2020, OPR&D - Quest For A COVID-19 Cure by Repurposing Small Molecule Drugs Mechanism PDF

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Review
Quest for a COVID-19 Cure by Repurposing Small Molecule Drugs: Mechanism
of Action, Clinical Development, Synthesis at Scale, and Outlook for Supply
Chris De Savi, David L Hughes, and Lisbet Kvaerno
Org. Process Res. Dev., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2020
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Quest for a COVID-19 Cure by Repurposing Small Molecule Drugs:
5 Mechanism of Action, Clinical Development, Synthesis at Scale, and Outlook
6 for Supply
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11 Chris De Savi*
12 Kymera Therapeutics, 300 Technology Square, 2nd Floor, Cambridge, Massachusetts, 02139,
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United States
15 E-mail of corresponding author: cdesavi@kymeratx.com
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18 David L. Hughes*
19 Cidara Therapeutics, Inc., 6310 Nancy Ridge Dr., Suite 101, San Diego, California 92121,
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United States
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E-mail of corresponding author: dhughes@cidara.com
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26 Lisbet Kvaerno
27 Hypha Discovery Ltd, 154C Brook Drive, Milton Park, OX14 4SD, United Kingdom
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Organic Process Research & Development Page 2 of 133
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6 Abstract:
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9 The outbreak of the COVID-19 pandemic has spurred an intense global effort to repurpose
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12 existing approved drugs for its treatment. In this review, we highlight the development of seven
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14 small molecule drugs that are currently being assessed in clinical trials for the treatment of
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16 COVID-19. Three sections are presented for each drug: (1) history, mechanism of action, and
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status of clinical trials; (2) scalable synthetic routes and final forms; and (3) outlook for supply
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21 should clinical trials show treatment efficacy. A brief overview of diagnostic testing and vaccine
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23 development is also presented.
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29 Key Words: COVID-19, repurposed drugs, mechanism of action, synthesis, supply
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5 Table of Contents
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8 1. Coronavirus disease 2019 COVID-19 outbreak.........................................................................
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2. SARS-CoV-2 diagnostic testing.................................................................................................
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11 3. COVID-19 vaccine treatments ...................................................................................................
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13 4. Re-purposed small molecule treatments for COVID-19 ............................................................
14 5. Remdesivir..................................................................................................................................
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16 5.1 History, Mechanism of Action, and Status of Clinical Trials .............................................
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18 5.2 Synthetic Routes to Remdesivir..........................................................................................
19 5.3 Final Forms and Formulations of Remdesivir ....................................................................
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21 5.4 Remdesivir - Outlook for Supply........................................................................................
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23 6. Hydroxychloroquine...................................................................................................................
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26 6.2 Synthesis of Hydroxychloroquine.......................................................................................
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28 6.3 Hydroxychloroquine Final Form and Formulation.............................................................
29 6.4 Hydroxychloroquine - Outlook for Supply.........................................................................
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31 7. Favipiravir ..................................................................................................................................
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34 7.2 Synthesis of Favipiravir ......................................................................................................
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36 7.2.1 Routes from the Innovator Company, Toyama Chemicals .........................................
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38 7.2.2 Summary of Innovator Routes to Favipiravir..............................................................
39 7.2.3 Alternate Routes to Favipiravir from Academic Labs and Generic Companies .........
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41 7.2.4 Summary of Alternate Routes to Favipiravir...............................................................
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7.3 Final Forms and Formulation of Favipiravir.......................................................................
44 7.4 Favipiravir – Outlook for Supply........................................................................................
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46 8. Pirfenidone .................................................................................................................................
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8.1 History, Mechanism of Action, and Status of Clinical Trials .............................................
49 8.3 Pirfenidone Final Form and Formulation............................................................................
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51 8.4 Outlook for Supply – Pirfenidone.......................................................................................
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9. Baricitinib...................................................................................................................................
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56 9.2 Synthetic Routes to Baricitinib ...........................................................................................
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3 9.3 Potential Manufacturing Route to Baricitinib .....................................................................
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5 9.4 Final Form and Formulation of Baricitinib.........................................................................
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7 9.5 Outlook for Supply of Baricitinib .......................................................................................
8 10. Camostat .................................................................................................................................
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12 10.2 Synthesis of Camostat.........................................................................................................
13 10.2 Formulations and Salt Forms of Camostat..........................................................................
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15 10.3 Outlook for Supply..............................................................................................................
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17 11. Lopinavir/Ritonavir ................................................................................................................
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20 11.2 Synthesis of Lopinavir ........................................................................................................
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22 11.3 Salt Forms of Lopinavir/Ritonavir......................................................................................
23 11.4 Outlook for Supply..............................................................................................................
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25 12. Summary and Conclusions .....................................................................................................
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13. References...............................................................................................................................
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6 1. Coronavirus disease 2019 COVID-19 outbreak
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9 Human coronaviruses were first discovered in the 1960s1 and are a large group of related viruses
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that are known to cause respiratory illness ranging in severity from common colds to diseases such
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14 as Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS).2
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17 The novel coronavirus is a previously unidentified strain of coronavirus named severe acute
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19 respiratory syndrome coronavirus 2 (SARS-CoV-2)3 by the World Health Organization (WHO)
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21 and the resulting coronavirus disease 2019 (COVID-19) is now a global, ongoing pandemic since
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24 its initial detection in Wuhan, China, in December 2019. SARS-CoV-2 is an enveloped, positive-
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26 sense, single-stranded RNA β-coronavirus which targets cells through the viral structural spike (S)
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28 protein that binds to the angiotensin-converting enzyme 2 (ACE2).4 Upon receptor binding, the
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virus particle uses host cell receptors and endosomes to enter cells. The host cellular protease
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33 transmembrane protease serine 2 (TMPRSS2) helps cell entry via the S protein. Viral polyproteins
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35 are then synthesized that encode for the replicase-transcriptase complex. Viral RNA synthesis is
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then achieved using RNA-dependent RNA polymerase. Key structural protein Epidemiological
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40 studies have shown that each infection can result in 1.4 to 3.9 new cases when no one is immune
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42 or has not taken preventative measures such as social distancing. It is primarily spread between
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44 humans through small droplets from the nose or mouth when an infected person coughs or
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47 sneezes.5
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50 As of May 30, 2020, there are over 6 million confirmed cases of COVID-19 of which have resulted
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52 in 370,000 deaths worldwide.6a Although the majority of cases of COVID-19 infection were first
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54 reported in China, the virus has now spread to 185 countries6b with the U.S. now leading the world
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3 in both total number of infections and deaths. Symptoms can show up anywhere between 2 to 14
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6 days after exposure and it also possible for an infected person to now be showing no symptoms so
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8 called asymptomatic disease. The disease is known to particularly afflict elderly people with pre-
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10 existing respiratory conditions or cardiovascular disease.
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16 Currently no drugs are available for the treatment of COVID-19. As such, the pandemic has
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spurred an intense, collaborative, and global effort to repurpose existing approved drugs for its
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21 treatment. In this review, we highlight the development of seven small molecule drugs that are
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23 currently being assessed in clinical trials for the treatment of COVID-19. Three sections are
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25 presented for each drug: (1) history, mechanism of action, and status of clinical trials; (2)
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28 scalable synthetic routes and final forms; and (3) outlook for supply should clinical trials show
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30 treatment efficacy. The review starts with a brief overview of diagnostic testing and vaccine
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32 development.
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38 2. SARS-CoV-2 diagnostic testing
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Testing for SARS-CoV-2 is becoming increasingly available with two broad categories of testing
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44 for the virus, nucleic acid amplification tests (NAAT), which can detect the SARS-CoV-2 genetic
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46 material from a patient’s respiratory system and a serology (or antibody) test which can measure
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the amount of antibodies present in the blood when the body is responding to the COVID-19
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51 infection (Table 1 – SARS-CoV-2 USA FDA EUA commercialized diagnostic tests).
