Microwave-assisted Organic Synthesis of a High-affinity Pyrazolo-pyrimidinyl TSPO Ligand

Abstract

We herein report a dramatically improved total synthesis of the high-affinity translocator protein (TSPO) ligand DPA-714, featuring microwave-assisted organic synthesis (MAOS). Compared with previously described approaches, our novel MAOS method dramatically reduces overall reaction time without adversely effecting reaction yields. We envision that the described MAOS protocol may be suitably applied to high-throughput, diversity-oriented synthesis of novel compounds based on the pyrazolo-pyrimidinyl scaffold. Such an approach could accelerate the development of focused libraries of novel TSPO ligands with potential for future development as molecular imaging and therapeutic agents.

Translocator protein (TSPO), formerly known as the peripheral benzodiazepine receptor (PBR), is an 18kDa outer-mitochondrial membrane protein that participates in the regulation of numerous cellular processes, including cholesterol metabolism, steroid biosynthesis, proliferation and apoptosis.^1a–c Under normal circumstances, TSPO expression tends to be highest in steroid producing tissues and mitochondrial enriched tissues such as skeletal muscle, kidney and heart. TSPO expression is also elevated in disease states such as neuroinflammation and cancer.^2a–c For this reason, TSPO is regarded as a potentially important target for drug and molecular imaging probe development. Our previous research involving TSPO resulted in the development of several labeled TSPO ligands for in vitro and in vivo fluorescence imaging and high-throughput screening applications.3a–e Presently, a diverse array of TSPO-targeted probes has been developed and radiolabeled with positron emitting isotopes such as carbon-11 and fluorine-18 for positron emission tomography (PET) imaging.⁴ One such compound, [¹⁸F]DPA-714 (N,N-diethyl-2-(2-(4-(2-[¹⁸F])fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide, is a high-affinity TSPO ligand that has shown promise for PET imaging of neuroinflammation.5a,5b Previous publications report total syntheses of this compound and its tosylate precursor, suitable for radiolabeling with fluorine-18, that incorporate lengthy, multi-step schema.^6a–d Our interest in evaluating pyrazolo-pyrimidinyl compounds as TSPO imaging ligands, such as [¹⁸F]DPA-714, led us to explore methodologies applicable to rapid, high-throughput synthesis of this compound and potentially novel analogues thereof. To this end, we have evaluated microwave assisted organic synthesis (MAOS) as an approach to accelerate the synthesis of DPA-714 and similar compounds. Compared with previously reported approaches, this study illustrates that MAOS applied to each reaction step dramatically reduces the overall time required to prepare the compound while achieving comparable, or in most cases, superior synthetic yields. According to the authors, this is the first MAOS-facilitated synthesis of DPA-714 and accordingly, this methodology can be extended to the synthesis of similar pyrazolo-pyrimidinyl-based compounds. As evidence of this, we illustrate application of MAOS to several additional pyrazolo-pyrimidinyl synthetic intermediates with good utility.

The overall synthetic methodology utilized in this study is shown in Scheme 1. Though the synthesis reported here utilized starting materials similar to previous methods, those syntheses were carried out either at room temperature or with conventional thermal heating. By applying MAOS to each step as further described below, the overall reaction time could be reduced from over 50 hours to approximately three, while maintaining consistently high yields. Full synthetic methodology and characterization data can be found in Supplementary Data.

Scheme 1.

Microwave-assisted organic synthesis of DPA-714 (7) and the tosylate precursor (8).

1. MAOS of 3-cyano-N,N-diethyl-4-(4-methoxyphenoyl)-4-oxobutanamide (3)

The consecutive five-step synthesis begins with formation of 3 by reaction of 3-(4-methoxyphenyl)-3-oxopropanenitrile (1) and 2-chloro-N,N-diethylacetamide (2) in 80% EtOH, NaI/KI and NaOH. Previous studies carried out this reaction at either room temperature or reflux over 8 h with reported yields ranging from 10 – 70%.6a,6b To reduce the reaction time required, microwave irradiation provided a reaction temperature of 80 °C. Brief irradiation for 40 min and flash chromatography on silica gel gave 3 with a slightly higher yield when compared with the best previously reported method.^6b (Table 1)

Table 1.

