Diastereoselective, Multicomponent Synthesis of Pyrrolopyrazinoquinazolinones via a Tandem Quinazolinone Rearrangement/ Intramolecular Ring Closure of Tautomeric (Z)-Benzamidines

Abstract

An expedient route to enantiopure, diastereomeric pyrrolopyrazinoquinazolinones was developed following the discovery of a domino quinazolinone rearrangement-intramolecular cyclization of N-H benzamidines. A Ugi-Mumm-Staudinger sequence employing an optically pure proline derivative gave quinazolinones that, upon N-Boc deprotection, rearranged to tautomeric Z-benzamidines. Subsequent spontaneous cyclization afforded 15 diastereomeric pyrazinoquinazolinone pairs in up to 83% overall yield and 89:11 d.r which were separated easily via routine chromatographic purification – the only one required in the entire process.

Graphical Abstract

Fungal metabolites featuring a pyrazino-[2,1-b]-quinazolinone framework represent a rich source of structural and pharmacological diversity (Fig. 1, blue motif).^{1, 2} For example, antimicrobial fumiquinazoline F 1 belongs to a class of more than 80 members that exhibits a broad range of bioactivities.^{1, 3} While aurantiomide B 2 shows moderate cytotoxic activity against multiple cancer cell lines,⁴ ardeemin 3 has been the subject of biological and synthetic studies due to its ability to block multidrug resistant tumor cell export pumps.^{5, 6} Recently, several antiviral pyrazinoquinazolinones, including quinadoline B 4, were identified as virtual hits against the novel coronavirus SARS-CoV2, the virus responsible for the worldwide COVID-19 pandemic.⁷ Access to these compounds and derivatives can be challenging due to complicated isolation or lengthy or low yielding synthetic approaches needed to incorporate chiral centers. Nonetheless, promising achiral derivatives also have been generated, highlighting the therapeutic potential of this framework. Wang and co-workers made pyrazinoquinazolinone 5 with potent in vivo activity against colon cancer,⁸ while pyrrolopyrazinoquinazolinones, exemplified by 6, have been patented as metabotropic glutamate receptor 5 (mGlu5) modulators which may be useful in treating neurological disorders.⁹

Figure 1.

Bioactive pyrazinoquinazolinone-containing natural products and analogs

We recognized an opportunity to generate chiral pyrazinoquinazolinones after discovering an unexpected tandem reaction involving our quinazolinone rearrangement chemistry (Scheme 1). We previously described a regiospecific quinazolinone-to-(E)-benzamidine rearrangement¹⁰ that employed secondary amines as part of the quinazolinone substrates (yellow highlight, Scheme 1, 7 to 9a). The (Z)-benzamidine 9b was not observed, an outcome that was reasoned to result by virtue of unfavorable allylic-like strain of the planar N-alkylated amidine moiety. We decided to then evaluate a primary amine 8 that might permit observation of an elusive (Z)-benzamidine 10b; however, instead of isolating either of the expected isomeric NH-benzamidines, pyrazinoquinazolinone 11 was isolated in 76% yield. While we suspected that the isomeric NH-benzamidines (E)-10a and (Z)-10b were likely both generated from 8 and that interconversion between the two NH-benzamidines was possible via tautomerization and carbon-nitrogen single bond rotation, the formation of 11 could only result from the intramolecular cyclization of Z-benzamidine-derived tautomer 10c with concomitant expulsion of aniline.

Scheme 1.