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54 Table 1. SARS-CoV-2 USA FDA EUA commercialized diagnostic tests
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3 Test name Company Test type Result Approval
4 time
5 (hr)
6 ID NOW COVID-19 Abbott Isothermal amp. <1 US FDA EUA
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8 Abbot RealTime SARS-CoV-2 EUA Test Abbott PCR 4-6 US FDA EUA
9 ANDi SARS-Cov-2 RT-qPCR Detection 3D Medicines PCR 4-6 US FDA EUA
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Kit
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12 Atila iAMP COVID-2019 Detection Kit Atila Biosystems, Inc Isothermal amp. 1 US FDA EUA
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BioGX SARS-CoV-2 Reagents for BD Becton Dickinson US FDA EUA
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15 MAX systems
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BioFire COVID-19 test Biofire Defense, LLC PCR <1 US FDA EUA
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18 Xpert Xpress SARS-CoV-2 Cepheid PCR <1 US FDA EUA
19 Simplexa COVID-19 Direct RT-PCR Kit DiaSorin PCR 1 US FDA EUA
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21 ePlex SARS-Cov-2 Test GenMark Diagnostics PCR 2 US FDA EUA
22 Panther Fusion SARS-CoV-2 assay Hologic PCR 3 US FDA EUA
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24 ARIES SARS-CoV-2 assay Luminex Corp PCR 2 US FDA EUA
25 New Cornavirus RT-PCR test Perkin Elmer PCR 4-6 US FDA EUA
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27 Quest SARS-CoV-2 RT-PCR Quest PCR 96-120 US FDA EUA
28 QIAstat-Dx Respiratory Panel 2019-nCoV QIAGEN GmbH PCR 1 US FDA EUA
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30 Lyra SARS-CoV-2 Assay Quidel PCR 4-6 US FDA EUA
31 Cobas SARS-Cov-2 Roche PCR 3-8 US FDA EUA
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33 TaqPath COVID-19 Combo Kit Thermo Fisher PCR 4 US FDA EUA
34 Scientific
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38 At the time of writing, currently 22 companies have been issued authorization by the FDA to
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distribute the NAAT and >50 additional companies have notified the FDA they will be submitting
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43 authorization requests. According to the Foundation for Innovative New Diagnostics (FIND) there
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45 are over 590 diagnostics, either developed or under development, for SARS-CoV-2 worldwide.7
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47 Estimates in the US suggest that 52/1000 people have been tested for COVID-19 as of May 30,
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50 2020, a number that continues to grow daily as more people are tested.6a The majority of NAAT
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52 focus on detecting SARS-CoV-2 viral RNA through quantitative reverse transcriptase PCR tests
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54 (RT-qPCR).8 One of the first tests to get approved by the FDA was the Abbott RealTime SARS-
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3 CoV-2 assay9 which is a real time RT-qPCR test intended for the qualitative detection of nucleic
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6 acid from SARS-CoV-2 either collected from nasal swabs, nasopharayngeal (NP) or
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8 oropharungeal swabs (OP). Results from these tests, which require in-house clinical amplification
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10 and detection machines, can take between 4-6 hours for a result. Most recently the same company
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has developed an even faster test called Abbott ID NOW COVID-19 cutting processing time from
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15 hours to as little as 5 minutes for a positive result and negative results in 13 minutes.10 This
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17 particular test utilizes isothermal nucleic acid amplification for qualitative detection of SAR-CoV-
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2 viral RNA collected from the same means above. The FDA has now authorized the first
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22 molecular test that uses saliva (instead of nose or throat swabs), being developed by Rutgers
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24 RUCDR Infinite Biologics.11
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27 An emerging group of tests can now detect antibodies to the disease (viz. serology tests), which
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29 can be used to better quantify the number of people infected with COVID-19, including those that
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32 could be asymptomatic or have recovered. These tests will also help authorities in both diagnosis,
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34 population surveillance and potential return-to-work actions. These blood derived tests fall into 3
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36 categories 1) Rapid diagnostic test (RDT); 2) Enzyme-linked immunosorbent assay (ELISA) and
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3) Neutralization assay.12 In most cases, these test for patient antibodies, Immunoglobulin M (IgM)
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41 and Immunoglobulin G (IgM) titer in a qualitative or quantitative readout (Table 2, Serology tests,
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43 courtesy John Hopkins Bloomberg School of Public Health).13
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46 Table 2. Serology assays
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48 Test type Result time (hr) Readout Limitations
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50 Rapid Diagnostic <1 Qualitative presence of absence Not a quantitative readout. Can’t
51 test (RDT) of antibodies against the virus in detect antibodies that are able to
52 patient serum protect against future infection
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Enzyme linked 1-5 Quantitative presence or absence Can’t detect if antibodies are able
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55 immunosorbent of antibodies against the virus to protect against future infection
56 assay (ELISA) present in patient serum
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3 Neutralization 72 - 120 The presence of active May miss antibodies to viral
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assay antibodies in patient series that protein that are not involved in
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6 are able to inhibit virus growth replication
7 ex vivo, in a cell culture system
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11 Currently, the CDC is evaluating two serological tests and multiple other manufacturers have tests
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13 under development. It is worth noting, there are still limitations associated with serology testing
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15 as it may take many days post infection to generate these initial IgM antibodies which are
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indicative of an active or recent infection. Over time, the body develops secondary antibodies, IgG,
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20 in response to the infection which are known to take approximately 4 weeks to develop.
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3. COVID-19 vaccine treatments
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26 There are diverse approaches being taken to develop vaccines for SAR-CoV-2 with multiple
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platforms under development. These include viral vector (replicating and non-replicating)
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31 vaccines, DNA and RNA vaccines, live attenuated vaccines and protein based vaccines (Table 3.
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33 SAR-CoV-2 vaccines in development).14 DNA and RNA vaccines have been delivered rapidly
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35 with Moderna testing its mRNA-1273 2 months after sequence identification. A phase I study (45
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38 healthy adults) is underway (NCT04283461) to evaluate both the safety and immunogenicity at 3
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40 dose levels of mRNA-1273 (25,100 and 250 g) administered on a 2-dose vaccination schedule
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28 days apart. The patients will be followed for 12 months after the second vaccination.
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INO-4800 is a DNA vaccine being tested in up to 40 healthy adults at University of Pennsylvania’s
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48 Perelman School of Medicines and at the Center of Pharmaceutical Research in the US
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50 (NCT04336410). Participants will be administered two doses of INO-4800 (1 and 2 mg) 4 weeks
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52 apart.
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3 Ad5-nCoV is a genetically engineered vaccine candidate with the replication-defective adenovirus
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6 type 5 as a vector to express SARS-CoV-2 spike protein. A single center, open-label, dose-
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8 escalating Phase I clinical trial will investigate the antibody response in healthy adults who will
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10 receive one of three doses.
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16 There are over 56 confirmed active COVID-19 vaccine candidates being developed across North
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America, Europe, China and Australasia and it is well known that vaccine development is a lengthy
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21 and expensive process with high attrition.15 Many of the vaccines in development are from small
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23 developers so it will be important that both manufacturing and supply will be there to meet
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25 demand.
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3 Table 3. SARS-CoV-2 Vaccines in Development
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6 Vaccine type General attributes Companies in Preclinical development Clinical candidate, Phase, company and
7 Single Licensed Speed Current clinical trial
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dose platform scale
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10 DNA No No Fast Medium Applied DNA Sciences, Takis Biotech and Evvivax; INO-4800, Ph I, Inovio Pharmaceuticals,
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Zydas Cadila NCT04336410
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13 RNA No No Fast Low to CureVac; Stermina Therapeutics, Tongji University mRNA-1273, Ph I Moderna
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Medium and Chinese Center for Disease Control and Pharmaceuticals, NCT04283461
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16 Prevention; Imperial College London
17 Inactivated No Yes Slow High Sinovac; Wuhan Institute of Biological Products
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19 (Sinopharm)
20 Live-attenuated Yes Yes Slow High Codegenix/Serum Institute of India
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22 Non-replicating Yes No High High GeoVax/BravoVax; Janssen Pharmaceutical Ad5-nCov, Ph II, CanSinoBio,
23 vector Companies; Altimmune; Greddex; Vaxart; NCT04341389
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25 ExpresS2ion ChAdOX1nCoV-19, Ph I, University of
26 Oxford, NCT04324606
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29 Protein-based No Yes Medium High Novavax; Clover Pharmaceuticals with GSK; Baylor
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to fast College of Medicine, University of Texas Medical
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32 Branch, New York Blood Center and Fundan
33 University, China; University of Saskatchewan,
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35 Canada; University of Queensland, Australia, and
36 Dynavax; Vaxart; Generex; ExpreS2ion; Vaxil Bio;
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38 Sanofi Pasteur; iBio/CC-Pharming
39 Replicating viral Yes Yes Medium High Zydus Cadlis
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vector to High
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6 4. Re-purposed small molecule treatments for COVID-19
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10 There are currently no treatments, drugs, or vaccines to treat or prevent COVID-19 and this has
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12 accelerated drug repurposing efforts, which is the investigation of approved, existing drugs for the
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14 treatment of new diseases. Multiple existing antiviral medications which have been used for SARS,
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MERS, HIV/AIDS and malaria are being tested in clinical trials today.16 Because of the immense
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19 pressure COVID-19 is placing on the world health systems, WHO has established an international
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21 “Solidarity” clinical trial to help find an effective treatment of this disease.17a The trials will
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compare four treatment options against standard of care to assess their relative effectiveness
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26 against COVID-19. WHO chose an experimental antiviral called remdesivir 4.1; the malaria
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28 medications hydroxychloroquine 4.2 /chloroquine 4.3, and the combination of HIV drugs Kaletra
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30 (lopinavir 4.4 and ritonavir 4.5) and other combinations including interferon beta-1a. (Figure 1).
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33 Most of the drugs being re-purposed in clinical trials interestingly inhibit key components of the
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36 coronavirus infection pathway including 1) viral entry into the host cell, inhibited by
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38 hydroxychloroquine 4.2, chloroquine 4.3, or interferon beta-1a, 2) viral replication, inhibited by
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40 lopinavir 4.4 and ritonavir 4.5 or darunavir 4.6 and cobicistat 4.7, which inhibit the 3C-like
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protease (3CLpro) and 3) viral RNA synthesis inhibited by RNA polymerase inhibitors such as
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45 remdesivir 4.1 and favipiravir 4.8 (Figure 2 – graphic courtesy V. Altounian/SCIENCE).
