Toward Secure Supply of Remdesivir via a 2-Pot Triazine

doi.org/10.26434/chemrxiv.12751124.v1

Toward Secure Supply of Remdesivir via a 2-Pot Triazine Synthesis:Supply Centered SynthesisDinesh J. Paymode, Flavio S. P. Cardoso, Joshua D. Sieber, John W. Tomlin, Daniel W. Cook, Justina Burns,Rodger W. Stringham, B. Frank Gupton, David Snead, Toolika Agrawal

Submitted date: 01/08/2020 • Posted date: 10/08/2020Licence: CC BY-NC-ND 4.0Citation information: Paymode, Dinesh J.; Cardoso, Flavio S. P.; Sieber, Joshua D.; Tomlin, John W.; Cook,Daniel W.; Burns, Justina; et al. (2020): Toward Secure Supply of Remdesivir via a 2-Pot Triazine Synthesis:Supply Centered Synthesis. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.12751124.v1

Pyrrolotriazine 1 is an important precursor to Remdesivir, and an efficient synthesis is disclosed. This routefeatures atom economy and reduced derivatization of starting materials, by making use of highly abundant,commoditized raw material inputs. The yield of triazine was doubled from 31% to 59%, and the synthetic stepcount was reduced from 4 to 2. A one-pot cascade sequence was developed for direct cyanation of pyrrole.Amination and cyclization with formamidine acetate complete the synthesis. The problematic nature oftypically dilute electrophilic aminations was solved with semi-continuous processing. Moreover, developmentof a continuous platform afforded access to the ideal yet non-commercial aminating reagent, monochloramine.These efforts help to secure the Remdesivir supply chain.

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https://chemrxiv.org/articles/preprint/Toward_Secure_Supply_of_Remdesivir_via_a_2-Pot_Triazine_Synthesis_Supply_Centered_Synthesis/12751124/1?file=24128462



Toward Secure Supply of Remdesivir via a 2-Pot Triazine Synthesis: Supply Centered Synthesis Paymode, Dinesh J.†a; Cardoso, Flavio S.P.†a; Agrawal, Toolikaa; Tomlin, John W.a; Cook, Daniel W.b; Burns, Justina M.b; Stringham, Rodger W.b; Sieber, Joshua D.a; Gupton, B. Franka; Snead, David R.*a

AUTHOR ADDRESS a) Chemical Development, Medicines for All Institute, 737 N. 5th St., Box 980100, Richmond VA, 23298-0100 b) Analytical Development, Medicines for All Institute, 737 N. 5th St., Box 980100, Richmond VA, 23298-0100

Supporting Information Placeholder

ABSTRACT: Pyrrolotriazine 1 is an important precursor to Remdesivir, and an efficient synthesis is disclosed. This route fea-tures atom economy and reduced derivatization of starting materi-als, by making use of highly abundant, commoditized raw material inputs. The yield of triazine was doubled from 31% to 59%, and the synthetic step count was reduced from 4 to 2. A one-pot cas-cade sequence was developed for direct cyanation of pyrrole. Ami-nation and cyclization with formamidine acetate complete the syn-thesis. The problematic nature of typically dilute electrophilic ami-nations was solved with semi-continuous processing. Moreover, development of a continuous platform afforded access to the ideal yet non-commercial aminating reagent, monochloramine. These efforts help to secure the Remdesivir supply chain.

■ Introduction: The Need for Supply Chain Improvements COVID-19’s emergence has greatly elevated awareness of the

pharmaceutical supply chain’s importance,1 and the desire to make remdesivir broadly available presents a case in point.2 Initial sup-ply is constrained2b-c after it emerged as a viable COVID-19 treat-ment,2a and Gilead subsequently donated the existing API stock.2d Production of final drug substance is a challenge and can take up to one year as a result of scarce, long lead-time raw materials2b-c and several low yielding steps.3 Due to issues of pricing and licensing, countries are left questioning who will or won’t have access to the drug.2f A more robust supply chain can be developed by inventing from inherently available building blocks (commodities) and in-creasing the route’s yields and throughput. We recently published on this topic which we term “Supply Centered Synthesis” (SCS) of API.4

We examined this topic of supply chain security within the con-struct of Remdesivir’s pyrrolotriazine synthesis (Fig. 1). The tria-zine passes through several low yielding steps, and as an early raw material, large quantities are required. We wondered if a preferred route could help overcome recent challenges related to supply and price. The only full route to the base triazine 1 was published by Bayer Healthcare5,6 despite the biological importance of the pyr-rolo[2,1-f][1,2,4]triazine framework.7 The sequence above nicely supplied preparative quantities of triazine (50 g scale); however, some aspects of the route warrant improvement.

1. The yield is 31% over four steps. Increasing yield would decrease consumption of raw materials.

2. Hydrazine is protected as a carbazate, and then carbamate 2 must be deprotected to reveal amine 3. This creates mass inefficiency, adds to the step-count, and decreases overall yield.

3. 2,5-Dimethoxytetrahydrofuran and tert-butyl carbazate are not commodity materials. 2,5-Dimethoxytetrahydrofuran is made in 2-steps from furan.8 tert-Butyl carbazate is made in 4 chemical steps from tert-butanol and ammonia.9 Deri-vatizing commodities requires additional chemical pro-cessing and creates waste.

OCNO

NN

N

NH2

HO OH

POH

NOPh

O

O

NNH2

CN

O

OMe

OMe 59%N

NH

CN

HCl/Dioxane

77%

85%

Literature Route to Triazine:

NN

N

NH2

K3PO4, EtOH81%

31% over 4 Steps

NC

OClO2S

Remdesivir

NH•HOAcHN

ON

NH O

H2NNH

O O

O

O

then NH2OHNH

1) POCl3, DMF

NH

C

NN

NCNH2

2) NH2Cl, then

NH2•HOAcHN

N

Isolated Intermediates (Step Count):Yield:Protecting Groups?

4 2

31 59Yes No

2,5-(MeO)2(OC4H6) Pyrrole1 50

Derivatized Raw Materials? Yes No

Market Volume(MT)10

Market Volume(MT)10Here:Lit.:

RawMaterial

RawMaterial

Proposed Route to Triazine:

93%

63%

tert-Butyl Carbazate

Sulfonyl Isocyanate 200

0.4

NaOCl

NH3

NH2OH•HClNH4Cl 11,200

1100

1,100,000384,000

4

1

13

2

Figure 1: An atom efficient route to triazine from abundant chem-icals.

The ideal synthesis would increase overall yield, proceed from commodity materials, and reduce step-count, thus strengthening the remdesivir supply chain. This work describes efforts to do so from pyrrole which is abundant and made commercially in 1 step from furan,11 Formyl groups are easily installed in the 2-position of pyr-roles,12 and aldehydes can be oxidized to nitriles via aldoxime in-termediates.13 Moreover, N-amination of pyrroles and indoles, though challenging, are known.14 The hypothetical synthesis

would intercept the penultimate intermediate 3 at this point, and the demonstrated literature condensation with formamidine acetate would render triazine 1. One could foresee how these transfor-mation could be executed with reagents having high atom effi-ciency and market availability. Perhaps efficiency could be further increased by reducing the step-count and thus eliminating associ-ated workups. ■ Results and Discussion I: A One-Pot Oxidative Vilsmeier Cascade

NH NH NH

CNNCl then

NH2OH, HOR

Ac2O, PyridineTemp., 16 hr

EntryScale

(g)Solvent

(Vol)Pyridine(Equiv.) 4, AY

4, IY (Purity)

Temp.(°C)

HOR(Vol)

1a 0.2 DMF (10)- EtOH, 3 90 84 -

2a 0.2 DMF (10)- H2O, 3 90 78 -

3 0.2 DMF (10) 5 EtOH, 3 90 93 -

4 0.2 DMF (10) 5 H2O, 3 90 92 -

5 0.2 MeCN (10) 5 EtOH, 3 90 88 -

6 0.2 MeCN (5) 3.5 EtOH, 3 90 85 -

7 5 MeCN (10) 3.5 H2O, 3 70 - 93 (80)8b 25 MeCN (10) 5 H2O, 3 70 76 (90)

10 100 DMF (10) 5 H2O, 3 90 94 (85)

-

-

4

a) Ac2O not added to reaction mixture. b) Purified by distillation.

POCl3, DMF

Solvent20 °C, 1 hr

9b 25 DMF (10) 5 H2O, 3 90 90 (89)-

Figure 2: A simple one-pot cyanation.

This effort began with functionalization of pyrrole (Fig. 2). We wondered whether isolation of the aldehyde or aldoxime interme-diate was necessary. 2-Formyl pyrrole is a low melting solid not easily distilled or recrystallized in good yield. Moreover, waste will be generated in the process of purifying the aldehyde, aldoxime or other intermediates. Perhaps the iminium chloride salt could be used directly to form nitrile 4 in a one-pot process. Residual POCl3 and related species would need to be quenched due to chemical in-compatibility with hydroxylamine; however, the HCl generated in the course of the quench could be used as catalyst for the dehydra-tion of aldoxime.15 This concept was validated experimentally with surprising simplicity by adding water or ethanol prior to adding the hydroxylamine salt to give the product in >80% AY (Entries 1-2). A recent paper nicely demonstrated use of DMPU·HCl as a key additive, but in our case, DMF as solvent worked sufficiently well.15c The yield was further improved to 90% AY by activating the oxime formed in situ with acetic anhydride and base (Entries 3-6). Distillation provided pure material for downstream investiga-tions (Entries 8-9). There are very few examples of one-pot nitrile formation via strategies that make use of an oxidative Vilsmeier cascade.16 This avoids the use of costly and high molecular weight iodine. II: Assessing Amination Feasibility The conditions of Hynes Jr. were used as a starting point to explore the critical amination (Fig. 3). Chloramine was made from bleach and ammonia then extracted into MTBE. Pyrrole 4 converted cleanly to the N-amino product (95% AY) despite reports of mod-erate yield. 14b,17 This serves as a drop-in replacement for 3 and improves yield to 88% as compared to 41% by the literature route. Unfortunately the reaction conditions are highly dilute (1 wt%) as a function of extracting monochloramine from water into MTBE, thus limiting overall throughput and jeopardizing supply. For this reason, we sought strategies which would increase throughput of N-amino-2-cyanopyrrole. This was also the likely

NNH2

CN1) NaH, DMF

2) NH2Cl in MTBE (100 Vol.)NH

CN

95% AYStrategies to Increase Throughput:

1) Use an alternative aminating reagent which is available in solid form2) Increase concentration of NH2Cl in MTBE or other solvent3) Run biphasic amination to continuously extract NH2Cl into organic media4) Generate gaseous NH2Cl to remove dependency upon organic extraction5) Continuously produce and consume NH2Cl with continuous MTBE recycle6) Substitute liquid NaOCl with solid Ca(OCl)27) Consider an alternative amination strategy such as Hofmann degradation or

nitrosylation/reduction

NH2Cl:

Extracted from mix of NH4Cl (s),

NH4OH (aq.), NaOCl (aq.)