2° or 1° amine-containing quinazolinones leading to (E)-amidines or pyrazinoquinazolinones, respectively

This result was significant as we recently reported a Ugi-Mumm-Staudinger sequence, coupled to a quinazolinone rearrangement, that generated racemic (E)-benzamidines like 9a but bearing substitution at C3 (c.f., Scheme 1).¹¹ Importantly, in that protocol, we had used achiral secondary amines to afford the desired benzamidines which would not have been expected to undergo cyclization. Given that primary amine tethered benzamidines spontaneously formed pyrazinoquinazolinones, we integrated this discovery with the aforementioned benzamidine assembly to afford a unique collection of enantiopure, diastereomeric pyrazinoquinazolinones. Furthermore, this strategy afforded products with enhanced fraction sp³ (Fsp³) character¹² that would be advantageous in our medicinal chemistry screening programs. To that end, we sought a reliable, diastereoselective protocol that would provide expedient access to pyrazinoquinazolinone derivatives with preservation of enantiopurity and harboring substitution patterns that are otherwise challenging to achieve by methods used to generate similar structures.^{9, 13, 14} Based on the preliminary results leading to compound 11, we anticipated that a quinazolinone tethered to a chiral N-Boc-protected primary alkylamine (e.g., 16a, Scheme 2), assembled by a Ugi-Mumm-Staudinger sequence,^{11, 15} would rearrange under basic conditions to afford analogous C4-substituted, NH-benzamidines 17a-c. Subsequent spontaneous cyclization would be expected to afford diastereomeric pyrazinoquinazolinones 18a-b. Furthermore, employing an enantiomerically pure chiral amine in the Ugi reaction, such as (S)- or (R)-proline derivative 12, was expected to provide access to enantiopure pyrrolopyrazinoquinazolinones rather than racemic mixtures which would benefit downstream medicinal chemistry work.

Scheme 2. Synthetic approach and rationale of diastereoselective formation of enantiopure pyrazinoquinazolinones^a.

^aOnly major isomers depicted from 15a to 17c for clarity.

This assumed that, due to the presence of a bulky NH-Boc amide moiety in iminium ion (E)-13a, the preference was strong for (a) formation of the (E)-isomer of the iminium ion in the Ugi assembly, (b) a biased approach of the isocyanide from the least hindered face, and (c) no epimerization of the newly formed chiral center in the process. Together, this would afford a nitrilium species 14a as a major isomer bearing (R,R)-stereochemistry. This intermediate, once intercepted by the carboxylic acid, would form an acylimidate intermediate (not shown) that undergoes spontaneous Mumm rearrangement to form imide 15a as the major diastereoisomer. The quinazolinone core of 16a would be constructed via a Staudinger/aza-Wittig reaction, with subsequent quinazolinone rearrangement leading to (E)- and (Z)-NH-benzamidines 17a-b. Unlike N-alkyl benzamidines, the NH variants would be expected to tautomerize and/or rotate about the carbon-nitrogen single bond to give 17c and then irreversibly cyclize to form the major ring-fused product 18a. A minor diastereomer 18b might also form through an analogous sequence originating from the minor nitrilium intermediate 14b. If successful, enantiopure, diastereomeric pyrazinoquinazolinones were expected to be generated and enable structural exploration of this privileged pharmacophore.

With this in mind, a pilot reaction was done using our reported conditions¹¹ for the preparation of the C3 and N-substituted benzamidines, but in place of achiral N-Boc protected secondary amines, we used (R)-t-butyl N-(pyrrolidin-2-ylmethyl) carbamate 12a, along with benzaldehyde, 2-azidobenzoic acid, and benzyl isocyanide (Scheme 2). Our original protocol leading to racemic, C3-substituted (E)-benzamidines was streamlined to avoid intermediate purification, so we attempted the same approach here. Hence, this new process was also telescoped, requiring only a single purification of the final products, and delivered diastereomeric, C5-phenyl-substituted, pyrroloquinazolinones (5R,13aR)-18a and (5S,13aR)-18b in an isolated, diastereomeric ratio of 89:11, respectively, and in 79% overall yield. When the reaction was done on 1 mmol scale, a similar diastereoselectivity (18a:18b = 84:16) and a slight loss in yield was observed (67% overall). Use of the chiral amine (S)-12b in place of the (R)-antipode delivered a 76% combined yield of two diastereomers, (5S,13aS)-19a and (5R,13aS)-19b, with an 86:14 d.r., respectively. Inspection of the NMR spectra for the minor isomers 18b and 19b revealed strong NOE resonances between the C5 and C13a hydrogen atoms, suggesting a 5,13a-cis-relationship. This was later unequivocally confirmed by X-ray crystallography of minor isomers, 20b and 21b, each obtained from separate reactions using either chiral amine, (R)-12a or (S)-12b, respectively (see Fig. S1, supplemental information). These results showed that all four stereoisomers of a given substrate could be isolated through a single achiral separation of diastereomers from two independent reactions.