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48 SARS-CoV-2 also uses angiotensin-converting enzyme 2 (ACE2) and the cellular protease
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50 transmembrane protease serine 2 (TMPRSS2) to enter target cells. A number of marketed
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52 TMPRSS2 inhibitors such as camostat mesylate 4.9 and nafamostat 4.10 block cellular entry of
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55 the SARS-CoV-2 virus. The Janus-associated kinase (JAK) inhibitor Olumiant (baricitinib 4.11),
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3 approved for rheumatoid arthritis, was identified using machine learning algorithms on the basis
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6 of its inhibition of ACE2-mediated endocytosis. Another JAK inhibitor, Jakafi (ruxolitinib 4.12),
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8 is also in clinical trials (combined with mesenchymal stem cell infusion) for COVID-19 (Figure
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10 1).
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13 In this article we review seven of the most promising small molecule drugs, representing different
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mechanistic approaches, that are currently in clinical trials for the treatment of COVID-19,
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18 including remdesivir, hydroxychloroquine, favipiravir, pirfenidone, baricitinib, camostat, and
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20 lopinavir/ritonavir. Except for hydroxychloroquine, none of these drugs has been intended for
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commercial use in large patient populations such as COVID-19. Therefore, the economics of
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25 production and supply considerations may become critical, including the availability of raw
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27 materials, number of steps, process efficiency, and available capacity.17b An estimate of the cost
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29 of manufacture of several drugs being repurposed for COVID-19 has been recently published by
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32 Hill et. al.17c
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58 14
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1
2
3 H 2N
4 N
Cl Cl
5 N N H H
6 N O O H O N N
HO N N
7 O N
P O N N
8
HO O
9 OH
10 Remdesivir, 4.1 Hydroxychloroquine, 4.2 Chloroquine, 4.3
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13 Ph Ph
14 O OH S O OH S
H H H
N N
15 N O N
N N N O
N
16 O N
H
O H
O O
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18 HN
19 Lopinavir, 4.4
Ritonavir, 4.5
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22 NH2 O
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24 N O
25 F N
NH2
26 O
H
O
O S O OH
27 H N
N N
N
N O N O
N N O H
28 O H H
S O
29 O O N Favipiravir, 4.8
30 S
31 Darunavir, 4.6 Cobicistat, 4.7
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33
34 O N NH2
35 O
O N O NH2
36
O
37 NH2 O H 2N O
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H 2N N
39 NH
40 Camostat, 4.9 Nafamostat, 4.10
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43 N O N
44 S
N
45 O O
N N
46 N N
N
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48 N
49 N
50 N N Pirfenidone, 4.13
N N H
51 H
52 Baricitinib, 4.11 Ruxolitinib, 4.12
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55 Figure 1. Re-purposed Small Molecules Drugs for the Potential Treatment of COVID-19
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Figure 2. Experimental Treatment Strategies to Interfere with Different Steps in the
48 Coronavirus Replication Cycle. (Kindly reproduced with permission by V.
49 Altounian/Science).
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6 5. Remdesivir
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10 5.1 History, Mechanism of Action, and Status of Clinical Trials
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Designed and developed by Gilead Sciences, remdesivir, formally known as GS-5734, is a broad
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15 spectrum antiviral agent that has been studied clinically, so far unsuccessfully, for the treatment
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17 of Ebola. According to the Summary on Compassionate Use document provided by the European
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Medicines Agency (EMA), remdesivir is a prodrug of a monophosphate nucleoside analog.18
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22 Remdesivir is seen as a promising potential therapy for COVID-19 due to its broad spectrum
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24 spectrum in vitro activity against several strains of coronavirus, including SAR-CoV-2 with EC50
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26 and EC90 values of 0.77 uM and 1.76 uM, respectively.19,20 Previously, in a mouse MERS-CoV
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29 lung infection model, remdesivir reduced viral lung titers more than lopinavir, ritonavir and
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31 interferon beta-1a.21 Remdesivir is administered via an IV infusion and the safety and
32
33 pharmacokinetics have been evaluated in both single- and multiple-dose Phase I clinical trials for
34
35
36
Ebola.22
37
38 In humans, remdesivir had a half-life of about one hour in plasma as it rapidly converts to the
39
40 intermediate mono-phosphate metabolite and the nucleoside metabolite GS-441524. Inside cells,
41
42
the monophosphate converts to the pharmacologically active analog of adenosine triphosphate
43
44
45 (GS-443902) that inhibits viral RNA polymerases. In vitro studies have shown that the
46
47 nucleoside triphosphate acts as an analog of adenosine triphosphate (ATP) and competes with
48
49 the natural ATP substrate to selectively inhibit RNA-dependent RNA polymerase.18
50
51
52
53
54
55
56
57
58 17
59
1
2
3
4
5
6 Scheme 1. Metabolism of Remdesivir
7 NH2
8
9 O N
10 H O N
N O
11 O P N
O CN
12
PhO
13 HO OH
14
15 remdesivir
16
17
NH2 NH2
18
19
N N
20 O N slow N
O O N
21 N
22 O P O CN
HO CN
23 O
HO OH HO OH
24
25 GS-704277 GS-441524
26
27 NH2
28
29 N
30 O O O N
O N
31 P P
O O O P O CN
32 O O O
33 HO OH
34
active triphosphate
35
36 GS-443902
37
38
39 Clinical trials are underway to evaluate both the safety and anti-viral activity in patients with
40
41
42
mild to moderate or severe COVID-19. Table 4 outlines a summary of seven clinical trials,
43
44 initiated in February and March 2020, that are planning to enroll about 9000 patients.23 The first
45
46 five studies listed compare five or ten days of remdesivir (200 mg on day one followed by 100
47
48
mg daily), dosed intravenously, either alone or on top of standard of care, versus placebo or
49
50
51 standard of care. The last two trials listed compare remdesivir against other anti-viral agents and
52
53 against standard of care. These trials are expected to enroll quickly with some readouts in May
54
55 and June 2020.
56
57
58 18
59
1
2
3
4
5 An early trial of open label remdesivir was published in the New England Journal of Medicine
6
7 on April 10, 2020.24 This was a small compassionate use study, lacking a placebo arm and
8
9
10 included 61 patients of which 53 patients remained on the study. Over a median follow-up of 18
11
12 days after receiving the first dose of remdesivir, 36 out of 53 patients (68%) showed an
13
14 improvement in the category of oxygen support whereas 8 of 53 patents (15%) showed
15
16
17 worsening.24
18
19 A recent unpublished PhIII clinical trial undertaken at the University of Chicago Medical Center,
20
21 which recruited 125 patients, most with severe COVID-19 disease has shown remdesivir can
22
23
quickly reduce both fever and respiratory symptoms associated with the infection. Again, this
24
25
26 study did not have a control arm, with all patients receiving daily IV infusions of the drug.25
27
28
29
30
31 The Adaptive COVID-19 Treatment Trial, a 1063-patient clinical trial sponsored by the National
32
33 Institute of Allergy and Infectious Diseases (NIAID), a well-controlled clinical trial with
34
35
36 remdesivir, reported out preliminary results on April 29, 2020.26a,b The independent data and
37
38 safety monitoring board (DSMB) overseeing the trial concluded that hospitalized patients with
39
40 advanced COVID-19 who received remdesivir recovered more quickly than patients who
41
42
received placebo. The remdesivir cohort had a statistically significant 31% faster time to
43
44
45 recovery than the placebo group (11 days vs 15 days). Recovery was defined as either hospital
46
47 discharge or return to normal activity. Mortality improved from 11.6% to 8.0 %, which was just
48
49 outside of statistical significance (p = 0.059).26a,b Based on this study, the U.S. FDA approved
50
51
52 use of remdesivir as an emergency treatment for COVID-19 on May 1, 2020.27
53
54
55
56
57
58 19
59
1
2
3 A smaller multi-center study in China that enrolled 237 severe adult COVID-19 patients,
4
5
6 published on April 29, 2020, found that remdesivir (10 days of treatment) numerically improved
7
8 clinical outcomes in hospitalized patients, but the results were not statistically significant.
9
10 Remdesivir treatment was terminated early because of adverse events in 12% of patients versus
11
12
13
5% of patients on the placebo treatment.26c
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 20
59
1
2
3 Table 4. A Sampling of Clinical Trials Underway With Remdesivir23
4
5 Clinical Trial Study Title Estimated Study Start/ Study Arms
6 Number Enrollment Estimated
7 Completion Active Control
8 Dates
9 NCT04257656 A Phase 3 Randomized, Double- 453 06Feb2020 – RDV 200 mg loading dose on day 1 is given, followed Placebo
10 blind, Placebo-controlled, 01May2020 by 100 mg iv once-daily maintenance doses for 9
11 Multicenter Study to Evaluate the days.
12 Efficacy and Safety of Remdesivir in
13 Hospitalized Adult Patients With
14 Severe 2019-nCoV Respiratory
15 Disease
16
17 NCT04252664 A Phase 3 Randomized, Double- 306 12Feb2020- RDV 200 mg loading dose on day 1 is given, followed Placebo
18 blind, Placebo-controlled 27Apr2020 by 100 mg iv once-daily maintenance doses for 9
19 Multicenter Study to Evaluate the days.
20 Efficacy and Safety of Remdesivir in
Hospitalized Adult Patients With
21
Mild and Moderate 2019-nCoV
22
Respiratory Disease.
