Facile access to potent antiviral quinazoline heterocycles with fluorescence properties via merging metal-free domino reactions

Abstract

Most of the known approved drugs comprise functionalized heterocyclic compounds as subunits. Among them, non-fluorescent quinazolines with four different substitution patterns are found in a variety of clinically used pharmaceuticals, while 4,5,7,8-substituted quinazolines and those displaying their own specific fluorescence, favourable for cellular uptake visualization, have not been described so far. Here we report the development of a one-pot synthetic strategy to access these 4,5,7,8-substituted quinazolines, which are fluorescent and feature strong antiviral properties (EC₅₀ down to 0.6±0.1 μM) against human cytomegalovirus (HCMV). Merging multistep domino processes in one-pot under fully metal-free conditions leads to sustainable, maximum efficient and high-yielding organic synthesis. Furthermore, generation of artesunic acid-quinazoline hybrids and their application against HCMV (EC₅₀ down to 0.1±0.0 μM) is demonstrated. Fluorescence of new antiviral hybrids and quinazolines has potential applications in molecular imaging in drug development and mechanistic studies, avoiding requirement of linkage to external fluorescent markers.

ARTICLE

Received 21 Jul 2016 | Accepted 27 Feb 2017 | Published 2 May 2017

Felix E. Held1, Anton A. Guryev1, Tony Frhlich1, Frank Hampel1, Axel Kahnt2, Corina Hutterer3,Mirjam Steingruber3, Hanife Bahsi3, Clemens von Bojnii-Kninski4, Daniela S. Mattes4, Tobias C. Foertsch4, Alexander Nesterov-Mueller4, Manfred Marschall3 & Svetlana B. Tsogoeva1

Most of the known approved drugs comprise functionalized heterocyclic compounds as subunits. Among them, non-uorescent quinazolines with four different substitution patterns are found in a variety of clinically used pharmaceuticals, while 4,5,7,8-substituted quinazo-lines and those displaying their own specic uorescence, favourable for cellular uptake visualization, have not been described so far. Here we report the development of a one-pot synthetic strategy to access these 4,5,7,8-substituted quinazolines, which are uorescent and feature strong antiviral properties (EC50 down to 0.60.1 mM) against human cytomegalo-virus (HCMV). Merging multistep domino processes in one-pot under fully metal-free conditions leads to sustainable, maximum efcient and high-yielding organic synthesis. Furthermore, generation of artesunic acidquinazoline hybrids and their application against HCMV (EC50 down to 0.10.0 mM) is demonstrated. Fluorescence of new antiviral hybrids and quinazolines has potential applications in molecular imaging in drug development and mechanistic studies, avoiding requirement of linkage to external uorescent markers.

1 Organic Chemistry Chair I and Interdisciplinary Center for Molecular Materials, Friedrich-Alexander University of Erlangen-Nrnberg, Henkestrasse 42, 91054 Erlangen, Germany. 2 Physical Chemistry Chair I, Friedrich-Alexander University of Erlangen-Nrnberg, Egerlandstrasse 3, 91058 Erlangen, Germany.

3 Institute for Clinical and Molecular Virology, Friedrich-Alexander University of Erlangen-Nrnberg, Schlossgarten 4, 91054 Erlangen, Germany. 4 Karlsruhe Institute of Technology, Institute of Microstructure Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany. Correspondence and requests for materials should be addressed to S.B.T. (email: mailto:[email protected]

Web End [email protected] ).

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Facile access to potent antiviral quinazoline heterocycles with uorescence properties via merging metal-free domino reactions

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15071

Quinazoline heterocycles are ubiquitous in pharmaceutical compounds and drugs. They are important subunits of a broad variety of natural products as well as synthetic

pharmaceuticals possessing anti-inammatory1, antiviral2, antimalarial3 and anticancer4 activities. The known quinazoline-based drugs, which are not demonstrating uorescence properties, can be divided into four groups AD (2,4,6,7-; 4,6,7-; 2,4,5,6- and 2,4,6,7,8-substituted quinazolines), representing four types of existing substitution patterns (Fig. 1a).

The synthesis of known quinazoline heterocycles by conventional ways is a hard work that implies many synthetic steps and expensive starting materials, and involves time-consuming and waste-producing isolation and purication of product intermediates48. The number of synthetic methods to afford quinazolines is, furthermore, restricted to the availability of the appropriate starting compounds. For this reason, development of new efcient and straightforward synthetic methods towards quinazoline heterocyclic compounds of value to medicinal chemistry, starting from simple precursors, is of high current demand. To our knowledge, the class of 4,5,7,8-substituted quinazolines (E-type, Fig. 1b) have not been prepared to date because of the lack of a synthetic route towards these compounds. Furthermore, no examples of uorescent quinazolines have been reported so far. External uorescent labels are usually employed to study the functions of quinazoline-based pharmacophores within cells. However, uorescent markers can inuence or change the properties of studied lead compounds and drugs. Therefore, the drug candidate should ideally display its own uorescence to allow its cellular uptake visualization and without incorporation of external uorescent labels. The motivation and desire to address these gaps prompted the research work we report here.