With these results in hand, we explored structural modifications of the Ugi-substrates. First, the Ugi-Mumm reaction employed benzyl isocyanide to install a benzylamine group that was eventually expelled in the final cyclization step of the synthetic sequence. Atom sparing alternatives were surveyed; however, both methyl- and ethyl isocyanide were inferior in the Ugi-Mumm reaction, and isopropyl- and cyclohexyl isocyanide eroded yield and diastereoselectivity compared to the benzyl isocyanide (see Table S1 in supplementary information). As such, we continued with the use of benzyl isocyanide. Next, we tried to improve the diastereoselectivity of the protocol. Achieving exceptional stereochemical control in the Ugi four-component reaction has historically been challenging without the use of specialized chiral carbohydrate-derived amines.^{16, 17} In fact, due to the minimal steric influence of isonitriles, a diastereoselectivity of 10:1 in isonitrile-based Ugi type reactions is typically considered excellent.¹⁸ Recently, an asymmetric Ugi reaction has been realized with the use of chiral phosphoric acids that advantageously coordinate through the hydrogen atom of a primary amine-derived iminium ion.¹⁹ Alternatively, our protocol requires a secondary amine (i.e., pyrrolidine) which changes the reaction outcome to produce imides (e.g., 15a, Scheme 2) as opposed to α-acylaminoamides. As such, our system lacks the necessary NH-coordination site through which an analogous chiral environment may be established. As an alternative, we investigated the use of pivaldehyde in place of benzaldehyde in the four-component coupling as a source of steric bulk to bias facial selectivity and synergistically compliment the pre-existing Boc-protected alkylamine chiral appendage of amines 12a-b. Unfortunately, stepwise analysis of each reaction in the protocol revealed that imide formation from the Ugi-Mumm reaction was impeded and promoted a competing Passerini reaction, generating an undesirable acyloxycarboxamide by-product. Increasing the concentration of amine (R)-12a (4 equiv.) suppressed this outcome, but the Mumm and quinazolinone rearrangements still suffered under the steric bulk of the tert-butyl group, the latter requiring heating at 150 °C and ultimately affording a single diastereomeric pyrrolopyrazinoquinazolinone in only 24% overall yield (not shown). As such, we switched our attention to examining the tolerance of the protocol to structural changes on the 2-azidobenzoic acid while employing the piloted reaction conditions (Scheme 3).

Scheme 3. Scope of diastereoselective sequence leading to pyrrolopyrazinoquinazolinones 18a-b through 32a-b^a–c.

^aIsolated, overall yields reported as an average of n > 2 experiments; d.r. was determined after isolation and purification of each diastereomer; ^bMajor isomer shown; minor isomers, omitted for clarity, have opposite C5 stereochemistry of major isomer; ^c(R)-12a used unless otherwise noted; ^dminor isomer stereochemistry established by X-ray, see SI; ^e1 mmol scale: 67% yield overall (84:16 d.r.); ^fUsed (S)-12b; ^gRequired heating at 120 °C in last step.

Generally, the synthetic sequence provided diastereomeric products in good overall yield for substituents surveyed on the azido acid core in the C7, C8, C9 positions (Scheme 3). The C8-fluorinated analogs 23a-b, obtained in 37% overall yield (74:26 d.r.), was an outlier due to the visibly poor solubility of the 2-azido-4-fluorobenzoic acid reagent; however, the sequence still averaged 78% yield per step. Notably, azido benzoic acids bearing electron donating methoxy substituents were competent in this protocol, generating methoxy compounds 28a-b (81%, 83:17 d.r.) and dimethoxy derivatives 29a-b (78%, 84:16), when the reaction temperature of the last transformation was increased to 120 °C. Exchange of benzaldehyde for other aldehydes in the first step was also surveyed. As previously noted, the bulky pivaldehyde gave a single diastereomer, but only in 24% yield due to poor iminium ion formation. Better results were obtained with 2,6-dimethylbenzaldehyde which afforded a 77:23 ratio of diastereomers 30a-b in 58% combined yield. The selectivity was eroded with 3-thiophenecarbaldehyde (31a-b, 57%. 63:37 d.r.) while the nonaromatic isobutyralde-hyde revealed a 80:20 d.r. of 32a-b in 64% yield.