23
24
NCT04280705 A Multicenter, Adaptive, 440 21Feb2020 – RDV 200 mg loading dose on day 1 is given, followed Placebo
25 Randomized Blinded Controlled Apr2023 by 100 mg iv once-daily maintenance doses for 9
26 Trial of the Safety and Efficacy of days.
27 Investigational Therapeutics for the
28 Treatment of COVID-19 in
29 Hospitalized Adults
30
31 NCT04292730 A Phase 3 Randomized Study to 1600 15Mar2020 – Part A. Standard of
32 Evaluate the Safety and Antiviral May2020 1) Standard of care therapy together with RDV 200 care
33 Activity of Remdesivir (GS-5734™) mg on Day 1 followed by RDV 100 mg daily on Days therapy
34 in Participants With Moderate 2-5
35 COVID-19 Compared to Standard of 2) Standard of care therapy together with RDV 200
36 Care Treatment mg on Day 1 followed by RDV 100 mg daily on Days
37 2-10
38
39 Part B, extension.
40
41
42
43 21
44
46
47
1
2
3 Continued standard of care therapy together with
4 RDV 200 mg on Day 1 followed by RDV 100 mg
5 daily on Days 2- 10.
6
7
8 NCT04292899 A Phase 3 Randomized Study to 2400 06Mar2020 – Part A. Standard of
9 Evaluate the Safety and Antiviral May2020 1) Participants who are not mechanically ventilated care
10 Activity of Remdesivir (GS-5734™) will receive continued standard of care therapy therapy
11 in Participants With Severe COVID- together with RDV 200 mg on Day 1 followed by
12 19 RDV 100 mg daily on Days 2 - 5.
13 2) Participants who are not mechanically ventilated
14 will receive continued standard of care therapy
15 together with RDV 200 mg on Day 1 followed by
16 RDV 100 mg daily on Days 2 - 10.
17
18 Part B.
19 1) Extension will enroll participants after enrollment
20 to Part A is complete. Participants will receive
continued standard of care therapy together with RDV
21
200 mg on Day 1 followed by RDV 100 mg on Days
22
2-10.
23
2) Participants on mechanical ventilation will receive
24 continued standard of care therapy together with RDV
25 200 mg on Day 1 followed by RDV 100 mg daily on
26 Days 2-10
27
28 NCT04321616 The (Norwegian) NOR Solidarity 700 26Mar2020 – 1) Hydroxychloroquine will be given orally (in the Standard of
29 Multicenter Trial on the Efficacy of Nov2020 ICU in gastrointestinal tubes) with 800 mg x 2 loading care
30 Different Anti-viral Drugs in SARS- dose followed by 400 mg x 2 every day for a total of
31 CoV-2 Infected Patients 10 days.
32 2) RDV 200 mg loading dose on day 1 is given,
33 followed by 100 mg iv once-daily maintenance doses
34 for 9 days.
35
36 NCT04315948 Multi-centre, Adaptive, Randomized 3100 22Mar2020 – 1) SoC + Remdesivir Standard of
37 Trial of the Safety and Efficacy of Mar2023 2) SoC + Lopinavir/Ritonavir care
38 Treatments of COVID-19 in 3) SoC + Lopinavir/Ritonavir plus interferon ß-1a
39 Hospitalized Adults 4) SoC + Hydroxychloroquine
40
41
42
43 22
44
46
47
1
2
3
4
5.2 Synthetic Routes to Remdesivir
5
6 An early medicinal chemistry route that has been further optimized has been published and
7
8 patented by Gilead.28-32 The first process to remdesivir generated both diastereomers (5.7-Rp
9
10
and 5.7-Sp), requiring separation by chiral chromatography (Scheme 2).28-33 The synthesis
11
12
13 started with the glycosylation reaction of benzyl-protected ribolactone 5.1 with bromide 5.2.
14
15 Two methods for metal-halogen exchange were reported. In the first method, 2 was treated
16
17 with TMS-Cl in THF at room temperature to generate silylated 5.2a, which was then cooled to
18
19
20 -78 oC and treated with n-BuLi followed by addition of lactone 5.1. The reaction was quenched
21
22 with HOAc. After aqueous workup and chromatography, 5.3 was isolated in 25% yield as a 1:1
23
24 mixture of anomers. The second method involved treatment of 5.2 with NaH and 2-
25
26
27
bis(chlorodimethylsilyl)ethane to generate silyl compound 5.2b followed by lithiation with n-
28
29 BuLi at -78 oC, then addition of lactone 5.1. This method provided 5.3 in 60% yield. The
30
31 authors noted that both methods were capricious with likely deprotonation occurring alpha to
32
33
the lactone moiety. Cyanation of hydroxyl nucleoside 5.3 was carried out using TMS-CN
34
35
36 mediated by boron trifluoride etherate in dichloromethane at -78 oC to afford cyano nucleoside
37
38 5.4, which was isolated as an 89:11 ratio of alpha/beta anomers after isolation by
39
40 chromatography in 58% yield. Deprotection of the benzyl groups was conducted with boron
41
42
43 trichloride in dichloromethane. The desired beta anomer 5.5 was isolated in 74% yield after
44
45 reverse phase chromatography. Coupling with the diastereomeric mixture of phosphoramidoyl
46
47 chloridate 5.6 was carried out in anhydrous trimethyl phosphate and THF with N-
48
49
50
methylimidazole (3 equiv) to furnish the diastereomeric mixture of 5.7-Sp and 5.7-Rp in 21%
51
52 yield after preparative HPLC. The two diastereomers were separated by chiral
53
54 chromatography using a Lux Cellulose-2 chiral column to afford, remdesivir, 4.1.
55
56
57
58 23
59
1
2
3 Scheme 2. First Gilead Route to Remdesivir
4
5
6 N(TMS)2
7 N
8 n-BuLi, -78 oC
TMS-Cl N 25%
9 NH2 N
10 Br NH2
THF 5.2a
11 N
O O N
12 N N
13 N BnO O TMSCN, BF3 OEt2
N
Br 5.2 NaH
14 BnO OBn BnO OH
15 Si Si 5.1 CH2Cl2, -78 oC
N BnO OBn 58%
16 Cl Si 5.3
17 Si Cl
N n-BuLi, THF
18 N -78 oC, 60%
19 N
20 Br
5.2b
21
O
22 H
Cl
23 NH2 NH2 N
O P
24 OPh
N BCl3, CH2Cl2 N O
25 N N 5.6
O O
26 N -78 oC, 74% N NMI, (MeO)3P=O
27 BnO CN HO CN 21%
28 BnO OBn HO OH
29 5.5
5.4
30
89:11 ()
31
32 NH2 NH2
33 O
34 H N O N
N O N H O N
35 O N + N O
O P O P N
36 O CN O CN
37 PhO PhO
HO OH HO OH
38
39 5.7-Rp 5.7-Sp
40 (remdesivir)
41
42 NH2
43
44 O N
H O N
45 chiral chromatography N O
O P N
46 O CN
47 PhO
48 HO OH
49 remdesivir, 4.1
50
51
52
53 Given the poor yields for several steps and the unreliability of the glycosylation step, an
54
55 improved route was required as the compound advanced from the discovery labs to
56
57
58 24
59
1
2
3 development.28-32 In addition to improving the early steps, the second generation route
4
5
6 achieved a diastereoselective synthesis via a selective crystallization of phosphorus coupling
7
8 partner 5.11-Sp, thus avoiding preparative scale chiral chromatography (Scheme 3). The
9
10 glycosylation step employed iodide 5.9 instead of bromide 5.2. Silyl compound 5.9 was
11
12
13
generated from the corresponding amine with PhMgCl and TMSCl followed by metal-halogen
14
15 exchange with i-PrMgCl-LiCl, then addition of lactone 5.1 at -20 oC, affording 5.3 after flash
16
17 chromatography in a modest but consistent yield of 40%. Installation of the cyano group was
18
19
conducted using TMSCN, TMSOTf, and TfOH at -78 oC to afford 5.4 in 85% yield with >95:5
20
21
22 anomeric ratio favoring the desired beta-anomer. When TFA was used instead of TfOH, the
23
24 anomeric ratio was only 3.8:1. Treatment of 5.4 with boron trichloride in dichloromethane at -
25
26 20 oC provided deprotected 5.5 in 86% yield after crystallization. To improve the yield in the
27
28
29 coupling with the phosphorus reagent, triol 5.5 was protected as an acetonide to afford 6 in
30
31 90% yield. Coupling with diastereomerically pure 5.11-Sp was then mediated by i-Pr2NEt and
32
33 MgCl2 in acetonitrile at 50 oC. After aqueous workup the product was concentrated, then
34
35
36
dissolved in THF and conc HCl added to cleave the acetonide and provide remdesivir in 48%
37
38 yield after purification by flash chromatography.