4 3

Figure 3: Clean amination of pyrrole from bleach and ammonia under highly dilute conditions. conclusion of Bristol Myers Squibb, whose subsequent disclosures focused on improved efficiencies via in situ chloramine production in a biphasic mixture18 and production of gaseous chloramine to negate the need for dilute organic solution.19

We did not have success with the biphasic conditions and did not have access to the degree of engineering required for gaseous chlo-ramine generation; however use of solid aminating reagents such as O-(4-Nitrobenzoyl)hydroxylamine provided a means to run re-actions at 15-20 volumes, thus greatly increasing product through-put.20 This reagent has been used at scale;14d however, neither aminating reagent is available at commodity levels, they are not atom efficient, and there are safety concerns with use of these rea-gents at increasing temperatures and concentrations.21 We thus fo-cused on producing solutions to complement these existing strate-gies. III: Increasing Amination Space-Time Yield

The remainder of our investigation centered on use of monochlo-ramine. It is an optimal reagent to install the nitrogen atom because atom efficiency is high, and it is made from simple ingredients which can be accessed anywhere in the world, bleach and ammo-nia. To render monochloramine accessible, the volumes of extrac-tion solvent (MTBE) must be mitigated. We sought better under-standing of chloramine preparation and use, and produced 0.5-0.9 M NH2Cl/MTBE solutions which were five to ten times stronger than the reported value. Our study suggested that the literature pro-cedure added NH2Cl in four-fold excess. Decreasing chloramine equivalents would greatly increase concentration by reducing MTBE consumption.

Probing the relationship between base and chloramine equiva-lents showed an interdependent nature, where as much chloramine as base is required (Fig. 4). Perhaps this is due to the acidic nature of chloramine which has an estimated pKa of 14. With 1.25 equiv-alents of base and 0.5 M NH2Cl, 25 volumes of MTBE are needed. In theory this can be decreased to a minimum of 17 volumes with a 0.9 M NH2Cl and 1.1 equivalents of NaH, which is an 80% re-duction in solvent usage. Still, dilution remains at levels higher than desired.

Perhaps, an effectively high concentration “gaseous” form of chloramine can be accessed by recycling MTBE solvent (Fig. 4). The reagent can be considered gaseous because at the extreme, in-finite recycle of MTBE, only chloramine, a gas, is consumed. Ad-dition of chloramine in fractional charges with subsequent evapo-ration and recycle of MTBE would seem to present one such option for achieving that aim. With this strategy in mind, the chloramine was added to 4 in four portions of 10 volumes with the MTBE re-moved and reused at the end of each addition. The effective chlo-ramine concentration becomes 2.0 M rather than 0.5 M. Assay yields of 3 matched those of the original procedure, and the total reaction volume was limited to 15 volumes (10 MTBE, 5 DMF) with an end-point of 5 volumes and 20 wt% pyrrole in DMF.

NNH2

CN1) NaH, DMF

2) NH2Cl in MTBENH

CN

4 3

Figure 4: a) Lower equivalents of chloramine can be used at lower loadings of NaH. b) Addition of NH2Cl in multiple charges with subsequent solvent recycle increases throughput and decreases sol-vent consumption.

Effective cycle time for this operation can be achieved because MTBE is easily removed from reaction solution due to its low boil-ing point (55 ﾟC), and because the amination of pyrrole is very rapid, occurring in less than 5 minutes. This presents a reasonable path toward manufacturing, and 90% of solvent was recycled. Aminated pyrrole was made in 90% assay yield, and this result was scaled 100x to 10 g without change in performance. We concluded our amination investigation by addressing the hazards surrounding use of NaH in conjunction with DMF.20,23 IV: On-Demand Chloramine

Even with a solution to the challenge of running dilute electro-philic aminations and despite the advantages of monochloramine, preparation and use of NH2Cl at scale poses further practical chal-lenges. The reagent needs to be made on site as it is not available for purchase. One reason for the lack of market presence is likely a result of the limited half-life of chloramine which is on the order of days,22 and also the difficulty in controlling titer of chloramine solutions. Chloramine is a dissolved gas and has a tendency to ef-

fervesce out of solution. The need for onsite production would re-quire a very large storage tank to hold the NH2Cl/MTBE solution—up to 40 reaction volumes are required.

Production of a chloramine generator24 via continuous pro-cessing presents a solution for on-demand production, reagent in-stability, and solvent recycle (Fig. 5). Creation of a captive solvent recycle loop prevents build up of large solvent volumes which can limit space time yield. Further, continuous chloramine generation fits within a semi-continuous amination motif which benefits from frequent volume reduction via application of vacuum. High con-centration aqueous chloramine solutions have been produced for on-site water treatment and manufacturing via a continuous stirred-tank reactor (CSTR) at concentrations up to 2 M.25 To demon-strate proof of concept, all that is needed is to modify the conditions of Hynes for continuous synthesis and separation. Chloramine was simultaneously made and extracted into MTBE in a CSTR with a 10 minute residence time. This biphasic mixture flowed into a gravity separator. Steady-state was reached within 30 minutes, and titration of the MTBE layer showed that ~0.45 M chloramine was produced as compared to 0.52 M solution as made in batch.

NNH2

CN1) NaH, DMF

2) NH2Cl in MTBE (0.48 M)NH

CN

4 3 Figure 5: Steady state operation of chloramine CSTR and sche-matic for chloramine generator.

The on-demand NH2Cl solution was flowed into a pot containing a solution of deprotonated pyrrole 4 in DMF. The system was placed under occasional vacuum to keep the reaction volume at a minimal level by splitting the charge of chloramine into four por-tions. The MTBE was collected by condensation in a separate pot, and then recycled to extract more chloramine. The total recycle rate of MTBE was 80-89%, and pyrrole 4 was aminated in 94% AY (1 g, 10 g scales). Optionally, the amination can also be conducted in flow. This demonstrates proof of concept for a chloramine gen-erator which helps overcome throughput issues related to dilute aminations. VI: Amination and Triazine Formation in One-Pot

For efficiency’s sake we elected to telescope the synthesis through to the triazine rather than isolate at this stage (Fig. 6). Our prior experience with hydroxypropyl adenine (HPA) suggested that DMF might be a good solvent for the condensation reaction of the amino nitrile with formamidinium acetate.26 Formamidine acetate was added to the amination reaction mixture and heated to form the triazine with this thought in mind. This provided material in 75% AY over two steps. A quick solubility study suggested that water or MTBE would be appropriate antisolvents. In situ concentration of reaction mixture followed by addition of water afforded the de-sired triazine in 60% IY over two steps.

NNH2

CN

NN

N

NH21) NaH, DMF

2) NH2Cl in MTBE rt, 30 min

NH

CN NH2•HOAcHN3)

EntryScale

(g)1,Purity

(%)1, IY (%)

1, AY (%)

1 2.8 74 62 82

2 10 76 64 78

3a 10 - 63 98

4a 10 75 60 99

DMF, 90 °C, 16 hr4 3 1

a) 2nd Stage purification by trituration with MTBE. See SI for details.

Figure 6: One-pot triazine synthesis from cyanopyrrole 4. ■ Conclusions

This work describes an efficient means to produce the aminotri-azine required for manufacturing remdesivir. Importantly, the syn-thesis makes use of highly abundant materials to bolster supply chain security. It is expected that these common materials will de-crease costs associated with chemical inputs, an important objec-tives given concerns around remdesivir supply and price. The syn-thesis has high atom economy and avoids derivatization and pro-tecting groups. Yield is approximately doubled over the prior pro-cedure described for the triazine while step count is cut in half. This should facilitate improved throughput of this important API inter-mediate. The chemical solution involves a novel one-pot cascade for nitrile formation, avoiding generation and isolation of aldehyde as an intermediate. Further, the work presents a solution to the challenge of limited space time yield posed by highly dilute elec-trophilic amination conditions and enables access to non-commer-cial monochloramine, an atom efficient and desirable reagent.

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimentals (PDF) Compound characterization (PDF)

AUTHOR INFORMATION

Corresponding Author David R. Snead 737 N. 5th St. Box 980100 Richmond, Virginia 23298-0100 email: [email protected]

Author Contributions †These authors contributed equally. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT

We thank the Bill and Melinda Gates Foundation for their-longstanding support of our research. In addition, we express grat-itude to Trevor Laird and John Dillon for their thoughtful commen-tary and discussion throughout this work. We also thank Silpa Sundaram and Dr. Susan Hershenson for fostering an ecosystem where rapid decisions on project direction can be made.

REFERENCES 1) a) Mullin, R. “COVID-19 is reshaping the pharmaceutical supply

chain”, Chem. Eng. News, 2020, 98. b) Mullin, R. “Bringing drug production back to the US”, Chem. Eng. News, 2020, 98. c) Wood-cock, J.; Safeguarding Pharmaceutical Supply Chains in a Global Economy, U.S. Food and Drug Administration. https://www.fda.gov/news-events/congressional-testimony/safe-guarding-pharmaceutical-supply-chains-global-economy-10302019 (Accessed July 10, 2020).

2) a) An Open Letter from our Chairman & CEO, Gilead Sciences, Inc. https://stories.gilead.com/articles/an-open-letter-from-our-chairman-and-ceo-april-29 (Accessed July 1, 2020). b) Working to Supply Remdesivir for COVID-19, Gilead Sciences, Inc. https://www.gilead.com/purpose/advancing-global-health/covid-19/working-to-supply-remdesivir-for-covid-19 (Accessed July 1, 2020). c) Gilead Sciences Statement on Expanding Global Supply of Investigational Antiviral Remdesivir, Gilead Sciences, Inc. https://www.gilead.com/news-and-press/company-statements/gil-ead-sciences-statement-on-remdesivir-global-supply (Accessed July 1, 2020). d) An Update on COVID-19 from our Chairman & CEO, Gilead Sciences, Inc. https://stories.gilead.com//articles/an-update-on-covid-19-from-our-chairman-and-ceo (Accessed July 1, 2020). e) An Open Letter from Daniel O’Day, Chairman & CEO, Gilead Sciences, Gilead Sciences, Inc. https://www.gil-ead.com/news-and-press/press-room/press-releases/2020/6/an-open-letter-from-daniel-oday-chairman--ceo-gilead-sciences (Ac-cessed July 1, 2020). f) Trump Administration Secures New Sup-plies of Remdesivir for the United States, U.S. Department of Health and Human Services. https://www.hhs.gov/about/news/2020/06/29/trump-administra-tion-secures-new-supplies-remdesivir-united-states.html (Ac-cessed July 10, 2020).