Recently, combined processes, where several fundamentally different catalytic reactions are joined in a one-pot protocol, were introduced9. Among known examples are combinations of (i) organocatalysis and transitionmetal catalysis10,(ii) organocatalysis and silver or gold catalysis1113 or(iii) organocatalysis and (photo)redox catalysis14,15. Even

more efcient and economical methods towards heterocyclic compounds are domino processes1622.

In terms of efciency and sustainability, generation of bioactive heterocycles through multicomponent multistep reactions avoiding intermediate isolation and purication steps, is unbeatable. While examples of organocatalysed linear domino reactions17,18,20 and of a single branched domino process21 are known, surprisingly, combining them in one-pot is unprecedented, although such a multistep one-pot process would further reduce costs and waste production in the synthesis of versatile heterocycles.

Herein, we describe development of a combined process, which joins a new ve-step branched domino reaction with two-step linear and subsequent three-step linear domino reactions. This new metal-free 10-step sequence, with only a single work up procedure and starting from simple and readily available compounds, results in new functionalized quinazolines of type E (Fig. 1b), which exceed the antiviral potency of the clinical reference drug ganciclovir23. Furthermore, selected uorescent quinazolines were applied for synthesis of rst artesunic acid quinazoline hybrids, which are, remarkably, also uorescent and display superior potency against HCMV (EC50 down to0.10.0 mM) compared to that of their parent quinazolines (EC50 down to 4.60.9 mM) and artesunic acid (EC503.80.4 mM), as well as ganciclovir (EC50 2.60.5 mM). For all quinazoline compounds, cytotoxicity for primary human broblasts (HFFs; CC50) was undetectable at concentrations up to 100 mM, indicating that the new quinazolines and hybrid compounds are selective. Importantly, the uorescent quinazolines could nicely be depicted both in extracellular and intracellular localizations when analysing primary HFFs and virus-infected cells using confocal laser-scanning microscopy. An accumulation of uorescent quinazolines was mostly observed in cytoplasmic areas, thus visually indicating their efcient cellular uptake. These results open up new perspectives for molecular imaging in the drug development process and mechanistic studies, avoiding the requirement of linkage of external uorescent labels to potential drug molecules.

a b

Known substitution patterns

A

NH2

R NH

2

N

N

N

R

N

N

R1

B

-Alfuzosin -Bunazosin -Prazosin -Terazosin

-Erlotinib -Gefitinib -Vandetanib

NH2

NH2

N

N

N

R

N

N

N

R

C

R1

D

R1

-Trimetrexate

-Trimazosin

Figure 1 | Quinazoline scaffolds with different substitution patterns. (a) List of the selected corresponding non-uorescent quinazoline-marketed drugs: Alfuzosin (adrenergic blocker), Bunazosin (antihypertensive agent), Prazosin (adrenergic blocker), Terazosin (adrenergic blocker), Erlotinib (anticancer agent), Getinib (anticancer agent), Vandetanib (tyrosine kinase inhibitor), Trimetrexane (dihydrofolate reductase inhibitor) and Trimazosin (antihypertensive agent). (b) Antiviral quinazolines with uorescence properties developed in this work.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15071 ARTICLE

ResultsProposed one-pot approach towards uorescent quinazolines. Inspired by the power of the domino concept introduced by Tietze16,22, we envisioned the possibility of designing a multicomponent one-pot process, involving domino reactions, which might allow to generate quinazolines starting from simple precursors: aldehydes, nitroalkenes and malononitrile (Fig. 2). The proposed unprecedented quinazolines, bearing amino (donor) and cyano (acceptor) groups at the same time, were expected to demonstrate desirable uorescence properties.

One-pot synthesis of cyclohexenes and 2,6-dicyanoanilines. We started by exploring a reaction of trans-b-nitrostyrene (1), benzaldehyde (2) and malononitrile (3; Figs 3 and 4a). Our most recent nding of an imidazole-catalysed six-step linear domino reaction of malononitrile with phenylethanal derivatives24 inspired us to apply imidazole as a catalyst also for this new transformation (Supplementary Fig. 1). To our delight, the domino process using dichloromethane as a solvent in presence of imidazole at room temperature resulted in highly functionalized cyclohexene derivative 4a. Structure and relative stereochemistry of isolated major diastereomer 4a was conrmed by X-ray analysis (Fig. 3).

We next screened further commercially available bases (Supplementary Fig. 1). A signicant improvement of product yields to 499% was observed in presence of hydroquinine 1,4-phthalazinediyl diether ((DHQ)2PHAL, Fig. 4a), well known as a ligand in osmium-catalysed Sharpless dihydroxylation reaction25.

The proposed mechanism of the branched domino reaction was conrmed by ESI- and APPI-MS studies (Fig. 3). In presence of a catalyst, we observed signals at m/z 154 [M] and m/z 218 [M 3H]3 , which correspond to products of two transforma

tions running in parallel, Knoevenagel (step 1) and nitro-Michael (step 2) reactions, respectively. A signal at m/z 370 [M H]

corresponds to product of a subsequent nitroalkane-Michael reaction (step 3) between previously formed nitroalkane derivative (donor) and a Knoevenagel product (Michael acceptor). The last steps, leading to target compound 4a, involve intramolecular addition (step 4) and tautomerization (step 5).