39
40
41
42
43
44 Scheme 3. Second Gilead Route to Remdesivir
45
46
47
48
49
50
51
52
53
54
55
56
57
58 25
59
1
2
3 N(TMS)2
4 NH2
5 N
6 N N
N N
7 O I 5.9a O
O N TMSCN, TfOH, TMSOTf
8 BnO BnO OH
9 i-PrMgCl LiCl CH2Cl2, -78 oC
10 BnO OBn BnO OBn
5.1 THF, -20 oC 85%
11 5.3
40%
12
13 NH2
14 NH2 NH2
MeO OMe
15 N
BCl3, CH2Cl2 N N
16 N N O N
N O
17 O N N HO
-20 oC, 86% H2SO4, rt CN
18 BnO HO CN
CN acetone O
O
19 BnO OBn HO OH 90%
20
5.4 5.5
21 >95:5 ()
5.10
22
23 O NO2
24 H O
N NH2
25 O P
O
26 PhO O
H N
27 O N
5.11-Sp 37% HCl N O
O P N
28 O
THF, rt CN
29 MgCl2, i-Pr2NEt, MeCN, 50 C o
PhO
69% HO OH
30 70%
31 remdesivir, 4.1
32
33
34 Alternate conditions for conversion of 5.1 to 5.3
35
NH2 1) TMSCl, PhMgCl, NH2
36
37 N i-PrMgCl, THF, -20 oC
N
38 N
N O N
39 N 2) O O BnO
I BnO OH
40 5.9b
41 BnO OBn
BnO OBn
42 5.1 5.3
43 NdCl3, n-Bu4NCl, THF, -20 oC
44
45
46 The routes to the two phosphoramidate chloridates used in the first and second generation
47
48
49 processes are outlined in Scheme 4.28-32 Chloridate 5.6 was prepared from alanate ester 5.8
50
51 with (PhO)P(O)Cl2 in dichloromethane mediated by triethylamine at 0 oC in 23% yield as a
52
53 mixture of diastereomers at the phosphorus center. Chloridate 5.11-rac was prepared in 80%
54
55
56
yield by first generating ester 8 at -78 oC then reacting with 4-nitrophenol at 0 oC. Given the
57
58 26
59
1
2
3 large difference in solubility of the two diastereomers in diisopropyl ether, the desired 5.11-Sp
4
5
6 diastereomer could be isolated with 39% recovery via crystallization in this solvent. The
7
8 desired diastereomer could also be crystallized from n-heptane. Based on x-ray crystal
9
10 structures of both 5.11-Sp and remdesivir, the coupling reaction of 5.10 with 5.11-Sp occurs
11
12
13
with inversion of stereochemistry at phosphorus.
14
15
Compared to the first generation route, the second generation route provided higher and more
16
17
18 consistent yields and afforded the single diastereomer of remdesivir without the need of chiral
19
20 HPLC separation. The route was used to prepare 200 g of drug for toxicity studies and other
21
22 preclinical studies.
23
24
25 Scheme 4. Routes to Phosphoramidoyl Chloridates 5.6 and 5.11-Sp
26
27
28 O
29 O O
Cl P OPh H
30 NH2 HCl Cl N Cl
O O P
31 OPh
Et3N, CH2Cl2 O
32 5.8 5.6
33 0 oC, 23%
34
35
36
37 O
38 O O NO2
Cl P OPh H
39 NH2 HCl Cl N O
40 O O P
O
41 a) Et3N, CH2Cl2 PhO
42 5.8 -78 oC 5.11-rac
43 b) 4-nitrophenol
44 Et3N, 0 oC, 80%
45
46
47 O NO2
i-Pr2O H O
48 N
O P
49 39% O
50 PhO
51 5.11-Sp
52
53
54
55
56
57
58 27
59
1
2
3 In a recently published paper, Gilead reported optimization of the cyanation step,
4
5
6 conversion of 5.3 to 5.4, as a batch process as well as the development of a
7
8
continuous flow process.33 The first goal was to determine if the cryogenic
9
10
11 conditions (reaction at -78 oC) could be avoided under batch conditions. A series of
12
13
14
14 experiments were reported with variation in temperature, equivalents of TFA,
15
16 TMSOTF, and TMSCN, and examination of other acids including HCO2H, HOAc,
17
18
19 MsOH, TsOH. The original conditions remained optimal with a 96:4 beta:alpha
20
21 ratio and 90% solution purity at -78 oC. The best alternative conditions were a
22
23
24 reaction temperature of -30 oC and addition of TFA (3 equiv) to a dichloromethane
25
26 solution of 5.3 followed by addition of a solution of TMSCN (6 equiv) and TMSOTf
27
28
29 (6 equiv), which afforded a solution purity of 5.4 of 83% and an 85:15 beta:alpha
30
31 ratio. Although these conditions afforded a higher level of the undesired alpha
32
33
34 isomer, the desired beta isomer could be isolated in >99% purity and overall 70%
35
36 yield by crystallization from EtOAc/heptane or toluene.33 This batch process was
37
38
39 described on a 23 kg scale and was used to prepare supplies of remdesivir for
40
41
clinical trials for the treatment of Ebola.
42
43
44
45
46
47
Key challenges remained, however, for further scaling of this step, including
48
49 operational hazards of handling large quantities of acidic mixtures of cyanide, and
50
51
52 controlling the diastereoselectivity and potential degradation during the increased
53
54 operational time at scale. To address these issues, a flow process was designed and
55
56
57
58 28
59
1
2
3 developed.33 The comprehensive account of the development of the flow process
4
5
6 from lab scale to manufacturing scale is very well written and provides an excellent
7
8
case study for development of a flow process for implementation at manufacturing
9
10
11 scale in the pharmaceutical industry. We commend Gilead scientists and
12
13
14
management for their willingness to publish the details of a key step in the
15
16 remdesivir process. The reader is encouraged to read the Gilead account for the
17
18
19 specifics of the development of the flow process. Herein, we only provide the final
20
21 manufacturing process, as presented in Figure 3.33 The equipment consisted of two
22
23
24 pre-cooling loops, reactor 1 (0.5 L) and reactor 2 (2.0 L), all stainless steel. In the
25
26 first step, a solution of compound 5.3 in dichloromethane (DCM) was pumped
27
28
29 through a cooling loop (-30 oC) into reactor 1, where it was mixed with a solution of
30
31 TFA (1.0 equiv) and TMSOTf (6 equiv) in DCM. The residence time in reactor 1
32
33
34 was 0.5 min. The outflow of reactor 1 was streamed into reactor 2 and mixed with a
35
36 pre-cooled solution of TMSCN in DCM, with a residence time of 2.0 min. The
37
38
39 outflow of reactor 2 was streamed into a quench tank containing aqueous KOH,
40
41
thereby neutralizing any HCN that may have been generated during the process. The
42
43
44 beta/alpha ratio was 94:6 with an overall solution purity of 90%. After workup and
45
46
47
solvent turnover, nitrile 5.4 was crystallized in 72-78% yield and >99.6% purity on
48
49 batch sizes of 86-280 kg. Reactor throughput was nearly 2 kg per h, meaning that
50
51
52 200 kg of 5.3 could be processed over a four day period. The flow process has
53
54 eliminated the need to cool large reaction vessels, has provided precise control of the
55
56
57
58 29
59
1
2
3 temperature and reaction time in order to improve diastereoselectivity and avoid
4
5
6 decomposition, and maintains a small volume of the toxic mixture that is
7
8
continuously quenched at the end of the reaction.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31 Figure 3. Flow Cyanation Step for Remdesivir
32
33
34
35
36 Although not discussed in the recent Gilead article, an improved process for
37
38
39 conversion of lactone 5.1 to the cyanation precursor 5.3 was described in the
40
41 experimental section on a 282 kg scale (Scheme 3).33 This process involved in situ
42
43
44 protection of 5.9b and use of 1.0 equiv of neodymium chloride to facilitate the
45
46 reaction with lactone 5.1. In vessel one, NdCl3 (1.0 equiv), n-Bu4NCl (1.0 equiv), and
47
48
49 THF were combined and azeotropically dried, followed by addition of lactone 5.1
50
51
(1.0 equiv). After dissolution the solution was cooled to -20 oC. In vessel two, iodide
52
53
54 5.9b (1.1 equiv) was dissolved in THF and cooled to 0 oC. Chlorotrimethylsilane (1.1
55
56
57
58 30
59
1
2
3 equiv) and the mixture was cooled to -10 oC, then PhMgCl (2.17 equiv) was added
4
5
6 and the mixture cooled to -20 oC, then i-PrMgCl (1.13 equiv) was added. After 2 h,
7
8
this mixture was added to the solution in vessel one and stirred for 8 h at -20 oC. After
9
10
11 aqueous workup, 5.3 was isolated in 69% yield after crystallization from 2-PrOAc,
12
13
14
MTBE, and n-heptane with a reported 100% purity.