Synthesis of 3-cyano-N,N-diethyl-4-(4-methoxyphenoyl)-4-oxobutanamide (3).

Entry	MAOS	Reaction Conditions	Temperature	Time	Yielda
16a	No	80% EtOH, NaI, NaOH	RT	8 h	10%
26b	No	80% EtOH, NaI, NaOH	RT	8 h	64%
3	Yes	80% EtOH, KI, NaOH	80 °C	40 min	70%

^aIsolated yield. See reference.6a,6b

2. Synthesis of 2(3-amino-5-(4-methoxyphenyl)-1H-pyrazol-4-yl)-N,N-diethylacetamide (4)

Synthesis of 4 features pyrazolo ring formation from the reaction of 3 with hydrazine. According to previous reports, this reaction can be carried out at room temperature or with traditional thermal heating for 4 – 6 h in ethanol/acetic acid to achieve yields ranging from 68 – 72%.6a,6b In this study, this reaction could be adapted to MAOS via microwave irradiation at 90 °C for 40 min, albeit with a somewhat lower yield (42%) than previous studies (Table 2). Efforts to increase the reaction yield above 42% while maintaining the rapid 40 min reaction time included elevated temperature (> 90 °C) and hydrazine concentration. However, these conditions resulted in increased byproduct formation and further diminished synthetic yields (data not shown). Despite the slightly reduced yield, the significantly increased speed with which this reaction could be performed using MAOS is a considerable advantage over traditional methods.

Table 2.

Synthesis of 2-(3-amino-5-(4-methoxyphenyl)-lH-pyrazol-4-yl)-N,N-diethylacetamide(4).

Entry	MAOS	Reaction Conditions	Temperature	Time	Yielda
16a	No	EtOH, acetic acid	RT	6 h	72%
26b	No	EtOH, acetic acid	Reflux	4 h	68%
3	Yes	EtOH, acetic acid	90 °C	40 min	42%

^aIsolated yield. See reference.6a,6b

3. MAOS of N,N-diethyl-2-(2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a] pyrimidin-3-yl) acetamide (5, DPA-713)

In the third step, 5 (DPA-713) was synthesized from the reaction of 4 and 2,4-pentanedione in ethanol. Previous studies have reported carrying out this reaction at room temperature or with conventional thermal heating.6a,6b Optimization with microwave irradiation resulted in final reaction conditions of 160 °C in ethanol for 30 min. Purification by flash chromatography on silica gel gave 5 in near quantitative yield (91%), comparable to the best previously reported synthesis while requiring only a fraction of the reaction time.^6b (Table 3)

Table 3.

Synthesis of N,N-diethyl-2-(2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[l,5-a] pynmidin-3-yl) acetamide (5, DPA-713).

Entry	MAOS	Reaction Conditions	Temperature	Time	Yielda
16a	No	EtOH	RT	4 h	44%
26b	No	EtOH	Reflux	12 h	93%
3	Yes	EtOH	160 °C	30 min	91%

^aIsolated yield. See reference.6a,6b

4. MAOS of N,N-diethyl-2-(2-(4-hydroxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl) acetamide (6)

The fourth reaction in the synthesis of DPA-714 features deprotection of 5, performed using either BBr₃ in CH₂Cl₂ at −60 °C or with aqueous HBr under conventional thermal conditions.^6b,6d To optimize this reaction with MAOS, examination of various reaction temperatures ranging from 100 to 140 °C demonstrated that temperatures > 120 °C resulted in byproduct formation. However, irradiation at 110 °C for 40 min in aqueous HBr with addition of hexadecyl tributyl phosphonium bromide (HTPB) proved successful. Compared to previously reported methods,^6b,6d this approach yielded 6 in higher yield (79%), with a significantly reduced reaction time (40 min versus 2 – 7 h) (Table 4).

Table 4.

Synthesis of N,N-diethyl-2-(2-(4-hydroxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyTimidin-3-yl) acetamide (6).

