Multistep Synthesis of 1,2,4-Oxadiazoles via DNA-Conjugated Aryl Nitrile Substrates

Abstract

A multistep protocol for the synthesis of 3,5-disubstituted 1,2,4-oxadiazoles on DNA-chemical conjugates has been developed. A set of six DNA-connected aryl nitriles were converted to corresponding amidoximes with hydroxylamine followed by the O-acylation with a series of aryl and aliphatic carboxylic acids. After cyclodehydration of the O-acyl amidoximes by heating at 90 °C in pH 9.5 borate buffer for two hours, the desired oxadiazole products were observed in 51─92% conversion with the cleavage of O-acylamidoximes as the major side-product. The reported protocol paves the way for the synthesis of oxadiazole core-focused DNA-encoded chemical libraries.

Graphical Abstracr

INTRODUCTION

DNA-encoded chemical libraries (DECLs) are collections of small molecules covalently linked to a unique, structure-identifying DNA tag which enables screens of large pools of library members for binders to biological targets.^1–7 DECLs constructed by a split-and-pool⁸ strategy (i.e., iterative rounds of molecule functionalization, DNA-tag ligation and pooling) may routinely contain millions of encoded small molecule structures, and if used with post-screen high-throughput DNA sequencing,^9–11 can interrogate enormous amounts of chemical space.¹² However the success of DECL screening campaigns is influenced by the inherent diversity and fidelity of small molecule structures available within the DECL collection^13,14 and many common synthetic transformations have not yet been demonstrated in solution-phase DECL production.^15,16 This is due in part to stability and solubility limitations imposed by the presence of DNA within partially aqueous medium,^13,14 and thus adaptation of synthetic transformations to DECL-friendly conditions is needed to further enhance the diversity of future DECL productions.

Oxadiazoles represent a privileged class of heterocycles that are frequently featured in discovery screening collections,^17,18 are present in several approved pharmaceutical drugs,^19,20 and have robust solution- and solid-phase synthetic methods.^21–31 In particular, the on-DNA formation of 1,2,4-oxadiazoles is an attractive DECL combinatorial methodology as 1,2,4-oxadiazoles may be constructed from nitriles and carboxylic acids, compound building block sets that have wide commercial-availability and diversity.³² A typical route to 1,2,4-oxadiazoles is through a three step process in which a nitrile 1 is converted to amidoxime 2 with hydroxylamine, coupled with a carboxylic acid to form O-acylamidoxime 3, and cyclodehydrated to the 1,2,4-oxadizole 4 (Figure 1).

Figure 1.

General synthetic route of 1,2,4-oxadiazoles in conventional chemistry.

RESULTS AND DISCUSSION

To the best of our knowledge, this transformation has not been demonstrated on DNA-chemical conjugates aimed towards use in a DECL production. This may be partly due to presumed difficulties associated with the ultimate dehydration step in aqueous medium, as well as potential competing hydrolysis of synthetic sequence intermediates.^33,34 To address these concerns, we initiated studies on the formation of 1,2,4-oxadiazoles from six DNA-attached aryl nitriles 1a–1f connected to a DNA construct through common functional handle transformations. We chose to utilize a DNA-connected nitrile rather than carboxylic acid to enable direct observation of all steps on a DNA-conjugate, to avoid the need to pre-form amidoximes, and to allow use of general carboxylic acid stocks as this is a common building block for many DECL designs.³² Nitriles may be converted to amidoximes through attack of hydroxylamine under basic conditions³⁵ and a survey of basic buffers (e.g. borate, phosphate) and bases (e.g., Na₂CO₃, NaOAc, Cs₂CO₃, NaOH, N,N-Diisopropylethylamine, triethylamine) generally showed comparable conversions of nitriles 1 to the amidoxime 2, albeit with prolonged reactions times (see Supporting Information for full details). Although heating could shorten the required reaction time, this led to mild decomposition of the DNA-chemical conjugates through formation of hydroxylamine DNA adducts and was avoided.³⁶ Ultimately, use of pH 8.2 borate buffer with addition of a small amount of Na₂CO₃ cleanly provided the amidoximes 2a–2f in excellent yield (Scheme 1).

Scheme 1.

Amidoxime formation from DNA-conjugated nitriles.

^aConversions determined by LC-MS.

With the on-DNA amidoximes in hand, we next sought to find conditions that enabled the formation of the O-acylamidoximes 3, using amidoxime 2b and benzoic acid as a model system. Unfortunately, use of water-soluble DMTMM, a common coupling agent in DECL synthesis,³⁷ resulted in negligible formation of the O-acylamidoxime 3b1. In other DECL studies, we have found DEPBT cleanly provides amidation products if the carboxylic acid is preactivated with coupling agent and Hunig’s base in acetonitrile.³⁸ Application of this preactivation condition to 2b in several different buffers all provided 3b1 in modest yield. Previously EDC/HOAt has been described as an efficient reagent system for this transformation³⁹ and applying this reagent combination provided 3b1 in 72–75% conversion (Table 1, entries 5–7). Switching to HATU under preactivation conditions further enhanced the conversion to 85–93% (Table 1, entries 8–10) but ultimately use of PyAOP without preactivation proved superior, providing 3b1 in excellent yield with pH 8.0 buffer and a slightly lessened yield with pH 9.5 buffer (Table 1, entries 11–12).