Next, optimal conditions for 2,6-dicyanoaniline derivative 5a formation (key intermediate for quinazoline synthesis according to Fig. 2) from cyclohexene 4a were studied (see Supplementary Table 1). Notably, 2,6-dicyanoaniline derivatives have recently attracted considerable attention, since they exhibit strong uorescence in ultraviolet light and may have utility in synthesis of uorescent materials, non-natural photosynthetic systems, materials with semiconducting or nonlinear optical properties, molecular electronic devices and light-emitting diodes2629.

Since direct aromatization of 4a was not successful using different oxidizing agents (see Supplementary Table 1), we assumed that hydrolysis of CN groups to COOH might facilitate the subsequent aromatization step through CO2 release and might also prevent generation of toxic hydrogen cyanide (HCN). Applying acetic acid (AcOH) and carrying out the reaction at

130 C for 6.5 h, product 4a0 with a quaternary carbon centre was isolated in 75% yield (Fig. 4b). Subsequently, we reacted 4a0 with Ac2O/Py, giving desired product 5a in 95% yield. Next, we turned our attention to nding the optimal conditions for two-step one-pot synthesis of 5a from 4a (Fig. 4c). Carrying out the reaction in presence of AcOH/Py (1:1) at 80 C for 15 h, desired product 5a was isolated in 41% yield. We obtained, furthermore, performing the same reaction under otherwise identical conditionsbut in presence of ammonium acetate (6) as an additivethe product 5a in a higher yield of 52% over two steps (Fig. 4c).

Another specic aim was to combine the ve-step branched domino reaction with the optimized two-step linear domino reaction in a seven-step one-pot process, which we successfully realized (Fig. 5a). Noteworthy, all reactions investigated can be effected in 3167% overall yield over seven steps. Generally, higher yields (6167% for 5a5e) were obtained using substrates with both neutral and electron-withdrawing moieties on benzene rings.

Laser-induced uorescence imaging with 2,6-dicyanoaniline. Using the combinatorial laser-induced forward transfer method30, we patterned selected new uorophore 5d in the form of an array consisting of 10 10 spots (Fig. 5b), and the logo of

Friedrich-Alexander University, consisting of single overlapping spots (Fig. 5c). Hereby, laser irradiation (wavelength of 532 nm) transferred the small amounts of 2,6-dicyanoaniline derivative 5d from the donor slide to an acceptor (see Supplementary Fig. 2 and Supplementary Note 1). In accord with the absorption spectrum of compound 5d, an excitation wavelength of 254 nm was chosen. The expected uorescent image was detected with an optical set-up developed for rapid wide-eld screening of chromophores (see Supplementary Fig. 2). The fact that the combinatorial laser-induced forward transfer procedure does not harm pushpull chromophore 5d opens up the possibility to test and optimize optical properties in a high-density array format by performing parallel screenings for different chromophores at the same time. In addition, 5d showed also solvatochromic properties (Fig. 5d).

One-pot 10-step synthesis of uorescent quinazolines. Consequently, anticipated one-pot synthesis of quinazolines 7ai was performed via combination of our new seven-step one-pot process with a three-step linear domino reaction using formamide as a suitable reagent in 2 h reaction time (Figs 3 and 6). Reaction of 5a with formamide can proceed through an imine formation (step 1), intramolecular condensation (step 2) and tautomerization (step 3) linear domino process (Fig. 3). We explored the substrate scope of the resulting 10-step one-pot process (Fig. 6). All applied aromatic substrates (carrying either neutral, electron-withdrawing or electron-donating groups) afforded high overall yields of 2050% over 10 steps. Remarkably, calculated average yields of every individual step in these multistep one-pot reactions, deduced from a total yield of up to 67% (seven-step, Fig. 5a) and up to 50% (10-step, Fig. 6) is 94% and 93%, respectively.

O

CN

NC

NO2

+ 2

NO2

CN

H2N

NC

CN

CN

CN

NH2

Cyclohexenes 2,6-Dicyano-anilines Quinazolines

Figure 2 | Proposed convenient synthesis of uorescent quinazolines. One-pot multistep synthetic approach towards 4,5,7,8-substituted quinazolines from simple compounds.

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Figure 3 | Merging metal-free domino reactions. Development of one-pot access to new 2,6-dicyanoaniline-based multichromophores and 4,5,7,8-substituted quinazolines. For detailed X-ray data of compounds 4a, 4a0 and 7a, see Supplementary Tables 24.

Ph NO2

a

O+ + 2

1 4a + ent, 99% yield

CN

CN

(DHQ)2PHAL (20 mol%)

0 C/6 h /CH2Cl2

2 3

Ph NC

H2N

NO2

Ph H

NC CN

Ph

b

Ph NC

H2N

NO2

AcOH

Ph NC

H2N

NO2

Ac2O/Py 80 C, 3 h

Ph

Ph NH2

CN

130 C, 6.5 h

NC CN

Ph

HOOC CN

Ph

CN

4a, 75% yield

4a 5a, 95% yield

c

AcOH/Py (1:1) 80 C, 15 h

AcOH/Py (1:1) NH4OAc (6, 1 equiv)

80 C, 15 h

5a

41% yield

4a

5a

52% yield

Figure 4 | Optimization of domino and one-pot processes. (a) Four-component (ABC2) ve-step branched domino reaction. (b,c) Optimization of one-pot two-step synthesis of 2,6-dicyanoaniline derivative 5a(see Supplementary Methods).