3) a)Siegel, D.; Hui, H.C.; Doerffler, E.; Clarke, M.O.; Chun, K.; Zhang, L.; Neville, S.; Carra, E.; Lew, W.; Ross, B.; Wang, Q.; Wolfe, L.; Jordan, R.; Soloveva, V.; Knox, J.; Perry, J.; Perron, M.; Stray, K.M.; Barauskas, O.; Feng, J.Y.; Xu, Y.; Lee, G.; Rheingold, A.L.; Ray, A.S.; Bannister, R.; Strickley, R.; Swaminathan, S.; Lee, W.A.; Bavari, S.; Cihlar, T.; Lo, M.K.; Warren, T.K.; Mack-man, R.L. J. Med. Chem., 2017, 60, 1648-1661. b) De Savi, C.; Hughes, D.L.; Kvaerno, L. Org. Process Res. Dev., 2020, 24, 940-976. c) Vieira, T.; Stevens, A.C.; Chtchemelinine, A.; Gao, D.; Ba-dalov, P.; Heumann, L. Org. Process Res. Dev., 2020, ASAP.

4) a) Snead, D.R.; McQuade, D.T.; Ahmad, S.; Krack, R.; Stringham, R.W.; Burns, J.M.; Abdiaj, I.; Gopalsamuthiram, V.; Nelson, R.C.;

mailto:[email protected]

https://www.fda.gov/news-events/congressional-testimony/safeguarding-pharmaceutical-supply-chains-global-economy-10302019



https://stories.gilead.com/articles/an-open-letter-from-our-chairman-and-ceo-april-29

https://stories.gilead.com/articles/an-open-letter-from-our-chairman-and-ceo-april-29

https://www.gilead.com/purpose/advancing-global-health/covid-19/working-to-supply-remdesivir-for-covid-19

https://www.gilead.com/purpose/advancing-global-health/covid-19/working-to-supply-remdesivir-for-covid-19

https://www.gilead.com/news-and-press/company-statements/gilead-sciences-statement-on-remdesivir-global-supply

https://www.gilead.com/news-and-press/company-statements/gilead-sciences-statement-on-remdesivir-global-supply

https://stories.gilead.com/articles/an-update-on-covid-19-from-our-chairman-and-ceo

https://stories.gilead.com/articles/an-update-on-covid-19-from-our-chairman-and-ceo

https://www.gilead.com/news-and-press/press-room/press-releases/2020/6/an-open-letter-from-daniel-oday-chairman--ceo-gilead-sciences



https://www.hhs.gov/about/news/2020/06/29/trump-administration-secures-new-supplies-remdesivir-united-states.html

https://www.hhs.gov/about/news/2020/06/29/trump-administration-secures-new-supplies-remdesivir-united-states.html

Gupton, B.F. Org. Process Res. Des., 2020, 24, 1194-1198. b) Kashinath, K.; Snead, D.R.; Burns, J.M.; Stringham, R.W.; Gup-ton, B.F.; McQuade, D.T.M. Org. Process Res. Dev., 2020, ASAP.

5) a) Dixon, J.A.; Phillips, B.; Achebe, F.; Kluender, H.C.E.; New-com, J.; Parcella, K.; Magnuson, S.; Hong, Z.; Zhang, Z.; Liu, Z.; Khire, U.; Wang, L.; Michels, M.; Chandler, B.; O’Connor, S. US8143393, 2006. b) O’Connor, S.; Dumas, J.; Lee, W.; Dixon, J.; Cantin, D.; Gunn, D.; Burke, J.; Phillips, B.; Lowe, D.; Shele-khin, T.; Wang, G.; Ma, X.; Ying, S.; McClure, A.; Achebe, F.; Lobell, M.; Ehrgott, F.; Iwuagwu, C.; Parcella, K. US8431695, 2006.

6) Private correspondence with generic manufacturers confirmed that this route is used to supply material for remdesivir.

7) a) Yu’Ning, S.; Peng, Z.; Qingzhu, A.; Xinyong, L. Curr. Pharm. Des., 2013, 19, 1528-1548. b) Cascioferro, S.; Parrino, B.; Spano, V.; Carbone, A.; Montalbano, A.; Barraja, P.; Diana, P.; Cirrin-cione, G. Eur. J. Med. Chem., 2017, 142, 328-375.

8) Hoydonckx, H.E.; Van Rhijn, W.M.; Van Rhijn, W.; DeVos, D.E.; Jacobs, P.E. “Furfural and Derivatives”, in Ullmann’s Encyclope-dia of Industrial Chemistry, Weinham, Germany: Wiley-VCH Ver-lag GmbH & Co. KGaA, 2000.

9) a) Tamura, M.; Yamada, N.; Ohta, M.; Yahata, T. EP0256559, 1987. b) Schirmann, J.-P.; Bourdauducq, P. “Hydrazine”, in Ullmann’s Encyclopedia of Industrial Chemistry, Weinham, Ger-many: Wiley-VCH Verlag GmbH & Co. KGaA, 2000.

10) Market volumes taken from Indian import records. 11) Harreus, A.L. “Pyrrole”, in Ullmann’s Encyclopedia of Industrial

Chemistry, Weinham, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2000.

12) a) Silverstein, R.M.; Ryskiewicz, E.E.; Willard, C.; Koehler, R.C. J. Org. Chem., 1955, 20, 668-672. b) ban den Broek, S.A.M.W.; Leliveld, J.R.; Becker, R.; Delville, M.M.E.; Nieuwland, P.J.; Koch, K.; Rutjes, F.P.J.T. Org. Process Res. Dev., 2012, 16, 934-938.

13) a) Capdevielle, P.; Lavigne, A.; Maumy, M. Synthesis, 1989, 6, 451-452. b) Wang, E.-C.; Lin, G.-J. Tetrahedron Letters, 1998, 39, 4047-4050. c) Dugar, S.; Chakravarty, S.; Conte, A.; Axon, J.; McEnroe, G.; Murphy, A. US7223766, 2004. d) Hwu, J.R.; Wong, F.F. Eur. J. Org. Chem., 2006, 11, 2513-2516. e) Aydin, A.E.; Yuksekdanaci, S. Tetrahedron: Asymmetry, 2013, 24, 14-22. f) Nandi, G.C.; Laali, K.K. Tetrahedron Letters, 2013, 54, 2177-2179. g) An, X.-D.; Yu, S. Org. Lett., 2015, 17, 5064-5067.

14) a) Somei, M.; Matsubara, M.; Kanda, Y.; Natsume, M. Chem. Pharm. Bull., 1978, 26, 2522-2534. b) Hynes, J.; Doubleday, W.W.; Dyckman, A.J.; Godfrey, J.D.; Grosso, J.A.; Kiau, S.; Leftheris, K. J. Org. Chem., 2004, 69, 1368-1371. c) Weiberth,

F.J.; Hanna, R.G.; Lee, G.E.; Polverine, Y.; Klein, J.T. Org. Pro-cess Res. Dev., 2011, 15, 704-709. d) Shi, Z.; Kiau, S.; Lobben, P.; Hynes Jr., J.; Wu, H.; Parlanti, L.; Discordia, R.; Doubleday, W.W.; Leftheris, K.; Dyckman, A.J.; Wrobleski, S.T.; Dambalas, K.; Tummala, S.; Leung, S.; Lo, E. Org. Process. Res. Dev., 2012, 16, 1618-1625.

15) a) Liebscher, J.; Neumann, B.; Hartmann, H. J. Prakt. Chem., 1983, 325, 915-918. b) Kumar, H.M.S.; Reddy, B.V.S.; Reddy, P.T.; Yadav, J.S. Synthesis, 1999, 4, 586-587. c) Mudshinge, S.R.; Potnis, C.S.; Xu, B.; Hammond, G.B. Green Chem., 2020, 22, 4161-4164.

16) Ushijima, S.; Moriyama, K.; Togo, H. Tetrahedron, 2012, 68, 4588-4595.

17) a) Patil, S.A.; Otter, B.A.; Klein, R.S. J. Het. Chem., 1994, 31, 781-786. b) Simmen, K.A.; Lin, T.-I.; Lenz, O.; Surleraux, D.L.N.G.; Raboisson, P. J.-M.B. WO2006035061, 2006. c) Xin,M.; Zhang, L.; Tang, F.; Tu, C.; Wen, J.; Zhao, X.; Liu, Z.; Cheng, L.; Shen, H. Bioorg. Med. Chem., 2014, 22, 1429-1440. d) Ning, X.; Li, M.; Hu, H.; Dai, W.; Li, X.; Wang, T.; Wu, Y. WO2016190847, 2015.

18) Bhattacharya, A.; Patel, N.C.; Plata, E.; Peddicord, M.; Ye, Q.; Par-lanti, L.; Palaniswamy, V.A.; Grosso, J.A. Tetrahedron Letters, 2006, 47, 5341-5343.

19) Tummala, S.; Leung, S.W.; Lo, E.T.; Alvarez, M.M. US7070751, 2003.

20) See Supporting Information for more details. 21) Tran, T.P.; Fisher, E.L.; Wright, A.S.; Yang, J. Org. Process Res.

Dev., 2018, 22, 166-172. 22) a) Ura, Y.; Sakata, G. “Chloramines”, in Ullmann’s Encyclopedia

of Industrial Chemistry, Weinham, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2000. b) Lutze, H.V. “Water, 6. Treatment by Oxidation Processes” in Ullmann’s Encyclopedia of Industrial Chemistry, Weinham, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2000.

23) Yang, Q.; Sheng, M.; Henkelis, J.J.; Tu, S.; Wiensch, E.; Zhang, H.; Zhang, Y.; Tucker, C.; Ejeh, D.E. Org. Process Res. Dev., 2019, 23, 2210-2217.

24) For examples of chemical generators see: Dallinger, D.; Gutmann, B.; Kappe, C.O. Acc. Chem. Res., 2020, 53, 1330-1341.

25) Delalu, H.; Peyrot, L.; Elkhatib, M.; Counioux, J.-J.; Cohen, A. US6222071, 2000.

26) Derstine, B.P.; Tomlin, J.W.; Peck, C.L.; Dietz, J.-P.; Herrera, B.T.; Cardoso, F.S.P.; Paymode, D.J.; Yue, A.C.; Arduengo, A.J.; Opatz, T.; Snead, D.R.; Stringham, R.W.; McQuade, D.T.M.; Gup-ton, B.F. Org. Process Res. Dev., 2020, ASAP.