Entry	MAOS	Reaction Conditions	Temperature	Time	Yielda
16b	No	HTPB, HBr, H2O	100 °C	7 h	54%
26d	No	BBr3, CH2Cl2	-60 °C	2 h	55%
3	Yes	HTPB, HBr, H2O	110 °C	40 min	79%

^aIsolated yield. See reference.6b,6d

5. MAOS of N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl) acetamide (7, DPA-714)

From compound 6, reported syntheses of 7 describe reactions carried out at room temperature with NaH in dry THF for 16 h with yields ranging from 60 – 80%.^6c,6d Employing microwave irradiation, an optimized reaction temperature of 120 °C was achieved with a subsequent reduction in reaction time from 16 h to 15 min, a factor of 64, while still achieving a comparable yield (79%) (Table 5)

Table 5.

Synthesis of N,Ndiethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[l,5-a]pyrimidin-3-yl)acetamide (7, DPA-714).

Entry	MAOS	Reaction Conditions	Temperature	Time	Yielda
16c	No	NaH, THF	RT	16 h	80%
26d	No	NaH, THF	RT	16 h	58%
3	Yes	NaH, THF	120 °C	15 min	79%

^aIsolated yield. See reference.6c,6d

6. MAOS of 2-(4-(3-(2-(diethylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-2-yl)phenoxy)ethyl-4-methylbenzenesulfonate (8), a precursor for radiosynthesis of [¹⁸F]DPA-714

In order to produce ¹⁸F-labeled DPA-714 as a PET tracer, 8 is commonly prepared as the precursor. Previous syntheses of this compound were carried out at room temperature in high yield.^6c,6d However, the prolonged reaction time reported (16 h) is a considerable disadvantage. We found that this reaction could be optimized using microwave irradiation for 30 min at 120 °C. Following purification, we obtained 8 in a yield of 65%, comparable to previous reports (Table 6).

Table 6.

Synthesis of 2-(4-(3-(2-(diethylamino)-2-oxoethyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-2-yl)phenoxy)ethyl 4-methylbenzenesulfonate (8).

Entry	MAOS	Reaction Conditions	Temperature	Time	Yielda
16c	No	NaH, THF	RT	16 h	59%
26d	No	NaH, THF	RT	16 h	77%
3	Yes	NaH, THF	120 °C	30 min	65%

^aIsolated yield. See reference.6c,6d

Extending the utility of MAOS to the synthesis of additional pyrazolo-pyrimidinyl-based compounds, we performed the first and third reactions shown in Scheme 1 using a variety of different reagents, mimicking a library-based synthetic approach. For example, substitution of 1 with the corresponding p-chloro or p-methyl reagent results in rapid synthesis of 3a and 3b in acceptable yields (Table 7). Similarly, application of MAOS to reactions featuring unique diones also appears feasible, enabling synthesis of potential TSPO ligands following the third reaction (Table 8). Interestingly, in evaluating somewhat bulkier diones, we observed significantly reduced reaction yield, presumably due to the geometric constraints afforded by groups in R₁, R₂ and R₃. We anticipate that synthetic optimization of the bulkier pyrazolo-pyrimidines is possible, though beyond the scope of this focused work.

Table 7.

Synthesis of pyrazolo-pyrimidine intermediates.

Entry	Product	Yield (%)a
3a		42
3b		40

^aIsolated yield.

Table 8.

Synthesis of pyrazolo-pyrimidines.

Entry	R1	R2	R3	Temperature (°C)	Time (min)	Yield (%)a
5a	-CH3	-CH3	-CH3	160	30	77
5b	-CH3	-CH2CH3	-CH3	160	30	24
5c	-CH(CH3)2		-CH(CH3)2	180	45	6.0
5d	-Ph		-Ph	180	45	5.0

^aIsolated Yield.

In summary, a detailed optimization of the total synthesis of a high-affinity TSPO ligand, DPA-714, utilizing MAOS, is described. The protocol reported here significantly improves overall reaction times while maintaining or even improving synthetic yields. We envision that this protocol can be extended to library synthesis of novel TSPO ligands with potential use for noninvasive visualization of TSPO expression in vivo as well as treatment of TSPO-expressing disease.