Photophysical study of selected uorescent quinazoline. On example of selected compound 7h we demonstrate the solvatochromy of new quinazolines (Fig. 6). Inspired by this interesting property, we conducted a basic photophysical study of 7h in acetonitrile (see Supplementary Fig. 3, Supplementary Discussion and Supplementary Methods). First insights into the excited-state properties came from steady-state uorescence measurements. Quinazoline compound 7h exhibits strong uorescence between 370 and 600 nm, with a maximum at 439 nm. The uorescence properties of potentially bioactive compounds, for example, antiviral, antimalarial and anticancer agents, might be very useful for an in vitro monitoring of compound-treated cultured cells by use of uorescence microscopy.

Synthesis of artesunic acidquinazoline hybrids. Human cytomegalovirus (HCMV) is a major human pathogen showing worldwide prevalence. Prevention and clinical interventions for HCMV disease are limited, since cross-resistance to all approved anti-HCMV drugs is increasingly observed31. As alternatives to standard drugs23,32, our ongoing search for highly active hybrid molecules3335, which exceed their parent compounds in activity

against HCMV, resulted in synthesis of new artesunic acidquinazoline hybrids 8 and 9 (Fig. 7a and Supplementary Methods). We chose compounds 7g and 7h (Fig. 6) as coupling partners, since their uorescence properties were considered benecial for biological investigations. Artesunic acid on the other hand was selected because it proved to be a valuable building block in order to get highly bioactive compounds with just a few chemical transformations and also because of its promising pharmacological properties23,32,35.

Antiviral activities of quinazolines and hybrid compounds. Subsequently, the selected quinazolines 7b, 7g, 7h, 7i and hybrids 8, 9 were quantitatively analysed for their antiviral activity against HCMV (Table 1). The new quinazolines 7b, 7g, 7h and 7i consistently demonstrated a high activity against HCMV, among which the quinazoline heterocycle 7i displayed the most potent antiviral efcacy with a lower in vitro EC50 value than the reference drug ganciclovir (Table 1).

Furthermore, our results clearly demonstrate the great potential of the hybridization concept. Artesunic acid quinazoline hybrids 8 and 9 (EC50 0.60.1 and 0.10.0 mM, respectively) are not only 6- to 46-fold more active than their parent compounds: artesunic acid, 7g and 7h (EC50 3.80.4,4.90.2 and 4.60.9 mM, respectively); they also exceed antiviral activity of the reference drug ganciclovir (EC50 2.60.5 mM) that is in use for standard treatment of HCMV infections. The measurement of antiviral activity was based on automated uorometry using a green uorescent protein (GFP)-expressing, recombinant HCMV (excitation 485 nm, emission 535 nm; see Supplementary Note 2, Supplementary Data 1 and Supplementary Fig. 4 for doseresponse curves).

In addition, EC50 values were conrmed using non-GFP-expressing HCMV (strain AD169) by a classical plaque formation assay on HFFs (n 4), providing data consistent with the

GFP-based data shown in Table 1, that is, compound 7h,7.30.7 mM; compound 8, 0.30.0 mM; compound 9,0.30.1 mM. Moreover, antiviral activity was clearly separated from putative cytotoxic effects because of the results obtained with a standard trypan blue exclusion assay. For all compounds, cytotoxicity for primary HFFs (CC50) was low or undetectable at concentrations up to 100 mM (that is, cytotoxicity values obtained at the highest test concentration of 100 mM remained under the

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Ar

Ar NH2

CN

a

Ar O

+ 2

CN

CN

Ar NO2

CN

Me

F

CF3

CN

CN

CN

CN

NH2

NH2

NH2

NH2

CN

CN

CN

CN

Me

F

F3C

5a

.

5b

5c

5d

67% overall yield[*] 67% overall yield[*] 61% overall yield[*] 61% overall yield[*] 55% overall yield[**]

OMe

OBn

Ph

Cl

CN

CN

CN

CN

NH2

NH2

NH2

NH2

CN

CN

Cl

CN

MeO

CN

BnO

Ph

5e

5f

5g

5h

63% overall yield[*] 31% overall yield[*] 34% overall yield[*] 32% overall yield[*]

b c d

CH2CI2 DMSO

Figure 5 | One-pot seven-step process and imaging experiments with 5d using combinatorial laser-induced forward transfer (cLIFT) method.(a) Synthesis of new 2,6-dicyanoaniline-based multichromophores via seven-step one-pot sequence. * (1) Catalyst (DHQ)2PHAL (5 mol%), CH2Cl2, RT, 3 h, (2) AcOH/Py 1/1, 1 eq. NH4OAc, 80 C, 15 h. ** (1) Catalyst (DHQ)2PHAL (20 mol%), CH2Cl2, 0 C, 6 h, (2) AcOH/Py 1/1, 1 eq. NH4OAc, 80 C,