Graphical Abstract

OCNO

NN

N

NH2

HO OH

POH

NOPh

O

O

Remdesivir

then NH2OHNH

1) POCl3, DMF

NH

C

NN

NCNH2

2) NH2Cl, then

NH•HOAcHN

N

Isolated Intermediates (Step Count):Yield:Protecting Groups?

4 2

31 59Yes No

2,5-(MeO)2(OC4H6) Pyrrole1 50

Derivatized Raw Materials? Yes No

Market Volume(MT)

Market Volume(MT)Here:Lit.:

RawMaterial

RawMaterial

Proposed Route to Triazine:

93%

63%

tert-Butyl Carbazate

Sulfonyl Isocyanate 200

0.4

NaOCl

NH3

NH2OH•HClNH4Cl 11,200

1100

1,100,000384,000

download fileview on ChemRxiv2020_07_24 Triazine ChemRXIV.pdf (400.46 KiB)



Toward Secure Supply of Remdesivir via a 2-Pot Triazine Synthesis: Supply Centered Synthesis Paymode, Dinesh J.†a; Cardoso, Flavio S. P.†a; Agrawal, Toolikaa; Tomlin, John W.a; Cook, Daniel W.b; Burns, Justina M.b; Stringham, Rodger W.b; Sieber, Joshua D.a; Gupton, B. Franka; Snead, David R.*a

Supplemental Information:

Contents General Remarks ..............................................................................................................................2

One-pot Synthesis of 2-Cyanopyrrole from Pyrrole ..........................................................................3

N-Amination of 2-Cyanopyrole Using Solid Aminating Reagents ................................................... 11

Investigation of NH2Cl Extraction .................................................................................................. 16

Continuous Production of Chloramine ........................................................................................... 18

Batch Amination in batch with NH2Cl from CSTR ......................................................................... .19

Continuous Amination in PFR with NH2Cl from CSTR ................................................................. 27

Batch Amination with Multicharge of Chloramine ......................................................................... 30

Amination and Triazine Formation in One-Pot .............................................................................. 32

Evaluation of Alternative Bases and Solvents for Amination .......................................................... 37

Analytical Method Section: ............................................................................................................. 39

References ...................................................................................................................................... 41

General Remarks

Instrumentation: For all compounds, H and C NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer. Chemical shifts were measured relative to the residual solvent resonance for 1H and 13C NMR (CDCl3 = 7.26 ppm for 1H and 77.2 ppm for 13C, DMSO-d6 = 2.50 ppm for 1H and 39.2 ppm for 13C). Coupling constants J are reported in hertz (Hz). The following abbreviations were used to designate signal multiplicity: s, singlet; d, doublet; t, triplet; q, quartet, p, pentet; dd, doublet of doublet; ddd, doublet of doublet of doublet; dt, double of triplet; ddt, doublet of doublet of triplet; m, multiplet; br, broad. Reactions were monitored by GC-MS or HPLC using the methods indicated. 2-Cyanopyrrole, N-amino-2-cyanopyrrole and triazine were monitored using identical HPLC methods (see Analytical Method Section for details). Glassware was oven-dried at 120 °C, assembled while hot, and cooled to ambient temperature under an inert atmosphere. Unless noted otherwise, reactions involving air sensitive reagents and/or requiring anhydrous conditions were performed under a nitrogen atmosphere.

Reagents and solvents. Reagents and solvents were purchased from Aldrich Chemical Company, Fisher Scientific, Alfa Aesar, Acros Organics, Oakwood, or TCI. Liquid reagents were purified by distillation when necessary. Unless otherwise noted, solid reagents were used without further purification. Methylene chloride (DCM) and dimethylformamide (DMF) taken from a solid-sorbant Solvent Dispensing System purchased from Pure Process Technologies or distilled as described in the literature.

One-pot Synthesis of 2-Cyanopyrrole from Pyrrole

Table S1: Optimization for synthesis of 2-cyanopyrrole from pyrrole under acidic conditions.

Entry Scale (g) Solvent (V) ROH (V) Temp. (°C) AY (%) IY (Purity)

(%)

1 0.5 DMF (15) - 125 58 -

2 0.2 DMF (10) - 90 25 -

3 0.2 DMF (10) H2O (3) 90 78 -

4 0.2 DMF (10) EtOH (3) 90 84 -

5 0.25 DMF (10) H2O (3) 80 74 -

6 0.25 DMF (10) H2O (5) 80 61 -

7 0.25 DMF (10) H2O (10) 80 51 -

8 0.25 DMF (15) H2O (3) 80 74 -

9 0.25 DMF (15) H2O (5) 80 94 -

10 0.25 DMF (15) H2O (10) 80 47 -

11 25 DMF (15) H2O (5) 80 93 72 (72)

The synthesis began with C-2 formylation of pyrrole and further its oxidation to nitrile. Both reactions are well known in literature, however, 2-formyl pyrrole is a low melting solid not easily distilled or recrystallized in good yield. Moreover, waste will be generated in the process of purifying the aldehyde, aldoxime or other intermediates. We wondered whether isolation of the aldehyde or aldoxime intermediate was necessary. Both reactions can be performed in same solvent, the iminium chloride salt could be used directly to form the nitrile in one-pot. Considering this, we began our efforts with formylation of pyrrole using POCl3 (1.1 eq.) in DMF (15V) (Table S1, entry 1). The complete conversion of pyrrole to iminium chloride salt was observed in 1h, analyzed by 1H NMR. According to the literature,1 the oxidation of 2-formylpyrrole to 2-cyanopyrrole is feasible at higher temperature (125 °C). Keeping this in mind, the hydroxylamine hydrochloride (1.1 equiv.) was added to the reaction mixture and heated at 125 °C for 16 h (overnight). The reaction was dark and sluggish, however, gave 58% assay yield by 1H NMR (mesitylene was used as internal standard). Further, the assay yield dropped to 25% by lowering the reaction temperature to 90 °C (Table S1, entry 2). The lower yields could be because of chemical incompatibility of hydroxylamine hydrochloride with residual POCl3 and related species which would need to be quenched. Thus, water or ethanol was added to the reaction mixture prior to addition of hydroxylamine hydrochloride.

The HCl generated in the course of the quench could be used as catalyst for the dehydration of aldoxime. Surprisingly, the reactions gave >80% assay yield under these conditions (Table S1, entry 3 and 4).

Exploring effect of amount of DMF and water on reaction:

The effect of amount of solvents like, DMF and water on reaction was examined (Table S1, entry 5-10). The reaction with 15V of DMF and 5V of water provided 94% assay yield. Importantly, the results were reproduced at 25 g batch under similar reaction conditions (Table S1, entry 9). The reaction gave 72 % isolated yield (adjusted with 72% purity). However, the extraction of product was found difficult due to emulsion formation and was not feasible at large scale. This led us to further optimize our reaction conditions to make product isolation easy.

Table S2: Optimization for synthesis of 2-cyanopyrrole from pyrrole under basic conditions.

Entry Scale (g)

Solvent (V) ROH (V) Base (eq.) Ac2O (eq.)

Temp. (°C)

AY (%)

IY (Purity)

(%)

1 0.2 DMF (10) H2O (3) Pyridine (5) 1.2 90 92 -

2 0.2 DMF (10) EtOH (3) Pyridine (5) 1.2 90 93 -

3 0.2 MeCN (10) EtOH (3) Pyridine (5) 1.2 90 88 -

4 0.2 MeCN (10) EtOH (3) Pyridine (3.5) 1.2 90 88 -

5 25 MeCN (10) EtOH (3) Pyridine (3.5) 1.1 70 - 81 (58)

6 5 MeCN (10) H2O (3) Pyridine (3.5) 1.2 70 - 93 (80)

7 25 MeCN (10) H2O (3) Pyridine (3.5) 1.1 70 - 76 (90)

7461

51

74

94

41

0

20

40

60

80

100

water (3V) water (5V) water (10V)

AY (%

)

Effect of amount of DMF and water

DMF (10V) DMF (15V)

8 5 DMF (10) H2O (3.5) Pyridine (10

mol%) + 50% NaOH (3.5 eq.)

1.2 90 - 59

9 50 DMF (5) H2O (3)

Pyridine (10 mol%) + 50% NaHCO3 (4

eq.)

1.1 90 - 64 (63)

10 25 DMF (10) H2O (3) Pyridine (5) 1.1 90 - 90 (89)

11 100 DMF (10) H2O (3) Pyridine (5) 1.1 90 - 94 (85)

Next, the reactions were attempted under basic conditions by activating the in situ formed oxime with acetic anhydride. Initially, the reactions were performed with pyridine (5 eq.) and acetic anhydride (1.2 equiv.) in DMF (Table 2S, entry 1 and 2). The reactions were smooth and provided excellent assay yields (>90%).

Reaction screening for alternative solvents and amount of ethanol required for reaction:

Exploring solvent volumes and amount of pyridine:

48

64

8086

66

8289 90

0

20

40

60

80

100

1 eq. 1.25V 2V 3V

AY (%

)

Amount of EtOH

Solvent screening vs quantity of EtOH

DMF MeCN

44

83 8885 88 8983 85 89

0

20

40

60

80

100

MeCN (5V) MeCN (10V) MeCN (15V)

AY(%

)

Pyridine

DOE for amount of MeCN and pyridine

2.5 eq. 3.5 eq. 5 eq.

Further, the reaction was screened for an alternative solvents, concentration and reagents. In this regard, few experiments were performed on 0.2 g scale, and it was found that DMF and acetonitrile works well. Acetonitrile would be better compared to DMF, as it could be easily evaporated from reaction which will be helpful for isolation of the product. With this results in hand, the reaction was scaled up to 25 g under similar reaction conditions (Table S2, entry 5). The reaction gave 81% yield with 58% purity, however, triethylphosphate generated by reaction of EtOH with residual POCl3 and its associated species was problematic for purification process. The addition of water instead of ethanol resolved this problem and reaction provided excellent yield and purity at 5 g and 25 g scale (Table S2, entry 6 and 7). In addition, inorganic bases, NaHCO3 and NaOH, were tried instead of pyridine giving lower yields (Table S2, entry 8 and 9). We ended up with pyridine as best base for the reaction. Importantly, the reaction reproducibility was demonstrated at 100 g scale (Table S2, entry 11). The product was purified by vacuum distillation and used for next step.