15
16 This recent publication from Gilead describing optimization of two manufacturing
17
18
19 steps33 indicates significant process development has occurred relative to the initial
20
21 publications and patents, 28-32 providing confidence that a robust manufacturing
22
23
24 process has been developed that can be further scaled internally as well as transferred
25
26 to contract manufacturing organizations to support the treatment of COVID-19.
27
28
29
30 5.3 Final Forms and Formulations of Remdesivir
31
32
33
34 Three crystalline forms of remdesivir free base have been disclosed by Gilead. Competition
35
36 experiments wherein the different forms were slurried together in various solvents demonstrated
37
38 conversion to form II, demonstrating that Form II was the thermodynamically most stable free
39
40
41 base form. A crystalline maleate salt was also reported. None of the forms was hygroscopic.34
42
43
44 Several experiments at the 10-20 g scale were described for the final two steps (reaction of 5.10
45
46 and 5.11-Sp followed by deprotection) and crystallization. The coupling reaction was carried out
47
48 in THF at 20 oC for 4 h followed by aqueous quench and turnover to acetonitrile. Addition of
49
50
51
concentrated aqueous HCl to this mixture at 0 oC effected deprotection of the acetonide. Workup
52
53 was carried out with 2-methyltetrahydrofuran followed by turnover to 2-PrOAc. Crystallization
54
55 provided a mixture of Forms II and IV.34
56
57
58 31
59
1
2
3 According to the EMA Summary on Compassionate Use document,35 the final form of the API
4
5
6 may be either Form II or a mixture of Forms II and IV, both of which have similar solubilities.
7
8 Remdesivir is provided in two dosage forms, a solution formulation (stored frozen) and a
9
10 lyophilized formulation, both of which are then diluted for intravenous administration.35 Betadex
11
12
13
sulfobutyl ether sodium is used in the formulation as a solubilizing agent due to the limited
14
15 aqueous solubility of remdesivir.35 Oral delivery of remdesivir was not feasible since due to
16
17 rapid first pass clearance in the liver.35 Since remdesivir is administered as an intravenous
18
19
solution, control of the final form is not required for bioavailability, but may be important for
20
21
22 purification and the final form must have the appropriate solubility to support IV administration.
23
24
25 5.4 Remdesivir - Outlook for Supply
26
27
28
29 According to CEO O’Day, Gilead’s existing supply as of April 4, 2020, was 1.5 million doses or
30
31 roughly 140,000 treatment courses, based on 10 day treatment.36 Much of the sterile formulation
32
33
34 of drug product was manufactured from February to April, 2020, at Gilead’s La Verne, CA,
35
36 facility.37 Gilead plans to have 500,000 treatment courses available by October and 1 million by
37
38 the end of 2020.36 Over the past few months the company has been able to half the end-to-end
39
40
41 manufacturing timeline from one year to about six months. In addition, the company has
42
43 repurposed internal facilities and also increased its network of external manufacturing partners.36
44
45 With a loading dose of 200 mg on Day 1 and 100 mg maintenance doses on Days 2-10, each
46
47
treatment will require 1.1 g of drug. A million treatments would thus require about 1100 kg of
48
49
50 API, not accounting for any losses during formulation.
51
52
53
54
55
56
57
58 32
59
1
2
3 Anticipating FDA approval, on the 2020 first quarter earnings call on April 30, 2020, Chief
4
5
6 Financial Officer Andrew Dickson stated that, in addition to the ramped up internal
7
8 manufacturing effort, Gilead was also working with other global companies to develop parallel
9
10 supply chains and licensing agreements to make remdesivir available on a larger scale.38a On
11
12
13
May 12, 2020, Gilead announced outlicensing of non-exclusive rights of remdesivir to five
14
15 generic drug manufacturers in India and Pakistan, including Jubilant, Cipla, Ferozsons, Hetero,
16
17 and Mylan, allowing manufacture as well as sales in 127 countries.38b
18
19
20
21
22
23 6. Hydroxychloroquine
24
26
27
28 Hydroxychloroquine (HCQ, 4.2) and chloroquine (CQ, 4.3) have been long-standing oral drugs
29
30 for the treatment of malaria and chronic inflammatory conditions including systemic lupus
31
32 erythematosus (SLE) and rheumatoid arthritis (RA).39 HCQ, a racemic mixture, was first approved
33
34
35
in the US in 1955 and is considered the first line treatment for SLE raising potential concerns of
36
37 drug shortages for these patients to treat COVID-19 during this crisis.
38
39 In malaria, CQ is known to inhibit the action of heme polymerase in malarial trophozites
40
41
preventing the conversion of heme to hemozin.40 This causes the buildup of toxic heme, killing
42
43
44 the parasite. If HCQ proves to be effective for SARS-CoV-2, it will not act by the same mechanism
45
46 by which the drug functions as an anti-malarial. HCQ and CQ are lipophilic weak bases and
47
48 passively diffuse through cell membranes and into endosomes, lysosomes and Golgi vesicles
49
50
51 where they become protonated trapping them in the organelles and raising the pH.20 It is postulated
52
53 that by raising the pH in endosomes this prevents virus particles from utilizing their activity for
54
55 fusion and entry into the cell.41 CQ is known to inhibit terminal glycosylation of ACE2, the
56
57
58 33
59
1
2
3 receptor that SARS-CoV-2 target for cell entry. When ACE2 is in its unglycosylated state it may
4
5
6 less efficiently interact with the SARS-CoV-2 spike protein (S), further inhibiting viral entry.41
7
8 CQ inhibits SARS-CoV-2 in vitro with a half-maximal effective concentration (EC50) in the low
9
10 micromolar range. HCQ has in vitro activity with a lower EC50 for SARS-CoV-2 compared with
11
12
13
chloroquine after 24 hours of growth (HCQ: EC50 = 6.14 μM and CQ: EC50 = 23.90 μM).42
14
15 HCQ and CQ are relatively well tolerated drugs and have been extensively tested in patients with
16
17 SLE and malaria, however both compounds can cause serious adverse effects, including QTc
18
19
prolongation, hypoglycemia, retinopathy and neuropsychiatric effects.43,44
20
21
22
23
24 There is currently no high quality clinical evidence that HCQ (or CQ) has efficacy vs SARS,
25
26 MERS or SARS-CoV-2. Currently there are over 142 trials that have been registered globally
27
28
29 involving either CQ or HCQ, alone, or in combination with other drugs such as azithromycin.
30
31 Clinicaltrials.gov currently have over 99 clinical trials registered for the use of HCQ to treat or
32
33 prevent COVID-19 (Table 5).45 Typical clinical doses vary between 200-400 mg orally either
34
35
36
once or twice daily ranging from 7-14 days (and up to 60 days). A 200 mg dose of HCQ has an
37
38 exceptional human half life of 537 hours in blood and 2963 hours in plasma.46 In the body, HCQ
39
40 is rapidly N-dealkylated by cytochrome P450 (CYP) 3A4 to its active metabolite
41
42
desethylhydroxychloroquine in addition to several inactive metabolites desethylchloroquine
43
44
45 (DCQ) and bidesethylchloroquine (BDCQ) (Scheme 5).47
46
47
48
49 Scheme 5. Metabolism of Hydroxychloroquine
50
51
52
53
54
55
56
57
58 34
59
1
2
3 Cl
4 H
5 N OH
N
6 N
7
8 Hydroxychloroquine (HCQ)
9
10
11
12
13
14
Cl Cl Cl
15
H H H
16 N N OH N
N N NH2
17 H
H N
18 N N
19
Desethylchloroquine (DCQ) Desethylhydroxychloroquine Bisdesethylchloroquine (BDCQ)
20
21
22
23
24
25 A recent open-label non-randomized study in France which published on March 20 in the
26
27
International Journal of Anti-microbial Agents described the treatment of 42 patients
28
29
30 hospitalized with COVID-19, 26 of whom received hydroxychloroquine and 16 of whom
31
32 received routine care.48 Twenty patients receiving 200 mg orally of hydroxychloroquine every 8
33
34 h reported improved virological clearance vs standard of care. By Day 6, virological clearance
35
36
37 was 70% (14/20) vs 12.5% (2/16) for the hydroxychloroquine and control groups, respectively. It
38
39 was also reported that the addition of azithromycin, an antibiotic, to the hydroxychloroquine arm
40
41 resulted in complete viral clearance (6/6, 100%) versus hydroxychloroquine alone (8/14, 57%).