15 h (see Supplementary Methods). (b) Fluorescent image of an array (10 10 spots) of the selected new uorophore 5d patterned via cLIFT. Excited at

254 nm, the uorescence was captured for 10 s at ISO 1,600 with a resolution of 1.25 mm per pixel (scale bar, 1 mm). (c) Imaging of Friedrich-Alexander University logo with the new uorophore 5d patterned on a glass slide using cLIFT (imaging parameters see b; scale bar, 2 mm), see Supplementary Fig. 2 and Supplementary Note 1. (d) Demonstration of the uorescence properties of 5d in two different solvents illuminated by ultraviolet light.

cutoff level for all compounds except artesunic acid also comprising a high CC50 of 82.00.9), while anti-HCMV activities (EC50) were in a sub- or single-digit micromolar range. Consistent with these ndings are results from a MTS-based cell proliferation assay, showing moderate or no antiproliferative effects on HFFs for all analysed compounds in concentrations up to 30 mM (see Supplementary Fig. 5 and Supplementary

Methods). Thus, the new quinazolines and hybrid compounds can be regarded as selective, which is one of the most important aspects in drug design.

DiscussionWe describe the operationally simple and expeditious one-pot synthetic methodmerging multistep domino reactions under fully metal-free conditionsproviding unprecedented convenient

access to a new class of antiviral 4,5,7,8-substituted quinazolines, bearing amino and cyano groups at the same time, in high overall yields, starting from simple and readily available compounds (Figs 3 and 6). Only one purication step is involved, leading to signicant reduction of costs, waste and labour input. The investigated quinazolines 7b, 7g, 7h, 7i exhibit signicant activity against HCMV. Furthermore, selected uorescent quinazolines 7g, 7h were applied for synthesis of rst artesunic acid quinazoline hybrids 8 and 9, which are, strikingly, also uorescent and display superior anti-HCMV activity (EC50 down

to 0.10.0 mM) compared to the parent compounds (artesunic acid and quinazolines) as well as the reference drug ganciclovir (EC50 2.60.5 mM).

When analysing primary HFFs, the uorescent compounds 7g, 7h and 9 could nicely be depicted both in extracellular (transparent arrows) and intracellular localizations (lled arrows;

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Fig. 7b,c). Interestingly, the extracellular signals included well-ordered crystalline structures (Fig. 7b, panel 8; Fig. 7c, panels 89) in the range of the applied concentrations (110 mM). The intracellular uorescence pattern of the compounds showed a speckled accumulation mostly in cytoplasmic areas, thus visually indicating their efcient cellular uptake (Fig. 7b, panels 25 and 710; Fig. 7c, panels 35 and 810). A nonuorescent artemisinin-derived control compound (Fig. 7c, panels 2 and 7; see also Supplementary Fig. 6), the DMSO panels (solvent controls) as well as the inspection of different excitation wavelengths of the confocal laser-scanning microscopy conrmed the specicity of signals and a lack of unwarranted cross-uorescence. Of note, the uorescent capacity of compounds 7h, 7g and 9 (depicted by microscopic imaging scored at a laser wavelength of 405 nm; Fig. 7bd) did not interfere with GFP

uorometry, so that control measurements with these compounds did not produce signals above background levels. Importantly, the evaluation of virus-infected cells could also be included in this analytic approach, opening new perspectives in the investigation of antiviral activity and mechanistic details of the compounds (the latter point might be supported by the study of intracellular drugtarget binding and localization in future applications). An indirect immunouorescence counterstaining of viral and cellular proteins36,37 led to the detection of nuclear markers, that is, the viral protein kinase pUL97 and cellular lamins type A/C (Fig. 7d, panels 4, 9, 14 or 5, 10, 15, respectively), in the context of extracellular and intracellular uorescence signals of compounds 7h and 9 (Fig. 7d, panels 15 or 615, respectively). The direct co-staining of putative target proteins of the compounds seems to be a further realistic goal and might be realizable at a later stage.

Ar

Ar N

CN

N

NH2

Ar O

+ 2

CN

CN

Ar NO2

Me

Ph

NH2

NH2

NH2

N

N

N

N

N

N

Me

Ph

7a

7b

7c

37% overall yield[*]

50% overall yield[**]

33% overall yield[**]

23% overall yield[] 20% overall yield[**]

t-Bu

NH2

NH2

NH2

N

N

N

N

N

N

t-Bu

Cl

7d

7e

Br

7f

25% overall yield[**] 40% overall yield[**] 40% overall yield[**]

F

CF3

OMe

NH2

NH2

NH2

N

MeCN

MeOH

N

N

N

N

N

F

F3C

MeO

7g

7h

7i

35% overall yield[**]

20% overall yield[] 25% overall yield[] 20% overall yield[**]

Figure 6 | Synthesis and uorescence properties of quinazolines. Synthesis of new quinazolines via 10-step one-pot process and demonstration of the uorescence properties of 7h in different solvents illuminated by ultraviolet light. * (1) Catalyst (DHQ)2PHAL (5 mol%), CH2Cl2, RT, 3 h, (2) AcOH/Py 1/