Procedure for synthesis of 2-cyanopyrrole (Table S2, entry 10):

A 1 L three necked round bottom flask was equipped with a J-KEM internal temperature probe, overhead stirrer and nitrogen line. The flask was charged with anhydrous DMF (250 mL) and cooled to an internal temperature of 0 – 5 °C under an atmosphere of nitrogen. With stirring (300 RPM), phosphoryl trichloride (38.3 mL, 409.9 mmol, 1.1 eq.) was slowly charged while maintaining internal temperature below 15 °C. The reaction mixture was stirred at 20 °C for another 30 minutes. The reaction was cooled to 0 – 5 °C and pyrrole (25.0 g, 372.6 mmol, 1.0 eq.) was slowly charged while maintaining internal temperature below 15 °C. The reaction mixture was stirred at 20 °C for another 1h. The light brown solution was cooled to 0 – 5 °C and water (75 mL) was added while maintaining internal temperature below 15 °C. The reaction was stirred for 5 minutes at same temperature. Solid hydroxylamine hydrochloride (28.48 g, 409.9 mmol, 1.1 eq.), acetic anhydride (38.7 mL, 409.9 mmol, 1.1 eq.) and pyridine (150 mL, 1.863 mol, 5.0 eq.) were added sequentially, the internal temperature was not allowed to exceed 15 °C. After the addition was complete, the reaction mixture was heated to 90 °C for overnight (16 hours). After complete consumption of the iminium chloride salt, as indicated by 1H NMR, the reaction mixture was diluted with water (250 mL) and transferred to the separating funnel. The product was extracted from dark brown reaction mixture by using ethyl acetate (3 X 400 mL). The combined organic layer was washed with 1M HCl (250 mL) and brine (250 mL), dried over sodium sulfate and concentrated using rotary evaporation under reduced pressure. The desired 2-cyanopyrrole (34.7 g, 90% (adjusted with 89% purity by HPLC)) was distilled out from dark brown oil under vacuum.

1H NMR of intermediate iminium chloride salt (600 MHz, DMSO-d6) δ 13.52 (br s, 1H), 9.03 (s, 1H), 7.49 – 7.50 (m, 1H), 7.17 – 7.17 (m, 1H), 6.53 – 6.54 (m, 1H) ppm.

1H NMR of 2-cyanopyrrole (600 MHz, DMSO-d6) δ 12.29 (br s, 1H), 7.13 – 7.15 (m, 1H), 6.89 – 6.91 (m, 1H), 6.21 – 6.23 (m, 1H) ppm.

13C NMR of 2-cyanopyrrole (150 MHz, DMSO-d6) δ 125.1, 119.9, 115.4, 110.0, 100.0 ppm.

=====================================================================Acq. Operator : SYSTEM Seq. Line : 38Sample Operator : SYSTEMAcq. Instrument : LC B Location : 17Injection Date : 7/6/2020 2:00:41 PM Inj : 1

Inj Volume : 1.000 µlAcq. Method : D:\Chem32\1\Data\Remdesivir\B070620 2020-07-06 10-51-13\Remd_Cyanopyrrole.MLast changed : 6/26/2020 9:22:56 AM by SYSTEMAnalysis Method : D:\Chem32\1\Data\Remdesivir\B070620 2020-07-06 10-51-13\Remd_Cyanopyrrole.M

(Sequence Method)Last changed : 7/7/2020 8:22:07 AM by SYSTEMMethod Info : Zorbax Eclipse Plus-C18 (4.6 x 100 x 3.5); 20% ACN, 30C,1.5 mL/min, pH 4.97

5 mM Phsophate Buffer

Additional Info : Peak(s) manually integrated

min0.5 1 1.5 2 2.5 3 3.5

mAU

0

100

200

300

400

500

600

700

800

DAD1 A, Sig=230,4 Ref=360,100 (Remdesivir\B070620 2020-07-06 10-51-13\038-17-DP-JT-WuXi-distil-5_3.D)

1.98

1

2.96

2

DAD1 B, Sig=260,4 Ref=360,100 (Remdesivir\B070620 2020-07-06 10-51-13\038-17-DP-JT-WuXi-distil-5_3.D)

2.96

2

=====================================================================Area Percent Report

=====================================================================

Sorted By : SignalMultiplier : 1.0000Dilution : 1.0000Do not use Multiplier & Dilution Factor with ISTDs

Signal 1: DAD1 A, Sig=230,4 Ref=360,100

Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.981 BB 0.0519 19.15703 5.89784 0.6284 2 2.962 BB 0.0655 3029.34009 714.35022 99.3716

Data File D:\Chem32\...mdesivir\B070620 2020-07-06 10-51-13\038-17-DP-JT-WuXi-distil-5_3.DSample Name: DP-JT-WuXi-distil-5_3

LC B 7/21/2020 10:05:38 AM SYSTEM Page 1 of 2

Totals : 3048.49712 720.24806

Signal 2: DAD1 B, Sig=260,4 Ref=360,100

Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 2.962 BB 0.0661 3559.29224 829.01477 100.0000

Totals : 3559.29224 829.01477

===================================================================== *** End of Report ***

Data File D:\Chem32\...mdesivir\B070620 2020-07-06 10-51-13\038-17-DP-JT-WuXi-distil-5_3.DSample Name: DP-JT-WuXi-distil-5_3

LC B 7/21/2020 10:05:38 AM SYSTEM Page 2 of 2

1H NMR Spectra of Intermediate Iminium Chloride Salt in DMSO-d6 at 600 MHz:

1H NMR Spectra 2-Cyanopyrrole in DMSO-d6 at 600 MHz:

13C NMR Spectra 2-Cyanopyrrole in DMSO-d6 at 151 MHz:

N-Amination of 2-Cyanopyrole Using Solid Aminating Reagents

Table S3: Optimization of N-amination of 2-cyanopyrrole with solid aminating reagents A and B.

Entry Scale

(g) Reagent

(eq.) Solvent (V) Base (eq.) Temp (°C)

Conversion (HPLC area %)

AY (%)

Type of Stirrer

1 0.1 A (1.5) DMF (150) NaH (1.5) 80 91 89 Magnetic

2 0.1 B (1.5) DMF (200) NaH (1.5) 80 92 94 Magnetic

3 0.5 A (1.5) DMF (25) NaH (1.5) 80 89 - Overhead

4 0.5 B (1.5) DMF (40) NaH (1.5) 80 90 - Overhead

5 0.1 A (1.2) NMP (100) KOtBu (1.2) 100 94 96 Magnetic

6 0.1 B (1.2) NMP (100) KOtBu (1.2) 100 94 97 Magnetic

7 0.5 A (1.2) NMP (25) KOtBu (1.2) 80 86 87 Overhead

8 0.5 B (1.2) NMP (25) KOtBu (1.2) 80 86 86 Overhead

9 1.0 A (1.1) NMP (10) KOtBu (1.1) 80 67 - Overhead

10 1.0 B (1.1) NMP (15) KOtBu (1.1) 80 95 95 Overhead

As discussed in manuscript, in order to avoid dilute reaction conditions and increase throughput for N-amination of 2-cyanopyrrole, the solid aminating reagents like, O-(4-nitrobenzoyl)hydroxylamine (A) and O-(diphenylphosphinyl)hydroxylamine (B) were planned to use. At the outset, in 10 mL glass vials, the solution of 2-cyanopyrrole (100 mg) in DMF (2 mL) was treated with aminating reagents (A and B; 1.5 eq.; individually) in presence of NaH (1.5 eq.). However, both reactions were very thick (almost dry) and unable to stir with magnetic stir bar. Then, the reactions were attempted with excess amount of DMF (Table S3, entry 1 and 2) and heated at 80 °C overnight (16 h). The reactions showed >90% conversion (by HPLC method). The assay yields were derived by using 1H NMR (mesitylene was used as internal standard). The assay yields showed well agreement with conversion. However, the reactions were highly diluted at this stage. Then, reactions were examined in EasyMax instrument with overhead stirrer employing lower amount of solvents. The reactions were carried out on 500 mg scale with both aminating reagents (A and

B) in DMF (25V and 40V) under similar reaction conditions (Table 3S, entry 3 and 4). The results were excellent, showing ~90% conversion in both reactions.

Exploring different solvents and bases with O-(4-nitrobenzoyl)hydroxylamine (A) reagent:

Exploring different solvents and bases with O-(diphenylphosphinyl)hydroxylamine (B) reagent:

Next, we screened the effect of solvents (DMF and NMP) and bases (NaH, KOtBu and KOH) with both aminating reagents (A and B). NMP and KOtBu combination gave the best result. Both reagents showed 94% conversion and assay yields were in good agreement with conversion (Table 3S, entry 5 and 6). Further, these reactions conditions were taken up forward for large scale reaction. The reactions were examined to investigate the lowest solvent volume that could be used on EasyMax (Table 3S, entry 7-10). The reactions were carried out on 1.0 g scale with lowest 10V and 15V of solvent. The reaction with 15V of solvent gave good conversion and assay yield.

Procedure for synthesis of N-amination of 2-cyanopyrrole:

A 50 mL glass reactor was equipped in EasyMax instrument with an internal temperature probe, overhead stirrer and nitrogen line. The flask was charged with anhydrous NMP (15 mL) and cooled to an internal temperature of 0 – 5 °C under an atmosphere of nitrogen. With stirring (300 RPM), 2-cyanopyrrole (1.0 g. 10.86 mmol, 1.0 eq.) was charged. Then, solid KOtBu (1.34 g, 11.94 mmol, 1.1 eq.) was added to reaction mixture. The temperature was raised to 10 °C, reactions stirred for 10 minutes. To the reaction mixture

8373

55

91 94

45

0

20

40

60

80

100

NaH KOtBu KOH

Conv

ersio

n (%

)

Solvent and base screening

DMF NMP

88

64

42

89 94

48

0

20

40

60

80

100

NaH KOtBu KOH

Conv

ersio

n (%

)

Solvent and base screening

DMF NMP

solid O-(diphenylphosphinyl)hydroxylamine (2.78 g, 11.94 mmol, 1.1 eq.) was charged in four portions over 10 minutes. The temperature of reaction was slowly increased to 80 °C and then reaction continued for overnight (16 hours). The consumption of the starting 2-cyanopyrrole was monitored with HPLC (95%) and assay yield (95%) was derived by using 1H NMR.

=====================================================================Acq. Operator : SYSTEM Seq. Line : 2Sample Operator : SYSTEMAcq. Instrument : LC 1 Location : 52Injection Date : 6/8/2020 9:47:37 AM Inj : 1

Inj Volume : 1.000 µlMethod : C:\Users\Public\Documents\ChemStation\2\Data\Dinesh\FC_CBRO3 2020-06-08 09-

41-30\Remd_Cyanopyrrole.M (Sequence Method)Last changed : 6/2/2020 6:09:12 PM by SYSTEMMethod Info : Zorbax Eclipse Plus-C18 (4.6 x 100 x 3.5); 20% ACN, 30C,1.5 mL/min, pH 4.97

5 mM Phsophate Buffer


min0.5 1 1.5 2 2.5 3 3.5

Norm.