42
43
44
45
46 A small placebo-controlled trial of 62 patients with mild COVID-19 in Remin Hospital, Wuhan
47
48 (Chinese Clinical trial ChiCTR2000029559)49 showed that 31 patients given HCQ reported a
49
50
normal body temperature and cessation of cough much quicker compared to the 31 patients given
51
52
53 the placebo. A large proportion of the patients on HCQ also demonstrated improvement in chest
54
55 CT scans.
56
57
58 35
59
1
2
3 In a non-peer reviewed study published on May 2, 2020, addition of zinc sulfate to
4
5
6 hydrochloroquine and azithromycin resulted in statistically significant increase in the frequency
7
8 of patients being discharged home (OR 1.53, 95% CI 1.12-2.09) and a reduction in mortality or
9
10 transfer to hospice.50a
11
12
13
14
15 In contrast, there are a number of very recent reports where the use of HCQ has not met the study
16
17 primary endpoints. One such multicenter, parallel, open-label randomized trial involved 150
18
19
patients hospitalized with COVID-19 in China.50b Half (75) of the patients were administered a
20
21
22 loading dose of HCQ 1200 mg daily for three days followed by a maintained dose of 800 mg for
23
24 2-3 weeks of total treatment dependent on severity of disease. The primary endpoint, which was
25
26 the number of negative SAR-CoV-2 tests at 28 days, did not show a statistically significant
27
28
29 difference between treated vs standard of care group. The secondary endpoints which were
30
31 negative SAR-CoV-2 conversion rates at earlier days (day 4, 7, 10, 14, 21) was also similar
32
33 between the two groups.
34
35
36
37
38 Despite these mixed reports, the US FDA issued an Emergency Use Authorization for the use of
39
40 HCQ to treat COVID-19 in the USA; nonetheless larger randomized clinical trials are underway
41
42
and will be necessary to determine how effective the drug is, including its risk/benefit ratio.
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 36
59
1
2
3 Table 5. Representative Phase III Clinical trials Underway with Hydroxychloroquine45
4
5
6
10 Dates
11 NCT04329611 A Randomized, Double-blind, 1660 13Apr2020 – HCQ 400 mg po bid loading dose for 1 day followed Placebo
12 Placebo-controlled Trial to Assess 31July2020 by 200 mg po twice daily for 4 days
13 the Efficacy and Safety of Oral
14 Hydroxychloroquine for the
15 Treatment of SARS-CoV-2 Positive
16 Patients for the Prevention of Severe
17 COVID-19 Disease.
18 NCT04342221 Randomized Controlled Trial of 220 29Mar2020- 800 mg. From 2nd day on, each patient will get 600 Placebo
19 Hydroxychloroquine Versus Placebo Mar2021 mg (3 capsules) once a day until day 7 (6 more does
20 for the Treatment of Adult Patients of 600 mg).
With Acute Coronavirus Disease
21
2019 - COVID-19
22
NCT04329923 The PATCH Trial (Prevention And 400 9Apr2020 – Cohort 1 HCQ Placebo
23
Treatment of COVID-19 With 1Apr2021 COVID-19 PCR+ patients quarantined at home
24
Hydroxychloroquine) randomized to this arm will be treated with HCQ 400
25 mg twice a day for up to 14 days
26 Cohort 2 HCQ high dose
27 Hospitalized COVID-19 PCR+ patients randomized to
28 this arm will be treated with HCQ 600 mg twice a day
29 for up to 14 days
30 Cohort 2 HCQ low dose
31 Hospitalized COVID-19 PCR+ patients randomized to
32 this arm will be treated with hydroxychloroquine 600
33 mg once a day for up to 7 days
34 Cohort 3 HCQ
35 Health care workers at high risk of contracting
36 COVID-19 randomized to this arm will be treated
37 with hydroxychloroquine 600 mg once a day for 2
38 months
39 NCT04345692 A Randomized, Controlled Clinical 350 26Mar2020 – Hydroxychloroquine 400 mg 2x day by mouth on day Standard of
40 Trial of the Safety and Efficacy of 30Dec2021 1, followed by 200 mg 2x day by mouth days care
41 Hydroxychloroquine for the
42
43 37
44
46
47
1
2
3 Treatment of COVID-19 in
4 Hospitalized Patients
5 NCT04334382 Hydroxychloroquine vs. 1550 2Apr2020 – 1) HCQ 400mg po BID x 1 day, then 200mg po None
6 Azithromycin for Outpatients in 31Dec2020 BID x 4 days (dose reductions for weight <
7 Utah With COVID-19 (HyAzOUT): 45kg)
8 A Prospective Pragmatic Trial 2) Azithromycin 500mg PO on day 1 plus
9 250mg PO daily on days 2-5
10
11 NCT04344379 Randomized Multicenter Study 900 15Apr2020 – 1) HCQ 200mg BID Placebo
12 Evaluating the Efficacy of 31Aug2020 2) Azithromycin 200mg per day
13 Azithromycin and
14 Hydroxychloroquine in the
15 Prevention of SARS-CoV-2
16 Infection in the Hospital Population
17 Exposed to Virus
18 NCT04328285 Chemoprophylaxis of SARS-CoV-2 1200 14Apr2020 – 1) HCQ 200 mg : 2 tablets on the evening at Day 1 Placebo
19 Infection (COVID-19) in Exposed Nov2020 and 2 tablets on the morning at Day 2 and 1
20 Healthcare Workers : A Randomized tablet once daily afterwards
21 Double-blind Placebo-controlled 2) Lopinavir/ritonavir 200/50mg, 2 tablets twice
22 Clinical Trial daily
23
NCT04331834 Pre-Exposure Prophylaxis With 440 3Apr2020 – HCQ 400 mg daily during the first 4 days, followed Placebo
24
Hydroxychloroquine for High-Risk Oct2020 by 400 mg weekly during 6 months
25
Healthcare Workers During the
26
COVID-19 Pandemic: A Unicentric,
27 Double-Blinded Randomized
28 Controlled Trial
29 NCT04315896 Hydroxychloroquine Treatment for 500 20Mar2020 – HCQ tablet 200mg every 12 hours for 10 days Placebo
30 Severe COVID-19 Respiratory Oct2020
31 Disease: Randomised Clinical Trial
32 (HYDRA Trial)
33 NCT04318015 Chemoprophylaxis With 400 14Apr2020 – HCQ 200 mg daily for 60 days Placebo
34 Hydroxychloroquine in Healthcare Dec2020
35 Personnel in Contact With COVID-
36 19 Patients: A Randomized
37 Controlled Trial (PHYDRA Trial)
38 NCT04341441 Will Hydroxychloroquine Impede or 3000 7April2020- 1) HCQ 200mg PO daily following day 1 dose Placebo
39 Prevent COVID-19: WHIP COVID- Jun2020 of 400 mg orally once
40 19 Study 2) HCQ for prophylaxis of malaria is 6.5 mg/kg
41 per dose (maximum of 400mg per dose)
42
43 38
44
46
47
1
2
3 administered orally weekly on the same day
4 of each week.
5 NCT04328012 Comparison Of Therapeutics for 4000 6April2020- 1) lopinavir/ritonavir 400mg/200mg mg po BID Placebo
6 Hospitalized Patients Infected With Jan2021 X 5-14 days depending on availability
7 SARS-CoV-2 In a Pragmatic 2) HCQ sulfate 400 mg BID on Day 0 200 mg
8 aDaptive randoMizED Clinical Trial BID Days 1-4, days 1-13 if available
9 During the COVID-19 Pandemic 3) losartan 25 mg po QD X 5-14 days
10 (COVID MED Trial) depending on availability
11 NCT04332991 Outcomes Related to COVID-19 510 2Apr2020- HCQ 400 mg twice daily, then 200 mg twice daily for Placebo
12 Treated With Hydroxychloroquine Apr2021 the next 4 days for a 5 day total course
13 Among In-patients With
14 Symptomatic Disease
15 NCT04325893 Hydroxychloroquine Versus Placebo 1300 Apr2020- First dose of 400 mg will be taken immediately after Placebo
16 in Patients Presenting COVID-19 Sep2020 inclusion at day 0, the second dose of 400 mg will be
17 Infection and at Risk of Secondary taken on the same evening and the treatment will then
18 Complication: a Prospective, be continued for the following eight days at a rate of
19 Multicentre, Randomised, Double- 200 mg in the morning and evening.
20 blind Study
21 NCT04341727 WU 352: Open-label, Randomized 500 4Apr2020- 1) HCQ 400mg orally twice a day for one day,
22 Controlled Trial of Apr2021 followed by 200mg twice a day for four
23 Hydroxychloroquine Alone or consecutive days (Five days in total)
Hydroxychloroquine Plus 2) HCQ 400mg orally twice a day for one day,
24
Azithromycin or Chloroquine Alone followed by 200mg twice a day for four
25
or Chloroquine Plus Azithromycin in consecutive days (Five days in total) AND
26
the Treatment of SARS CoV-2 Azithromycin 500mg orally once, followed
27 Infection by 250mg daily for four consecutive days
28 (five days total)
29 3) Chloroquine phosphate 1000mg orally once,
30 followed in 12 hours by 500mg, then 500mg
31 orally twice daily for 4 days (Five days in
32 total)
33 4) Chloroquine phosphate 1000mg orally once,
34 followed in 12 hours by 500mg, then 500mg
35 orally twice daily for 4 days (Five days in
36 total) AND Azithromycin 500mg orally
37 once, followed by 250mg daily for 4
38 consecutive days (5 days total).The drug will
39 be supplied in 250mg tablets.