1, 1 eq. NH4OAc, 80 C, 15 h; (3) formamide, reux, 2 h. ** (1) Catalyst (DHQ)2PHAL (20 mol%), CH2Cl2, 0 C, 6 h, (2) AcOH, 130 C, 15 h, (3) formamide, reux, 2 h. z (1) Catalyst (DHQ)2PHAL (5 mol%), CH2Cl2, RT, 3 h, (2) AcOH, 130 C, 15 h, (3) formamide, reux, 2 h. y (1) Pyrrolidine (20 mol%), CH2Cl2,

0 C, 6 h, (2) AcOH, 130 C, 15 h, (3) formamide, reux, 2 h.

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a

H

H

O

O

O

H

H

O O

O

F

CF3

O

O

O

HN O

8 9

HN

O

O

O

O

O

O

N

N

N

N

F CN

CN

F3C

b

Compounds

DMSO

7h

7h

9

9

HCMV-inf.

Insets

Mock-inf.

Insets

7h

9

9

1 2 3 4 5

6 7 8 9 10

c

DMSO

Control cmp

7h

7g

9

1 2 3 4 5

6 7 8 9 10

d

Insets

HCMV-Inf. Lamin A/C

Merge

Compounds pUL97

1 2 3 4 5

6 7 8 9 10

11 12 13 14 15

Figure 7 | Microscopy-based visualization of cellular uptake of uorescent quinazoline compounds. (a) Artesunic acidquinazoline hybrids 8 and 9. (bd) Compounds 7h, 7g and 9 were analysed for properties of uorescence imaging using confocal laser-scanning microscopy. HFFs were cultivated in six-well plates on cover slips and were either directly incubated with compounds by addition to the culture media for 20 h (c) or were added after HCMV infection (b,d; compound addition at 50 h post infection, xation and analysis of cells 20 h later; scale bar, 10 mm). The chosen concentrations were 10 mM for compounds 7h, 7g and 9, or 1 mM for compound 9 in panels 4, 9 (b), panels 5, 10 (c) and panels 610 (d), referring to the compounds EC50 values of anti-HCMV activity (see Table 1). The depicted images are representative for three independent experiments. Lack of mycoplasm contamination was veried by routine 4,6-diamidino-2-phenylindole staining. General procedures of cell xation, indirect immunouorescence staining of proteins (d) and microscopic analysis have been described elsewhere36,37.

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Table 1 | EC50 values of anti-HCMV activity (AD169-GFP) determined in primary HFFs.

Compound HCMV EC50 (lM)*,w HFFs CC50 (lM)*,z Ganciclovir 2.60.5 4100

Artesunic acid 3.80.4 82.00.9 Artemisinin 410 4100 7b 9.54.8 4100 7g 4.90.2 4100 7h 4.60.9 4100 7i 0.60.1 41008 0.60.1 41009 0.10.0 4100

*The cell culture-based systems for the determination of EC and CC values (trypan blue exclusion assay) has been previously reported38.

wn 4. zn 6 (except artesunic acid: n 3).

GFP, green uorescent protein; HCMV, human cytomegalovirus; HFF, human broblast.

The fact that the studied antiviral quinazoline compounds are uorescent opens up a new vista of molecular imaging in drug development and mechanistic studies, avoiding the requirement of linkage of external uorescent markers to quinazoline drugs. Through uorescence these new compounds might then be analysed for details of putative biological activities, such as in vitro inhibitory properties against HCMV as demonstrated in the present study and also for the aspects of drug delivery in single cells and whole tissue.

In general, these unprecedented ndings have important future implications for the development of efcient drugs (for example, antiviral, anticancer and antimalarial) for clinics based on uorescent bioactive heterocycles as lead compounds.

Methods

General methods. For details of the reaction optimization procedures and product characterizations, see Supplementary Fig. 1, Supplementary Table 1 and Supplementary Methods. For X-ray data of compounds 4a, 4a0 and 7a, see

Supplementary Tables 24. For uorescent imaging with 5d, see Supplementary Fig. 2 and Supplementary Note 1. For photophysical studies of compound 7h, see Supplementary Fig. 3, Supplementary Methods and Supplementary Discussion. For antiviral studies of selected compounds in this manuscript, see Supplementary Figs 4 and 5, Supplementary Methods and Supplementary Note 2. For 1H, 13C NMR spectra of the compounds in this article, see Supplementary Figs 652.

Data availability. CCDC 1472467 (4a), 1472469 (4a0) and 1472470 (7a) contain the supplementary crystallographic data for this paper. These data are provided free of charge by The Cambridge Crystallographic Data Centre. The authors declare that all other data supporting the ndings of this study are available within the article and its Supplementary Information le.

References

1. Alafeefy, A. M., Kadi, A. A., Al-Deeb, O. A., El-Tahir, K. E. & Al-Jaber, N. A. Synthesis, analgesic and anti-inammatory evaluation of some novel quinazoline derivatives. Eur. J. Med. Chem. 45, 49474952 (2010).

2. Hutterer, C. et al. The chemical class of quinazoline compounds provides a core structure for the design of anticytomegaloviral kinase inhibitors. Antiviral Res. 134, 130143 (2016).