0

100

200

300

400

500

600

700

DAD1 A, Sig=230,4 Ref=360,100 (Dinesh\FC_CBRO3 2020-06-08 09-41-30\002-52-DP-81-2.D)

1.92

0

2.17

7

DAD1 B, Sig=260,4 Ref=360,100 (Dinesh\FC_CBRO3 2020-06-08 09-41-30\002-52-DP-81-2.D)

1.92

0

2.17

7

=====================================================================Area Percent Report

=====================================================================



Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.920 BV 0.0546 2030.24060 583.89984 94.4255 2 2.177 VB 0.0600 119.85724 30.41911 5.5745

Totals : 2150.09784 614.31895

Data File C:\Users\P...Station\2\Data\Dinesh\FC_CBRO3 2020-06-08 09-41-30\002-52-DP-81-2.DSample Name: DP-81-2

LC 1 7/23/2020 2:19:51 PM SYSTEM Page 1 of 2



Totals : 2478.50374 705.98047

=====================================================================*** End of Report ***

Data File C:\Users\P...Station\2\Data\Dinesh\FC_CBRO3 2020-06-08 09-41-30\002-52-DP-81-2.DSample Name: DP-81-2


Investigation of NH2Cl Extraction

3.00 g of NH4Cl (57.2 mmol) was added to a 250 mL Erlenmeyer flask along with 4.70 mL of 14.5 M NH4OH (68.1 mmol). The mixture was cooled to -5 °C and stirred on a magnetic hotplate. 72 mL of bleach was added via addition funnel over the course of 15 minutes. The concentration of the bleach was stated as 7.5% NaOCl (7.1% active chlorine) which translates to 15.8 wt% NaOCl and 2.2 M NaOCl as found by iodometric titration. Some of this apparent difference in concentration exists as a result of ambiguity in tradeterms of the bleaching industry. The same procedure was used with more concentrated bleach, but the portions of reagent were changed as follows: 4.08 g NH4Cl, 6.39 mL NH4OH, and 72 mL of 10.6% NaOCl were mixed. Care should be taken working with monochloramine. It is a reactive oxidant.

The mixture reacted for 10 minutes at -5 °C prior to sampling. The aqueous feed was then held at this temperature through course of the study. 8 mL aliquots were taken to explore extraction of NH2Cl into organic solvent under various conditions. The aqueous chloramine was added to a 20 mL scintillation vial and vigorously shaken with organic solvent for 30 seconds. The biphasic mixture was allowed to separate and 1 mL of the organic phase was removed and titrated by the iodometric titration (See below). The results are listed in the table below.

Iodometric Titration: 6.20 g of Na2S2O3·5(H2O) was dissolved in 250 mL of water and set aside. 0.5 g of starch was dissolved in 50 mL of water by heating to 80 °C for 10 minutes and then set aside. 0.8 g of NaI was dissolved in 200 mL of water, and 10 mL of AcOH and 10 mL of the starch indicator were added. 20 mL of the iodide solution was transferred to a 50 mL Erlenmeyer flask with stir bar. 1.00 mL of NH2Cl in organic solvent was added to the iodide solution which turned a purplish brown color. The oxidant solution was stirred rapidly on a magnetic hotplate. The thiosulphate solution was added dropwise until the solution became clear. The volume required to quench the oxidant was recorded and molarity of NH2Cl was recorded.

Perhaps the difference between the reported and observed values can be explained as follows. A significant amount of volatile NH2Cl could be lost as the literature procedure evaporates the organic layer prior to filtration.2 Also, the concentration of NaOCl was not stated. Strength of commercial bleach varies widely.

Table S4: Chloramine concentration from different solvents and bleach sources

Entry Solvent (Volume) NaOCl (%) NH2Cl (M)

1 Reported Literature Value unknown 0.09

2 MTBE (11 mL)—(Repeat of Literature Procedure) 7.5 (2.2 M) 0.53

3 MTBE (5.5 mL, 50% MTBE Charge) ” 0.66

4 MTBE (3.7 mL, 25% MTBE Charge) ” 0.89

5 MTBE (11 mL) 10.6 (2.99 M) 0.65

6 MTBE (5.5 mL) “ 0.83

7 MTBE (5.5 mL)—organic saturated with NaOAc “ 0.81

8 MTBE (5.5 mL)—aqueous layer saturated with NaCl “ 0.84

9 MTBE (3.7 mL) ” 0.90

10 Et2O (3.7 mL) ” 0.79

11 2-Methyl THF (3.7 mL) ” 0.80

12 Dioxane (6 mL) ” 0.86

13 CPME “ 0.98

14 DEGDBE (3.7 mL) ” 1.05

15 THF (3.7 mL) ” 1.19

Continuous Production of Chloramine

A continuous stirred tank reactor (CSTR) was constructed from a 100 mL Schlenk flask with a liquid fill level set at 100 mL. Bleach, NH4Cl/NH4OH, and MTBE feeds were positioned below the liquid level surface, and the dip tubes removing reaction fluids were placed at the top of the fluid surface level, the 100 mL fill level volume. The CSTR was equipped with a large oval shaped stir bar, stirred at 800 rpm, and cooled to -5 °C. The total flow rate of fluids entering the CSTR was set at 10 mL/min to give a residence time of 10 min. The exit flow was set at 12 mL/min, faster than the entering flow to ensure a reactor volume of 100 mL/min. The exit stream flowed into the bottom of a gravity liquid-liquid settler made from a simple pressure-equalizing addition funnel (25 mL). The funnel was filled to a level of 25 mL, allowing the organic and aqueous layers to separate, and then the aqueous layer (bottom) was drained from the addition funnel, while the MTBE layer containing NH2Cl was pumped off the top layer. Steady state was reached 30 minutes after the start of pumping (0.45 M NH2Cl in MTBE).

The input feeds for the CSTR are as follows. 75.0 g of NH4Cl (1.43 mol), 117.5 mL of concentrated NH4OH and 125 mL of H2O were combined and stirred until the mixture was homogeneous (Stream A). The total volume was 300 mL, and pumped at a rate of 0.619 mL/min (Stream A). Aqueous NaOCl was pumped at a rate of 3.71 mL/min (Stream B). Some ambiguity exists in the terminology describing concentration of NaOCl. Our bleach was 7.5% NaOCl (7.1% active chlorine, 15.8 wt% NaOCl, 2.2 M NaOCl as found by iodometric titration). MTBE was pumped at a rate of 5.67 mL/min (Stream C). The exit dip tubes were programmed to remove solution at a rate of 12 mL/min (2 pumps flowing at 5 and 7 mL/min). Peristaltic pumps from Vapourtec were used for fluid transport.

0.54

0.5

0.44 0.440.46

0.44 0.440.46 0.46 0.46

0.44

0.3

0.4

0.5

0.6

15 25 35 45 55 65 75

NH 2

Cl in

MTB

E (M

)

Time (min)

Titration of NH2Cl from CSTR

Batch Amination with NH2Cl from CSTR

2-Cyanopyrrole (9.21 g, 10.0 mmol) was added to a 500 mL 3-neck round-bottom flask along with 100 mL of DMF. The solution was cooled to 0 °C with an ice-bath. 5.00 g of 60 wt% NaH in mineral oil was added slowly with stirring, keeping temperature below 35 °C. The round-bottom was connected to two condensers connected in series and cooled to -20 °C with a chiller. The condensers were configured to drain into a 250 mL receiving flask which was used to recycle the MTBE.

After the CSTR reached steady state, the stream of NH2Cl in MTBE was connected to the basified pot of 2-cyanopyrrole. The NH2Cl was added at a rate of 5.67 mL/min. It was added in 5 portions, where each fraction was flowed into the cyanopyrrole for 15 minutes. After every 15 minute addition, the chloramine feed was removed from the amination pot. The reaction vessel was set to 30 °C and placed under vacuum. The MTBE distilled and was collected in the receiving flask. After 14 minutes of evaporation, vacuum was turned off and the vessel was returned to atmospheric pressure. The MTBE was recycled and returned to the feedline used to extract NH2Cl in the CSTR (Stream C).

After the fifth addition of chloramine, conversion was measured as 93%, and 360 mL of MTBE was recovered (84% recovery).

=====================================================================Acq. Operator : SYSTEM Seq. Line : 1Sample Operator : SYSTEMAcq. Instrument : LC 1 Location : 54Injection Date : 7/2/2020 4:59:46 PM Inj : 1 Inj Volume : 1.000 µlMethod : C:\Users\Public\Documents\ChemStation\2\Data\Dinesh\FC_remd 2020-07-02 16- 58-34\Remd_Cyanopyrrole.M (Sequence Method)Last changed : 6/2/2020 6:09:12 PM by SYSTEMMethod Info : Zorbax Eclipse Plus-C18 (4.6 x 100 x 3.5); 20% ACN, 30C,1.5 mL/min, pH 4.97 5 mM Phsophate Buffer


min0.5 1 1.5 2 2.5 3 3.5

Norm.

0

200

400

600

800

1000

DAD1 A, Sig=230,4 Ref=360,100 (Dinesh\FC_remd 2020-07-02 16-58-34\FC_remd.D)

2.04

4

2.35

2

DAD1 B, Sig=260,4 Ref=360,100 (Dinesh\FC_remd 2020-07-02 16-58-34\FC_remd.D)

2.04

4

2.35

2

===================================================================== Area Percent Report=====================================================================




Totals : 3531.13327 992.76380

Data File C:\Users\P...nts\ChemStation\2\Data\Dinesh\FC_remd 2020-07-02 16-58-34\FC_remd.DSample Name: DP-CSTR-4




Totals : 4071.42122 1138.85769

===================================================================== *** End of Report ***

Data File C:\Users\P...nts\ChemStation\2\Data\Dinesh\FC_remd 2020-07-02 16-58-34\FC_remd.DSample Name: DP-CSTR-4


=====================================================================Acq. Operator : SYSTEM Seq. Line : 1Sample Operator : SYSTEMAcq. Instrument : LC 1 Location : 65Injection Date : 7/7/2020 11:04:38 AM Inj : 1 Inj Volume : 1.000 µlMethod : C:\Users\Public\Documents\ChemStation\2\Data\Dinesh\FC_remd 2020-07-07 11- 03-23\Remd_Cyanopyrrole.M (Sequence Method)Last changed : 6/2/2020 6:09:12 PM by SYSTEMMethod Info : Zorbax Eclipse Plus-C18 (4.6 x 100 x 3.5); 20% ACN, 30C,1.5 mL/min, pH 4.97 5 mM Phsophate Buffer


min0.5 1 1.5 2 2.5 3 3.5

Norm.