Table 1.

Optimization of o-acylation of amidoxime 2b.

entry	buffer	coupling reagent	3b1a (%)
1	pH 8.0 phosphate	DMTMMb	< 5%
2c	pH 8.0 phosphate	DEPBTd	61%
3c	pH 8.2 borate	DEPBT	54%
4c	pH 9.5 borate	DEPBT	63%
5c	pH 8.0 phosphate	EDCe/HOAtf	75%
6c	pH 8.2 borate	EDC/HOAt	72%
7c	pH 9.5 borate	EDC/HOAt	74%
8c	pH 8.0 phosphate H	HATUg	85%
9c	pH 8.2 borate	HATU	90%
10c	pH 9.5 borate	HATU	93%
11	pH 8.0 phosphate	PyAOPh	95%
12	pH 9.5 borate	PyAOP	85%

^aConversions determined by LC-MS.

^b4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride.

^cCarboxylic acid was preactivated by mixing with DIPEA (200 equiv) and coupling agent (200 equiv) in CH₃CN for 15 min at 25 °C prior to use.

^d3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one.

^e1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide.

^f1-Hydroxy-7-azabenzotri-azole.

^g1-[Bis-(di-methylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate.

^h(7-Azaben-zotriazol-1-yloxy) tripyrrolidinophophonium hexafluorophosphate.

Using the optimal PyAOP/pH 8.0 condition (Table 1, entry 11), we investigated the O-acylation of amidoximes 2a–2f with a range of aryl and aliphatic carboxylic acids (Table 2). Gratifyingly, nearly all of the O-acylamidoximes were formed in very good to excellent conversion, with only slightly decreased conversions observed with electron-deficient amidoxime nucleophiles 2d–2f. Notably, both aryl and aliphatic carboxylic acids could be coupled, including electron deficient pyridyl carboxylic acids and sterically-encumbered secondary and tertiary aliphatic carboxylic acids (Table 2). With this collection of O-acylamidoximes, we next turned towards optimizing the final cyclodehydration step of amidoxime formation on the model substrate 3b1. Typically, this cyclodehydration has been performed under basic, anhydrous, aprotic conditions at prolonged elevated temperature.²⁶ Although stable under ambient conditions,⁴⁰ heating O-acylamidoximes 3 in basic buffers may induce hydrolytic cleavage or elimination to ultimately provide a mixture of unreacted acylamidoxime 3, desired oxadiazole 4, amidoxime 2, and cyanide 1.

Table 2.

Substrate scope of o-acylation with carboxylic acids.^a

	2a	2b	2c	2d	2e	2f
1	3a1: 94%	3b1: 95%	3c1: 95%	3d1: 92%	3e1: 99%	3f1: 92%
2	3a2: 97%	3b2: 98%	3c2: 95%	3d2: 92%	3e2: 99%	3f2: 90%
3	3a3: 94%	3b3: 95%	3c3: 94%	3d3: 92%	3e3: 98%	3f3: 96%
4	3a4: 89%	3b4: 96%	3c4: 94%	3d4: 90%	3e4: 96%	3f4: 88%
5	3a5: 91%	3b5: 97%	3c5: 95%	3d5: 84%	3e5: 97%	3f5: 81%
6	3a6: 90%	3b6: 99%	3c6: 94%	3d6: 89%	3e6: 98%	3f6: 87%
7	3a7: 81%	3b7: 90%	3c7: 94%	3d7: 88%	3e7: 90%	3f7: 73%
8	3a8: 97%	3b8: 97%	3c8: 96%	3d8: 94%	3e8: 99%	3f8: 91%
9	3a9: 97%	3b9: 99%	3c9: 94%	3d9: 93%	3e9: 98%	3f9: 93%

^aConversions determined by LC-MS.

Our initial tests concentrated on the use of pH 9.5 borate buffer at elevated temperatures, as we have found this to be an optimal basic buffer system that maintains DNA and amide bond integrity for other reactions which require prolonged heating.⁴¹ Use of this buffer alone or with a variety of additives produced the desired oxadiazole in fair yields, although with significant amounts of hydrolysis product 2b (Table 3, entries 1–6). Lowering the starting pH of the buffer system suppressed hydrolysis to 2b but led to large amounts of unreacted 3b even after 16 h (Table 3, entries 7–8). Increasing the temperature to 80 °C in pH 9.5 borate buffer with N,N-diisopropylethylamine as an additive⁴² boosted formation of 4b to 83% after only 1.5 h, and ultimately application of the same system at 90 °C resulted in slightly improved conversion to the desired oxadiazole (Table 3, entries 9–10). Encouraged by this result, we applied this cyclodehydration condition to our collection of O-acylamidoximes 3a1–3f9 which produced all oxadiazoles 4a1–4f9 in fair to very good yields (Table 4). Although lowered conversion and significant hydrolysis was observed for all electron-deficient pyridyl substrates 3d1–3d9, overall a variety of electronically-diverse aryl and alkyl substituted oxadiazoles were prepared through this multistep protocol.