3. Madapa, S. et al. Search for new pharmacophores for antimalarial activity. Part II: synthesis and antimalarial activity of new 6-ureido-4-anilinoquinazolines. Bioorg. Med. Chem. 17, 222234 (2009).

4. Zhang, Y. et al. Synthesis and anticancer activities of 5,6,7-trimethoxy-N-phenyl(ethyl)-4-aminoquinazoline derivatives. Eur. J. Med. Chem. 66, 335344 (2013).

5. Erba, E., Pocar, D. & Valle, M. n-Triazolines. Part 41.1 A new synthesis of 2-alkylquinazolines and 2,9-dialkylpyrimido[4,5-b] indoles. J.Chem. Soc. Perkin Trans. 1, 421426 (1999).

6. Bedi, P. M., Kumar, V. & Mahajan, M. P. Synthesis and biological activity of novel antibacterial quinazolines. Bioorg. Med. Chem. Lett. 14, 52115213 (2004).

7. Zhang, Z.-H., Zhang, X.-N., Mo, L.-P., Li, Y.-X. & Ma, F.-P. Catalyst-free synthesis of quinazoline derivatives using low melting sugarureasalt mixture as a solvent. Green Chem. 14, 1502 (2012).

8. Karnakar, K., Kumar, A. V., Murthy, S. N., Ramesh, K. & Nageswar, Y. V. D. Recyclable graphite oxide promoted efcient synthesis of 2-phenyl quinazoline derivatives in the presence of TBHP as an oxidant. Tetrahedron Lett. 53, 46134617 (2012).

9. Du, Z. & Shao, Z. Combining transition metal catalysis and organocatalysis an update. Chem. Soc. Rev. 42, 13371378 (2013).

10. Deiana, L. et al. Highly enantioselective cascade transformations by merging heterogeneous transition metal catalysis with asymmetric aminocatalysis. Sci. Rep. 2, 851 (2012).

11. Loh, C. C., Badorrek, J., Raabe, G. & Enders, D. Merging organocatalysis and gold catalysis: enantioselective synthesis of tetracyclic indole derivatives through a sequential double Friedel-Crafts type reaction. Chem. Eur. J. 17, 1340913414 (2011).

12. Loh, C. C. & Enders, D. Merging organocatalysis and gold catalysisa critical evaluation of the underlying concepts. Chem. Eur. J. 18, 1021210225 (2012).

13. Hack, D. et al. Combining silver catalysis and organocatalysis: a sequential Michael addition/hydroalkoxylation one-pot approach to annulated coumarins. Org. Lett. 16, 51885191 (2014).

14. Tejedor, D., Mendez-Abt, G., Cotos, L. & Garcia-Tellado, F. Merging domino and redox chemistry: stereoselective access to di- and trisubstituted b,g-unsaturated acids and esters. Chem. Eur. J. 18, 34683472 (2012).

15. Nicewicz, D. A. & MacMillan, D. W. Merging photoredox catalysis with organocatalysis: the direct asymmetric alkylation of aldehydes. Science 322, 7780 (2008).

16. Tietze, L. F. Domino reactions in organic synthesis. Chem. Rev. 96, 115136 (1996).

17. Enders, D., Huttl, M. R., Grondal, C. & Raabe, G. Control of four stereocentres in a triple cascade organocatalytic reaction. Nature 441, 861863 (2006).18. Enders, D., Grondal, C. & Huttl, M. R. Asymmetric organocatalytic domino reactions. Angew. Chem. Int. Ed. 46, 15701581 (2007).

19. Grondal, C., Jeanty, M. & Enders, D. Organocatalytic cascade reactions as a new tool in total synthesis. Nat. Chem. 2, 167178 (2010).

20. Albrecht, L., Jiang, H. & Jorgensen, K. A. A simple recipe for sophisticated cocktails: organocatalytic one-pot reactionsconcept, nomenclature, and future perspectives. Angew. Chem. Int. Ed. 50, 84928509 (2011).

21. Zeng, X., Ni, Q., Raabe, G. & Enders, D. A branched domino reaction: asymmetric organocatalytic two-component four-step synthesis of polyfunctionalized cyclohexene derivatives. Angew. Chem. Int. Ed. 52, 29772980 (2013).

22. Tietze, L. F. Domino Reactions: Concepts for Efcient Organic Synthesis (Wiley-VCH, 2014).

23. Reiter, C. et al. Highly potent artemisinin-derived dimers and trimers: synthesis and evaluation of their antimalarial, antileukemia and antiviral activities. Bioorg. Med. Chem. 23, 54525458 (2015).

24. Bock, C. M. et al. Generation of complex azabicycles and carbobicycles from two simple compounds in a single operation through a metal-free six-step domino reaction. Chem. Eur. J. 22, 51895197 (2016).

25. Dupau, P., Epple, R., Thomas, A. A., Fokin, V. V. & Sharpless, K. B. Osmiumcatalyzed dihydroxylation of olens in acidic media: old process, new tricks. Adv. Synth. Catal. 344, 421433 (2002).