0

500

1000

1500

2000

DAD1 A, Sig=230,4 Ref=360,100 (Dinesh\FC_remd 2020-07-07 11-03-23\FC_remd.D)

2.07

9

2.38

0

DAD1 B, Sig=260,4 Ref=360,100 (Dinesh\FC_remd 2020-07-07 11-03-23\FC_remd.D)

2.07

9

2.38

0





Totals : 8536.02615 2314.79264

Data File C:\Users\P...nts\ChemStation\2\Data\Dinesh\FC_remd 2020-07-07 11-03-23\FC_remd.DSample Name: DP-CSTR-2-5



Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 2.079 BV R 0.0610 8552.48535 2215.67578 96.3123 2 2.380 VB E 0.0625 327.46469 82.22898 3.6877

Totals : 8879.95004 2297.90476

===================================================================== *** End of Report ***

Data File C:\Users\P...nts\ChemStation\2\Data\Dinesh\FC_remd 2020-07-07 11-03-23\FC_remd.DSample Name: DP-CSTR-2-5


=====================================================================Acq. Operator : SYSTEM Seq. Line : 4Sample Operator : SYSTEMAcq. Instrument : LC 1 Location : 61Injection Date : 7/9/2020 8:53:52 PM Inj : 1 Inj Volume : 1.000 µlDifferent Inj Volume from Sample Entry! Actual Inj Volume : 0.200 µlMethod : C:\Users\Public\Documents\ChemStation\2\Data\Dinesh\FC_remd 2020-07-09 20- 37-57\Remd_Cyanopyrrole.M (Sequence Method)Last changed : 6/2/2020 6:09:12 PM by SYSTEMMethod Info : Zorbax Eclipse Plus-C18 (4.6 x 100 x 3.5); 20% ACN, 30C,1.5 mL/min, pH 4.97 5 mM Phsophate Buffer


min0.5 1 1.5 2 2.5 3 3.5

Norm.

0

100

200

300

400

500

600

DAD1 A, Sig=230,4 Ref=360,100 (Dinesh\FC_remd 2020-07-09 20-37-57\FC_remd3.D)

0.25

4

1.17

2

1.32

5

1.48

2

1.70

1

2.01

4

2.30

9

DAD1 B, Sig=260,4 Ref=360,100 (Dinesh\FC_remd 2020-07-09 20-37-57\FC_remd3.D)

Area: 2

113.9

5

2.01

4

Area: 2

34.72

9

2.31

0




Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 0.254 BB 0.0951 7.39442 1.21373 0.2890 2 1.172 BV E 0.1151 67.19736 9.00551 2.6262 3 1.325 VV R 0.0598 409.72327 104.44360 16.0128 4 1.482 VB E 0.0770 18.64740 3.45442 0.7288

Data File C:\Users\P...ts\ChemStation\2\Data\Dinesh\FC_remd 2020-07-09 20-37-57\FC_remd3.DSample Name: DP-CSTR-14


Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 5 1.701 BB 0.0646 14.08705 3.25077 0.5505 6 2.014 BV 0.0562 1817.92456 502.10944 71.0480 7 2.309 VB 0.0616 223.75229 57.24744 8.7447

Totals : 2558.72634 680.72489


Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 2.014 FM 0.0601 2113.95459 586.64136 90.0059 2 2.310 MF 0.0649 234.72888 60.26175 9.9941

Totals : 2348.68347 646.90311

===================================================================== *** End of Report ***

Data File C:\Users\P...ts\ChemStation\2\Data\Dinesh\FC_remd 2020-07-09 20-37-57\FC_remd3.DSample Name: DP-CSTR-14


Continuous Amination in PFR with NH2Cl from CSTR

A CSTR was made as described above in a 22 mL Scintillation vial with a fill volume of 20 mL. The total flow rate in was 2 mL/min, the exit was programmed at 3 mL/min, and the residence time was 10 min.

The input feeds for the CSTR are as follows. 15.0 g of NH4Cl (286 mol), 23.5 mL of concentrated NH4OH and 25 mL of H2O were combined and stirred until the mixture was homogeneous (Stream A). The total volume was 60 mL, and pumped at a rate of 0.124 mL/min by syringe pump (Stream A). Aqueous NaOCl was pumped at a rate of 0.742 mL/min (Stream B). MTBE was pumped at a rate of 1.13 mL/min (Stream C). The exit dip tube was programmed to remove solution at a rate of 3 mL/min, and it transported the biphasic mixture to the gravity separator as described previously. Peristaltic pumps from Vapourtec were used for fluid transport.

The NH2Cl in MTBE was pumped at a rate of 1.13 from the gravity separator, and mixed with a solution of 2-cyanopyrrole anion (0.609 M in DMF) via a T-Mixer (Idexx, 0.02 “ ID). The 2-cyanopyrrole mixture was flowed at 0.129 mL/min by syringe pump. The solution was made by dissolving 1.40 g of 2-cyanopyrrole (15.2 mmol, 1.00 equiv.), 1.22 g NaH (60 wt%, 30.4 mmol, 2.00 equiv.) in DMF to reach a total volume of 25 mL. The reaction mixture flowed into a PFR constructed from PFA tubing (0.06” ID, 5.04 mL, 4 min tR). The reaction ran for 12 minutes before collecting sample for analysis. 89% conversion was observed.

=====================================================================Acq. Operator : SYSTEM Seq. Line : 1Sample Operator : SYSTEMAcq. Instrument : LC 1 Location : 2Injection Date : 5/28/2020 3:11:00 PM Inj : 1 Inj Volume : 1.000 µlMethod : C:\Users\Public\Documents\ChemStation\2\Data\Dinesh\FC_CBRO3 2020-05-28 15- 09-51\Remd_Cyanopyrrole.M (Sequence Method)Last changed : 5/26/2020 4:16:12 PM by SYSTEMMethod Info : Zorbax Eclipse Plus-C18 (4.6 x 100 x 3.5); 20% ACN, 30C,1.5 mL/min, pH 4.97 5 mM Phsophate Buffer


min0.5 1 1.5 2 2.5 3 3.5

Norm.

0

100

200

300

400

500

600

DAD1 A, Sig=230,4 Ref=360,100 (Dinesh\FC_CBRO3 2020-05-28 15-09-51\001-2-TA1051-1.D)

Area: 1

637.3

7

1.89

3

Area: 2

05.34

6

2.95

9

DAD1 B, Sig=260,4 Ref=360,100 (Dinesh\FC_CBRO3 2020-05-28 15-09-51\001-2-TA1051-1.D)

Area: 1

990.9

9

1.89

3

Area: 2

45.02

7

2.95

9




Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.893 MF 0.0549 1637.36975 497.25723 88.8563 2 2.959 MF 0.0731 205.34579 46.79128 11.1437

Totals : 1842.71555 544.04852

Data File C:\Users\P...Station\2\Data\Dinesh\FC_CBRO3 2020-05-28 15-09-51\001-2-TA1051-1.DSample Name: TA1051-1



Peak RetTime Type Width Area Height Area # [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------| 1 1.893 MF 0.0549 1990.98730 604.32129 89.0418 2 2.959 MF 0.0733 245.02650 55.69839 10.9582

Totals : 2236.01381 660.01968

===================================================================== *** End of Report ***

Data File C:\Users\P...Station\2\Data\Dinesh\FC_CBRO3 2020-05-28 15-09-51\001-2-TA1051-1.DSample Name: TA1051-1


Batch Amination with Multicharge of Chloramine

HN

NN

N

NH2NaH (2 eq.),

DMF, RT

Multicharge of NH2Cl in MTBE: - Add 1/4, then vacuum

distillation of MTBE at 30 oC

- Repeat cycle for 3 more additions

Typical multi-charge amination procedure:

To a solution of 1H-pyrrole-2-carbonitrile (0.46 g, 5 mmol) in MTBE (5 mL) was added NaH (0.40 g, 10.0 mmol, 60% in mineral oil) in portions, and the reaction was stirred for 20 min at room temperature. DMF (5 mL) was added and MTBE was distilled under vacuum at 20-30 °C. NH2Cl in MTBE (5 mL, 0.59M solution) was added via syringe. The mixture was let to stir between 20-30 °C for 5 min and HPLC analysis was carried out. MTBE was distilled under vacuum at 20-30 °C and NH2Cl in MTBE (5 mL, 0.59M solution) was added via syringe. MTBE was distilled under vacuum at 20-30 °C and this cycle was repeated 2 more times. Mesitylene was added as internal standard and the reaction was assayed by quantitative 1H NMR. This procedure was consistently repeated in 1, 5 and 30 mmol scale (Table S5).

Table S5. Conversion and assay yield of chloramine multicharge reactions

Chloramine charge Conversion (HPLC area %)

1 mmol (0.092 g)

5 mmol (0.460 g)

30 mmol (2.8 g)

1 43 38 43 2 70 72 70 3 86 92 89 4 98 98 98

Assay Yield (NMR) 87% 91% 89%

Typical 1H NMR spectrum after amination reaction:

Monochloramine (0.59M in MTBE) preparation procedure:

NH4Cl (3.0 g) in MTBE (55 mL) was cooled to -5 °C (internal temperature), and concentrated NH4OH (4.7 mL) was added. Commercial bleach (72 mL, Clorox ~7.5% NaOCl) was then added via addition funnel over 15 min. The mixture was stirred for 15 min, the layers were separated, and the organic layer was washed with brine (1 × 30 mL). The organic layer was dried over powdered CaCl2 in the freezer for at least 1 h and kept at the same temperature. Approximate concentration is 0.59 M.

Amination and Triazine Formation in One-Pot

For scale-up to 10 g, a few changes were implemented: 1) In order to increase the throughput of the overall process, DMF was reduced from 10V to 5V in relation to 1H-pyrrole-2-carbonitrile with no impact to the reaction profile; 2) Based on our findings on the relation of NaH vs chloramine (see manuscript for details), NaH was reduced from 2 to 1.5 equiv.; 3) The number of chloramine charges were reduced from 4 to 3. This procedure was employed at in 2.8 g and 10 g scale furnishing the N-amino-2-cyanopyrrole product in 92% assay yield (Table S6, entries 1 and 2). The N-amino-2-cyanopyrrole product was cyclized by adding 3 equiv. of formamidine acetate and heating at 90-95 °C for 16 h. Assay 1H NMR using 1,3,5-trimethoxybenzene as internal standard showed 76% assay yield to triazine, which was isolated in 64% isolated yield after reducing the DMF volume and addition of water (78% purity). Preliminary purification studies showed that >97% pure triazine can be obtained by recrystallizing the crude solid from boiling water/EtOH (1:1), however, further studies showed that trituration with MTBE was more efficient for mass recovery in good purity.