Table 3.

Optimization of cyclodehydration of acylamidoximes conditions.

entry	buffera	baseb	temperature	time	4b1c (%)	2bc (%)	3b1c (%)	1bc (%)
1	pH 9.5 borate	None	60 °C	16 h	54%	28%	9%	Trace
2	pH 9.5 borate	NaOAcd	60 °C	16 h	53%	26%	10%	Trace
3	pH 9.5 borate	Cs2CO3e	60 °C	16 h	59%	24%	9%	Trace
4	pH 9.5 borate	NaHCO3f	60 °C	16 h	54%	25%	10%	Trace
5	pH 9.5 borate	Et3Ng	60 °C	16 h	57%	23%	20%	Trace
6	pH 9.5 borate	DIPEAh	60 °C	16 h	60%	27%	13%	Trace
7	pH 8.2 borate	DIPEA	60 °C	16 h	52%	11%	37%	Trace
8	pH 8.0 phosphate H	DIPEA	60 °C	16 h	53%	8%	39%	Trace
9	pH 9.5 borate	DIPEA	80 °C	1.5 h	83%	17%	Trace	Trace
10	pH 9.5 borate	DIPEA	90 °C	1.5 h	86%	14%	Trace	Trace

^a500 equiv used.

^b100 equiv used.

^cConversions determined by LC-MS.

^dSodium acetate.

^eCesium carbonate.

^fSodium bicarbonate.

^gTriethylamine.

^hN,N-diisopropylethylamine.

Table 4.

The synthesis of 1,2,4-oxadiazoles via cyclodehydration of acylamidoximes.^a,b

R1=
Products
	4a1: 79%	4a2: 69%	4a3: 69%	4a4: 66%	4a5: 82%	4a6: 69%	4a7: 78%	4a8: 79%	4a9: 65%
	4b1: 82%	4b2: 78%	4b3: 77%	4b4: 74%	4b5: 82%	4b6: 71%	4b7: 75%	4b8: 87%	4b9: 69%
	4c1: 89%	4c2: 86%	4c3: 81%	4c4: 83%	4c5: 92%	4c6: 81%	4c7: 70%	4c8: 88%	4c9: 79%
	4d1: 68%	4d2: 62%	4d3: 60%	4d4: 63%	4d5: 67%	4d6: 56%	4d7: 53%	4d8: 61%	4d9: 51%
	4e1: 81%	4e2: 77%	4e3: 76%	4e4: 77%	4e5: 85%	4e6: 74%	4e7: 75%	4e8: 78%	4e9: 71%
	4f1: 86%	4f2: 84%	4f3: 80%	4f4: 85%	4f5: 92%	4f6: 80%	4f7: 81%	4f8: 87%	4f9: 72%

^aConversions determined by LC-MS.

^bTrace amount to 10% of cyanides 1a─1f were also observed.

Finally to further corroborate the formation of the DNA-conjugated oxadiazole product, we sought to compare the synthesis of amide-bound 1,2,4-oxadizole ataluren derivative 6b prepared through our four-step process with the ataluren derivative 6a prepared through direct acylation of commercially-obtained ataluren (Scheme 2). Formation of 6a and 6b proceeded smoothly through both synthetic routes and both were found to have identical retention on LC-MS during co-injection studies. To simulate the late-stage use of this process in a DECL production, these studies were performed on elongated DNA 5 (56 b.p.). No DNA degradation was detected and post-reaction 6b efficiently ligated with a duplexed pair of 12 b.p. DNA oligomers (see Supporting Information for full details).

Scheme 2.

(a) Synthesis of DNA-conjugate 6a via acylation with ataluren. (b) Multistep synthesis of 1,2,4-oxadiazole 6b and enzymatic ligation.

CONCLUSIONS

In summary, we have demonstrated an efficient multistep synthesis of diverse 3,5-disubstituted 1,2,4-oxadiazoles on DNA-chemical conjugates. DNA-conjugated aryl nitriles were converted to 1,2,4-oxadiazoles through a four step process featuring conversion to the amidoxime, amidoxime O-acylation with a variety of aryl and aliphatic carboxylic acids, and cyclodehydration of O-acylamidoxime intermediates. This protocol produced a variety of electronically- and sterically-substituted oxadiazoles, utilizes building blocks that are widely commercially-available, and preserves the integrity of DNA. Efforts to apply this method within a full-scale DECL synthesis are ongoing and will be reported in due course.