26. Kanis, D. R., Ratner, M. A. & Marks, T. J. Design and construction of molecular assemblies with large second-order optical nonlinearities. Quantum chemical aspects. Chem. Rev. 94, 195242 (1994).

27. Bendikov, M., Wudl, F. & Perepichka, D. F. Tetrathiafulvalenes, oligoacenenes, and their buckminsterfullerene derivatives: the brick and mortar of organic electronics. Chem. Rev. 104, 48914946 (2004).

8 NATURE COMMUNICATIONS | 8:15071 | DOI: 10.1038/ncomms15071 | http://www.nature.com/naturecommunications

Web End =www.nature.com/naturecommunications

NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15071 ARTICLE

28. Kulkarni, A. P., Kong, X. & Jenekhe, S. A. High-performance organic light-emitting diodes based on intramolecular charge-transfer emission from donor acceptor molecules: signicance of electron-donor strength and molecular geometry. Adv. Funct. Mater. 16, 10571066 (2006).

29. Das, P., Butcher, R. J. & Mukhopadhyay, C. Zinc titanate nanopowder: an advanced nanotechnology based recyclable heterogeneous catalyst for the onepot selective synthesis of self-aggregated low-molecular mass acceptordonor acceptoracceptor systems and acceptordonoracceptor triads. Green Chem. 14, 1376 (2012).

30. Loefer, F. F. et al. High-exibility combinatorial peptide synthesis with laser-based transfer of monomers in solid matrix material. Nat. Commun. 7, 11844 (2016).

31. Lurain, N. S. & Chou, S. Antiviral drug resistance of human cytomegalovirus. Clin. Microbiol. Rev. 23, 689712 (2010).

32. Reiter, C. et al. New efcient artemisinin derived agents against human leukemia cells, human cytomegalovirus and Plasmodium falciparum: 2nd generation 1,2,4-trioxane-ferrocene hybrids. Eur. J. Med. Chem. 97, 164172 (2015).

33. Tietze, L. F., Bell, H. P. & Chandrasekhar, S. Natural product hybrids as new leads for drug discovery. Angew. Chem. Int. Ed. 42, 39964028 (2003).34. Tsogoeva, S. B. Recent progress in the development of synthetic hybrids of natural or unnatural bioactive compounds for medicinal chemistry. Mini-Rev. Med. Chem. 10, 773793 (2010).

35. Frhlich, T., Capci Karagz, A., Reiter, C. & Tsogoeva, S. B. Artemisinin-derived dimers: potent antimalarial and anticancer agents. J. Med. Chem. 59, 73607388 (2016).

36. Steingruber, M. et al. The interaction between cyclin B1 and cytomegalovirus protein kinase pUL97 is determined by an qctive kinase domain. Viruses 7, 45824601 (2015).

37. Milbradt, J. et al. The prolyl isomerase Pin1 promotes the herpesvirus-induced phosphorylation-dependent disassembly of the nuclear lamina required for nucleocytoplasmic egress. PLoS Pathog. 12, e1005825 (2016).

38. Hutterer, C. et al. A novel CDK7 inhibitor of the Pyrazolotriazine class exerts broad-spectrum antiviral activity at nanomolar concentrations. Antimicrob. Agents Chemother. 59, 20622071 (2015).

Acknowledgements

We gratefully acknowledge the nancial support from Deutsche Forschungsgemeinschaft (DFG) by grants TS 87/17-1 (SPP 1807, Priority Programme Control of London Dispersion Interactions in Molecular Chemistry), TS 87/15-1, TS 87/16-3 and MM 1289/7-1/ 7-3. We also thank the Graduate School Molecular Science (GSMS), Interdisciplinary Center for Molecular Materials (ICMM), the Wilhelm Sander-Stiftung (Grants Nr. 2014.019.1 and 2011.085.1-2), BMBF (Grant Nr. 031A095C) and Emerging Fields

Initiative (EFI) Chemistry in Live Cells supported by Friedrich-Alexander-Universitat Erlangen-Nrnberg for research funding.

Author contributions

F.E.H. and A.A.G. conducted the domino reactions and one-pot multistep transformations. F.E.H. contributed to the development and evaluation of the scope of 2,6-dicyanoanilines and quinazolines synthesis. A.A.G. contributed to the scope of 2,6-dicyanoanilines synthesis. T.F. performed the synthesis of artesunic acidquinazoline hybrid molecules. F.H. conducted the X-ray analysis. A.K. performed the photophysical measurements. H.B. and C.H. performed antiviral tests and cytotoxicity studies. M.S. performed confocal microscopy analyses. M.M. and C.H. supervised the biological investigations of new quinazoline molecules, evaluated data and included the relevant sections into the manuscript. C.v.B.-K., D.S.M., T.F. conducted and A.N.-M. supervised the laser-induced imaging with selected new uorophore. S.B.T. conceived and directed the research, supervised the synthetic experiments and wrote the manuscript; all authors discussed the results and commented on the manuscript.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Competing interests: The authors declare no competing nancial interests.

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How to cite this article: Held, F. E. et al. Facile access to potent antiviral quinazoline heterocycles with uorescence properties via merging metal-free domino reactions. Nat. Commun. 8, 15071 doi: 10.1038/ncomms15071 (2017).

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