Aiming at further increasing the throughput, more concentrated solutions of chloramine were investigated. Instead of using Clorox (~7.5% NaOCl), a sodium hypochlorite 10-15% solution from Sigma-Aldrich was used to prepare a ca. 0.74M chloramine solution in MTBE. By the addition of 170 mL (~1.2 equiv.) of this solution to deprotonated 1H-pyrrole-2-carbonitrile (10.0 g scale) in DMF gave 98% conversion to the N-amino-2-cyanopyrrole product (Table S6, Entry 4). It is important to mention that this batch employed a total of 22 volumes of solvent (5V of DMF + 17V of chloramine solution in MTBE), a reduction of approximately 5-fold compared to the Hynes Jr. procedure3 (20V of DMF + 80V of chloramine solution). The N-amino-2-cyanopyrrole solution was subjected to cyclization with formamidine acetate. The crude solid obtained from this batch was further purified by trituration with MTBE to give triazine in 60% isolated yield over the two steps.

N NN

NH2

HN NH2HOAc (3 eq.)

90-95 oC (internal temp.)

Concentrate, then add water

78-82% purity

N NN

NH2

HN

NN

N

NH2

- NaH (1.5 eq.), MTBE/DMF- Add NH2Cl in MTBE, then vacuum distillation of MTBE at 20-30 oC

- Repeat cycle until >95% conversionTriazine

>97% purity

Trituration with MTBE

Table S6: Scale-up to 10g and evaluation of concentrated chloramine solution

Entry Scale (g)

Chloramine in MTBE charge (concentration)

Amination conversion (HPLC)

Cyclization AY (1H

NMR)

Purity (HPL

C)

IY yield Comments

1 2.8 3 charges

(3 x 40mL) Conc.: 0.56M

97% 74% 82% 62%

2 10.0 3 charges

(3 x 140mL) Conc.: 0.56M

98% (92% AY) 76% 78% 64%

Analysis of the mother liquor showed ~2 g of triazine (~14%

yield)

3 10.0 2 charges

(220 + 50 mL) Conc.: 0.68M

99% NA 98% 63% Crude solid was

resuspended/triturated in MTBE to give 98% purity triazine.

4 10.0 1 charge (170 mL)

Conc.: 0.74M 98% 75% 99% 60%

Crude solid was resuspended/triturated in MTBE

to give 99% purity triazine.

Typical Amination/Cyclization procedure (Table S6, Entry 4):

A 0.5 L three necked round bottom flask was equipped with a J-KEM internal temperature probe and a stirring bar. A solution of 1H-pyrrole-2-carbonitrile (10.0 g, 105.3 mmol, 97% purity) in MTBE (100 mL) was added, cooled in an ice bath (internal temperature between 5-10 °C) and NaH (6.3 g, 158.0 mmol, 60% in mineral oil Sigma-Aldrich) was added in portions. The reaction was stirred for 30 min at room temperature. DMF (50 mL) was added and MTBE was distilled under vacuum at 20-30 °C (internal temperature). Chloramine in MTBE (170 mL, 125.8 mmol, 0.74 M solution) was added. The reaction was monitored by HPLC until conversion is >95% (less than 30 minutes). To the reaction mixture was added formamidine acetate (32.89 g, 315.9 mmol, 99% Chem-Impex). The mixture was heated while distilling MTBE at atmospheric pressure until internal temperature reaches 90-95 °C. The reaction was stirred at the same temperature for 16 hours. Assay 1H NMR using 1,3,5-trimethoxybenzene as internal standard showed 75% assay yield to triazine. The mixture was concentrated through distillation under vacuum (40-45 mL of DMF was recovered) and 100 mL of water was added washing the vessel walls. The flask was placed in an ice-bath and the internal temperature was monitored until it reached ~5 °C. The mixture was filtered and washed with 20 mL of water. The brown solids were transferred to 250 mL round bottom flask, dried under vacuum and 100 mL of MTBE was added. The mixture was vigorously stirred until a uniform suspension is observed. The suspension was filtered giving the triazine as a brown powder which was dried under vacuum (8.53 g, 99.0% purity by HPLC, 60% yield). 1H NMR (600 MHz, DMSO-d6) δ 6.61 (s, 1H), 6.91 (s, 1H), 7.61 (s, 1H), 7.66-8.01 (m, 3H); 13C NMR (151 MHz, DMSO-d6) δ 155.7, 148.1, 118.2, 114.2, 110.2, 101.5. Data match those previously reported.4

Monochloramine (0.74M in MTBE) preparation procedure:

NH4Cl (24.6 g) in MTBE (330 mL) was cooled to -5 °C (internal temperature) in a 2L-round bottom flask, and concentrated NH4OH (38.4 mL) was added. Sodium hypochlorite (10-15% solution, Sigma-Aldrich, 432 mL) was then added via addition funnel over 30 min. The mixture was stirred for 30 min, the layers were separated, and the organic layer was washed with brine (1 × 180 mL). The organic layer was dried over powdered CaCl2 (15 g) in the freezer for at least 1 h and kept at the same temperature. Approximate concentration is 0.74 M.

1H NMR spectrum after cyclization reaction in DMSO-d6 (600 MHz):

1H NMR spectrum of triazine in DMSO-d6 at 600 MHz:

13C NMR spectrum of triazine in DMSO-d6 at 151 MHz:

HPLC trace of commercial Triazine (red) and M4ALL triazine (green):

Evaluation of Alternative Bases and Solvents for Amination

We concluded our amination investigation by addressing the hazards surrounding use of NaH in conjunction with DMF. An interesting solution is the deprotonation of 1H-pyrrole-2-carbonitrile in MTBE, then solvent swap to DMF before adding the chloramine (Table S7, entry 2). This alternative was employed in the 10 g batches previously discussed. Alternative solvents and bases were also evaluated. Prior experience at M4ALL Institute5 suggested that NaH deprotonation can be successful in “glyme type” solvents. Diglyme and diethylene glycol dibutyl ether (DEDGBE) were explored furnishing high conversion to the desired N-aminated product (Table S7, Entries 3 and 4). THF and MTBE were also investigated, however, lower conversion was obtained (Table S7, entries 5 and 6). These results confirm the importance of the coordinating nature of the “glyme type” ethers which can be used as an alternative to DMF. Different bases like, NaHMDS, NaOtBu and KOtBu were examined, however the product is formed in reasonable conversion, the reaction profiles did not excel the use of NaH as base (Table S7, Entries 7-9).

HN

NN

N

NH2Base Solvent

1 h, RTNH2Cl in MTBE

Typical amination screening procedure:

To a solution of 1H-pyrrole-2-carbonitrile (1 mmol) in DMF or alternative solvent (1 mL) was added NaH or alternative base, and the reaction was stirred for 30 min at room temperature. NH2Cl (4 mL, ca. 0.56 M in MTBE) was added via syringe. The reaction conversion was monitored by HPLC after 1 hour.

Table S7: Evaluation of alternative bases and solvents for the amination reaction

Entry Solvent Base (equiv.) Conversion (HPLC)

AY (NMR) Comments

1 DMF NaH (1.1 to 2) >95% 89-95% See the manuscript for relation between NaH and Chloramine

2 MTBE/DMF NaH (2) >95% -

Deprotonation was carried out in MTBE, then DMF was added and

MTBE was distilled before adding chloramine solution

3 Diglyme NaH (1.5) >95% 97% -

4

Diethylene glycol

dibutyl ether (DEGDBE)

NaH (2) >95% 92% -

5 MTBE NaH (2) 45% - - 6 THF NaH (1.5) 26% - -

7 DMF NaHMDS (1.5) 73% - -

8 DMF NaOtBu (1.5) 71% - For a full screening of sodium and potassium tert-butoxide

equivalents, see table S8 9 DMF KOtBu (1.5) 81% -

Table S8: Evaluation of Sodium and Potassium tert-butoxide equivalents for the amination reaction

Entry Equiv. Base Conversion (HPLC area %)

1 1 KOtBu 69 2 NaOtBu 80 3 1.2 KOtBu 85 4 NaOtBu 80 5 1.5 KOtBu 81 6 NaOtBu 71 7 2 KOtBu 78 8 NaOtBu 66 9 2.5 KOtBu 62

10 NaOtBu 46 11 3 KOtBu 31 12 NaOtBu 19 13 4 KOtBu 0 14 NaOtBu 0

Analytical Method

Structures & IDs :

2

34

5

HN

1

1H-pyrroleExact Mass: 67.04

predicted pka = 17.0

5

43

2

HN

1

O

1H-pyrrole-2-carbaldehydeExact Mass: 95.04


5

43

2

HN

1

CN

2-cyanopyrroleExact Mass: 92.04


5

43

2

N 1

CN

NH2

1-amino-2-cyanopyrroleExact Mass: 107.05

predicted pka = -4.62

N3

2N1

N8

4a4

7

6

5

NH2

pyrrolo[2,1-f][1,2,4]triazin-4-amineExact Mass: 134.06

predicted pka = 4.28 Conditions Mobile Phase A: 5 mM KH2PO4 in Water, pH ~5.0 (0.680g HPLC grade KH2PO4 in 1000 mL HPLC grade water)

Isocratic Table Time (min)

%A

%B

0 80%

20%

4.0 80%

20%

Post-run equilibration: Off

Mobile Phase B: Acetonitrile Flow rate: 1.5 mL/min Injection volume: 1 µL Column: Agilent Zorbax Eclipse C18 (4.6 x 100 mm; 3.5 µm) Column temp: 30 °C Detector wavelength: 230 nm (Pyrrole to Carbaldehyde), 260 nm (Carbaldehyde to Triazine) Sample preparation: Prepare samples at approximately 1.0 mg/mL in MeOH (preferred).

Retention Times Compound Time (min)

Pyrrolo[2,1-f][1,2,4]triazin-4-amine 1.301 min 1H-Pyrrole-2-Carbaldehyde 1.542 min 1-Amino-2-Cyanopyrrole 1.894 min

Pyrrole 2.052 min 2-Cyanopyrrole 2.961 min

Representative Chromatogram(s) (attach additional chromatograms and spectra as needed)

References

1) Xi, N.; Li, M.; Hu, H.; Dai, W.; Li, X.; Wang, T.; Wu, Y. WO 2016/190847A1, 2016. 2) Jaffari, G. A.; Nunn, A. J. J. Chem. Soc. C, 1971, 823−826. 3) Hynes, J.; Doubleday, W. W.; Dyckman, A. J.; Godfrey, J. D.; Grosso, J. A.; Kiau, S.; Leftheris,

K. J. Org. Chem., 2004, 69, 1368−1371. 4) Patil, S. A.; Otter, B. A.; Klein, R. S. J. Het. Chem., 1994, 31, 781−786. 5) Verghese, J.; Kong, C. J.; Rivalti, D.; Yu, E. C.; Krack, R.; Alcazar, J.; Manley, J. B.; McQuade,

D. T.; Ahmad, S.; Belecki, K.; Gupton, B. F. Green Chem. 2017, 19, 2986−2991.

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