 has a relatively long biological half-life, necessitating multiday
imaging protocols, where injection occurs on one day with the patient returning on
a later day for scanning, which is not ideal for the patient and limits the extent
of quantitation that can be performed with the imaging data. It is common for fluorine
analogues to have improved metabolic stability and other pharmacokinetic parameters.
For instance, the biological half-life of metaiodobenzylguanidine is estimated at
34 hours, whereas its fluorine analogue metafluorobenzylguanidine has a 2-hour biological
half-life.[5 ]
Reflecting these advantages as well as a wider interest in the use of radiofluorinated
steroids for imaging purposes,[6 ] there have been efforts for decades to prepare a fluorinated analogue of NP-59,
as well as the corresponding 18 F-isotopologue. However, common fluorinating reagents have overwhelmingly led to elimination
(e.g., cesium fluoride, (2-chloro-1,1,2-trifluoroethyl)diethylamine (FAR), diethylaminosulfur
trifluoride (DAST), and hexafluoropropene diethylamine (FPA)), ring expansion, rearrangement,
and other undesired products.[7 ] While other steroids prone to unwanted side reactions have been successfully fluorinated
with 1-butyl-3-methylimidazolium tetrafluoroborate, such as 7α-(fluoromethyl)dihydrotestosterone,
these methods gave low-single-digit yields of the fluorinated products.[8 ] Recently, the coordination of fluoride species with various alcohols, and the effects
of hydrogen bonding on their reactivity have given rise to milder fluorinating reagents,
which are less apt to produce unwanted byproducts. One of these alcohol coordinated
reagents, tetra-N -butylammonium fluoride bis-pinacol (TBAF(pinacol)2 ), proved particularly promising at producing primary fluorides while minimizing byproduct
formation.[9 ] However, this reagent was evaluated on only one test substrate, so it was necessary
to vet it on a series of model compounds prior to its application as the penultimate
step of a multistep steroid synthesis.
To determine the potential utility of TBAF(pinacol)2 , including for the fluorination of NP-59, a representative group of primary, secondary,
and tertiary tosylates and alkyl bromides were prepared. Each was then stirred with
TBAF(pinacol)2 at 70 °C in acetonitrile for 2 hours, and an extract was removed. To these extracts,
an equimolar amount of 4-fluorobenzonitrile was added as an internal standard, and
the conversion into the fluorinated product was determined by 19 F NMR spectroscopy. The process was then repeated with a second reaction, and extracts
were taken after 18 hours to determine the time dependency of the reactions (Table
[1 ]).
Table 1 TBAF(pinacol)2 Substrates and 19 F NMR Yieldsa
Entry
Substrate
Conversion (%)b
2 h
18 h
1
63
57
2
68
64
3
61
72
4
21
9
5
2
10
6
7
7
7
5
4
8
19
44
a Starting material (0.2 mmol) was dissolved in acetonitrile (0.8 mL), TBAF(Pinacol)2 (0.4 mmol) was then added. The reaction was heated at 70 °C for 2 or 18 hours.
b Non-isolated conversion determined by 19 F NMR spectroscopic analysis.
Comparing the substrates by their degree of substitution shows that TBAF(pinacol)2 performs best in the synthesis of primary fluorides (Table [1 ], entries 1–3), although secondary (entries 4–6) and tertiary (entries 7 and 8) fluorides
were also accessible, albeit with lower conversions into fluoride product. Alkyl bromides
showed higher conversions into product after 18 hours compared with 2 hours, whereas
there were no significant differences between 2- and 18-hour conversions when using
tosylates. This substrate scope study suggests tosylates are better leaving groups
for use with TBAF(pinacol)2 .
To prepare NP-59, we started from cholesterol (Scheme [1 ]). The synthesis of 1 –4 was conducted according to reported procedures, with some optimization for scale
and time.[10 ] Cholesterol was protected at the 3-position by treating it with acetic anhydride
in the presence of pyridine to give 1 , the acetylated intermediate. Compound 1 was then stirred with N -bromoacetamide under acidic conditions under foil to block light to give bromohydrin
2 . Compound 2 was heated with lead tetraacetate and iodine to give 3 , the cyclized intermediate, which was then treated with zinc powder in acetic acid
to give alcohol 4 . Intermediate 4 was then treated with p -toluenesulfonsyl chloride in the presence of dimethylaminopyridine to give tosylate
5 . While there are various methods for accessing NP-59 from protected tosylate intermediate
5 , we expected that deprotection and a subsequent one-step iodination/rearrangement
would be the most straightforward and reliable.[2 ]
[11 ] Thus, compound 5 was deprotected at the 3-position by stirring it in a solution of K2 CO3 to yield 6 , which was immediately heated with KI to promote the iodination/rearrangement reported
by Maeda and colleagues.[2b ] Analysis showed that, after 7 h, the product was approximately a 1:1 mixture of
the unrearranged 19-iodocholesterol and NP-59. As such, the mixture was resuspended
in MeCN and heated for an additional 2 h to give only NP-59.
Scheme 1 Synthesis of NP-59 from cholesterol
Lastly, we investigated the conversion of NP-59 into FNP-59 (Scheme [2 ]). To produce the intermediate for fluorination, NP-59 was initially protected as
the acetate at the 3-position by treating it with acetic anhydride in the presence
of 4-(dimethylamino)pyridine to form 7 . We initially explored whether treating 7 directly with TBAF(pinacol)2 could produce the desired product, but this resulted in a complex mixture. Therefore,
7 was instead heated with AgOTs to yield 8 . In the penultimate step 8 was heated with TBAF(pinacol)2 in acetonitrile to give 9 , the protected fluoride, in 67% yield. Treatment of 9 with K2 CO3 in a mixture of MeOH/CH2 Cl2 (1:1) yielded FNP-59.
In summary, an updated synthesis of NP-59, along with spectroscopic characterization
of all intermediates has been conducted. NP-59 was then converted into FNP-59 via
a four-step synthesis in an overall yield of 16% using TBAF(pinacol)2 in the key fluorination step. With FNP-59 in hand, toxicity studies are under way,
and a method for the radiosynthesis of [18 F]FNP-59 is being developed.
Scheme 2 Synthesis of FNP-59 from NP-59