As noted above, we anticipated that the relative C5/C13a stereochemistry was established in the Ugi reaction by virtue of preferred iminium ion geometry and facial selectivity of isonitrile attack; however, it was possible that some epimerization of the newly formed stereocenter occurred at a subsequent point in the synthetic process leading to pyrrolopyrazinoquinazolinone formation. In our work involving quinazolinone formation and subsequent rearrangement to the benzamidines, we determined that the 4N HCl conditions used for N-Boc-deprotection of quinazolinones and the subsequent free-base reaction using triethylamine (necessary for promoting the benzamidine formation) does not erode the stereochemical integrity of similar quinazolinone substrates.²⁰ We did not know, however, if the resulting pyrrolopyrazinoquinazolinones were susceptible to epimerization at the activated C5 position once the new scaffold was assembled, as some structurally similar quinazolinediones have been reported to racemize spontaneously.²¹ As such, purified minor product, iodo-derivative 21b was resubmitted to the last step of the reaction sequence employing NEt₃ in methanol at 100 °C for 1 h. If the C5 stereocenter was susceptible to racemization, we expected that the minor isomer might thermodynamically equilibrate to afford the diastereomeric ratio that was observed for 21a:21b when the entire synthetic sequence leading to these products was implemented. However, we did not detect any formation of the major isomer 21a by either ¹H NMR or LCMS of the crude reaction mixture. As a result, we concluded that the diastereomeric outcomes were predominately a result of the Ugi assembly and less likely due to subsequent racemization during the synthetic sequence.

In summary, while exploring the fate of achiral primary alkylamine-appended quinazolinones in a historically regiospecific rearrangement, we discovered that the resulting NH-benzamidines underwent a spontaneous cyclization that afforded pyrazinoquinazolinones. This discovery and the pyrazinoquinazolinone architecture were subsequently elaborated by pairing a diastereoselective, four-component Ugi-Mumm-Staudinger sequence with a new domino quinazolinone rearrangement-intramolecular ring closure to smoothly generate diastereomeric pyrrolopyrazinoquinazolinones. Several features of this protocol are notable. First, the approach leverages the efficiency of a multicomponent Ugi-based assembly that establishes two stereocenters at an early stage through use of chiral N-Boc-protected primary amines. Second, the domino quinazolinone rearrangement-intramolecular cyclization requires the intermediacy of tautomeric Z-benzamidines which we have not previously encountered in the quinazolinone rearrangement chemistry. Third, the process integrates three structural rearrangements and telescopes a minimum of seven transformations into one sequence. As a result, simple building blocks were transformed into enantiomerically pure, diastereomeric products that were easily isolated by a normal phase, achiral chromatographic separation. Furthermore, the approach only required this single, final purification, obviating the need for isolation of any intermediates. A survey of azidobenzoic acids, aldehydes and isonitriles was done to demonstrate the scope and limitations of the procedure, revealing overall yields ranging from 53–83% and diastereomeric ratios that maximally reached 89:11. This tandem quinazolinone rearrangement-intra-molecular cyclization represents another post-Ugi transformation^22–24 that affords heterocyclic scaffolds of pharmacological interest.²⁵ The novel diastereomeric pyrazinoquinazolinones described herein are being assessed in silico and in biological assays, the results of which will be pursued and reported in due course. Further, we are exploring the in-situ capture of chiral tautomeric Z-benzamidines in other bond forming reactions to build molecular complexity and heterocyclic diversity.