40
41
42
43 39
44
46
47
1
2
3
4
6.2 Synthesis of Hydroxychloroquine
5
6 The synthesis of hydroxychloroquine was disclosed by Sterling-Winthrop in a 1950 publication
7
8 and 1951 patent.51 The route involves three steps, as outlined in Scheme 6. In the first step 1-
9
10
chloro-4-pentanone (6.1) was alkylated with N-ethyl-N-hydroxyethylamine (6.2) (2.0 equiv) for
11
12
13 3 h in refluxing xylene. The resulting tertiary amine 6.3 was isolated in 44% yield after vacuum
14
15 distillation. The reductive amination of 6.3 was carried out in methanolic ammonia with 1000 psi
16
17 hydrogen for 24 h at room temperature catalyzed by Raney nickel. Amine 6.4 was isolated in
18
19
20 89% yield after vacuum distillation. For the third step, amine 6.4 (1.7 equiv), 4,7-
21
22 dichloroquinoline (6.5)(1.0 equiv), phenol, and catalytic KI were heated to 125-130 oC for 18 h.
23
24 When the reaction was completed, the mixture was diluted with MeOH and phosphoric acid was
25
26
27
added, then the side of the flask scratched with a glass rod to induce crystallization. The
28
29 diphosphate salt of hydroxychloroquine was isolated in 42% yield.51
30
31
32 Scheme 6. Initial Route to Hydroxychloroquine
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 40
59
1
2
3 OH
4 N
H OH
5 Cl 6.2 N
6 xylene, NaCl
O O
7 6.1 reflux 6.3
8 44%
9
10
11 H2 (1000 psi)
12 28% NH3 in MeOH OH
N
13
RaNi, rt, 24 h NH2
14
15 89% 6.4
16
17 Cl
18
19 HN
20
Cl N
21 6.5 H3PO4
22 N
MeOH Cl N
23 PhOH, KI
42%
24 125-130 oC 2 H3PO4 OH
25 hydrochloroquine phosphate
26
27
28
29
30
31
Protection of ketone 6.1 as its ketal (not shown) prior to displacement with amine 6.2 improved
32
33
34 the yield to 83% (110 oC in toluene) but requires two additional steps, ketalization and
35
36 deprotection. Overall yield for the 3-step sequence was 67%.52
37
38
39 The yield for the displacement reaction of 4,7-dichloroquinoline (6.5) with 6.4 was subsequently
40
41 improved as follows:
42
43
44 1) Carrying out the reaction neat under a pressure of 10 – 20 bars allowed for a reduced
45
46
47
reaction temperature of 100-110 oC for 4 h. Hydroxychloroquine free base was isolated in
48
49 78% after aqueous workup, pH swings, carbon and alumina treatment, then
50
51 crystallization from dichloroethane.53
52
53
2) A 78% yield was reported using a supported cobalt nitrate catalyst and carrying out the
54
55
56 reaction at 90-110 oC in xylenes for 1.5-2.5 h.54
57
58 41
59
1
2
3 3) The reaction was carried out using sodium ethoxide in 2-PrOAc, then slowly distilling off
4
5
6 2-PrOAc and increasing the temperature to 120-122 oC. After aqueous workup and
7
8 crystallization from 2-PrOAc, hydroxychloroquine free base was isolated in 88% yield
9
10 and 99.3% purity.55
11
12
13
4) The reaction was carried out neat at 120-130 oC for 13-24 h followed by aqueous workup
14
15 and crystallization of the crude free base from EtOAc or 1,2-dichloroethane in 88-89%
16
17 yield and 95-96% purity. Crystallization of the sulfate salt from EtOH improved the
18
19
purity to >99%.56
20
21
22 5) A yield of 78% was achieved by carrying out the reaction in i-Pr2NEt at 125-135 oC for
23
24 three to four days. The product was isolated by silica chromatography.57 We note also in
25
26 this paper that resolution of 6.4 was described using each enantiomer to mandelic acid to
27
28
29 provide gram quantities of each enantiomer of 6.4,57 although hydroxychloroquine is
30
31 approved as a racemate.
32
33
34 Gupton’s group at Virginia Commonwealth has recently adapted the hydroxychloroquine
35
36 synthesis to continuous flow.58 A few modifications were made to the original synthesis to
37
38
39
render it practical for flow synthesis: (1) iodide 18 was used instead of chloride 12 to accelerate
40
41 the displacement reaction and minimize side reactions; (2) to facilitate telescoping reactions,
42
43 THF was chosen as the primary solvent; and (3) instead of direct reductive amination of ketone,
44
45
a two- step process was developed via oxime 19, allowing both steps to be carried out in THF
46
47
48 (Scheme 7).
49
50
51 Scheme 7. Hydroxychloroquine Route Adapted to Flow Synthesis
52
53
54
55
56
57
58 42
59
1
2
3 OH
4 O N
O H 6.2
5 55% HI
O I N
6 -CO2 THF, K2CO3
O O
7 6.6 89% 6.7 6.3
100 oC, 86%
8 OH
9
10
11 NH2OH-H2O N RaNi, H2 (10 bar)
12
N
13 K2CO3, THF HO 6.8 THF, 80 oC
14 100 oC OH 84%
15 78 %
16 Cl
17
18 HN
19
20 N Cl N
6.5 N
21 NH2 Cl N
22 o
6.4 Et3N, K2CO3, 125 C
23 OH 78% OH
24 hydroxychloroquine
25
26
27 For the first step in flow, lactone 6.6 and 55% aqueous HI were flowed into a tubular reactor at
28
29 80 oC with a residence time of 5 min (Figure 4). The crude mixture of 6.7 was advanced into a
30
31 mixer along with streams of sodium bicarbonate and hexanes/MTBE. The two phases were then
32
33
34 passed through a hydrophobic membrane separator (Zaiput). The product in the organic layer
35
36 was concentrated to dryness and used without purification in the next sequence.
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53 Figure 4. Flow Schematic for Conversion of Lactone 6.6 to 1-Iodo-4-pentanone (6.7)
54
55
56
57
58 43
59
1
2
3 The next three steps were telescoped (Figure 5). Streams of iodide 6.7 and amine 6.2 in THF
4
5
6 were combined and pre-heated prior to flowing into a packed bed of potassium carbonate at 100
7
8 oC. The output stream of 6.3 was combined with hydroxylamine in THF and flowed through a
9
10 packed bed of potassium carbonate at 100 oC to afford a solution of oxime 6.8. This solution
11
12
13
was then advanced into a continuous stirred tank reactor (CSTR) where it was combined with
14
15 Raney Nickel and hydrogen gas (10 bar) at a temperature of 80 oC. The product was collected
16
17 and worked up in batch mode. After concentration and vacuum distillation, amine 6.4 was
18
19
isolated in 68% yield for the three steps.
20
21
22
The final step, reaction of 6.4 with 4,7-dichloroquinoline (6.5) was carried out in batch mode and
23
24
25 only described on a 200 mg scale. The best conditions were mixing triethylamine and potassium
26
27 carbonate in EtOH, then removing the EtOH and warming to 125 oC, which afforded
28
29 hydroxychloroquine in 78% yield after purification by silica chromatography.58
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
Figure 5. Flow Schematic for Conversion of 1-Iodo-4-pentanone (6.7) to Amine 6.4
53
54
55
56
57
58 44
59
1
2
3
4
5
6
7 6.3 Hydroxychloroquine Final Form and Formulation
8
9
10
11 The final form of hydroxychloroquine is a mono-sulfate salt. Both the tertiary amine and
12
13 quinoline nitrogens are protonated in the crystalline sulfate salt.59 Two polymorphs have been
14
15 reported, one melting at 240°, the other ~198°.60
16
17
18 The oral formulation is provided as a film coated tablet containing 200 mg of the sulfate salt,
19
20
21
equivalent to 155 mg of the free base.61
22
23
24 6.4 Hydroxychloroquine - Outlook for Supply
25
26
27
28 Substantial quantities of hydroxychloroquine have been promised over the next few months from
29
30 seven manufacturers.62
31
32
33 In March 20, 2020, press release, the Sandoz division of Novartis reported that it has 50 million
34
35
doses (200 mg tablet) in stock and is planning to donate up to 130 million doses by the end of
36
37
38 May. The company is also evaluating increasing production capacity internally and with external
39
40 partners.63
41
42
43 In a March 19, 2020, press release, Mylan stated that the company had restarted production of
44
45 hydroxychloroquine sulfate tablets at its West Virginia facility and is exploring options to initiate
46
47
48 production ex-US. With the API currently available, the company expects to begin supplying
49
50 drug by mid-April with the goal to ramp up manufacture to supply 50 million doses.64
51
52
53 On March 19, 2020, Teva announced they will provide 6 million doses of hydroxychloroquine
54
55 sulfate tablets by March 31, 2020 and more than 10 million by the end of April, 2020.65
56
57
58 45
59
1
2
3 On March 20, 2020 Amneal announced it is increasing production of hydroxychloroquine sulfate
4
5
6 and expects to provide 20 million tablets by mid-April.66
7
8
9 On April 10, 2020, Sanofi announced its intent to donate 100 million doses of hydroxychloroquine
10
11 across 50 countries. Sanofi has doubled its incremental production capacity across its eight
12
13 hydroxychloroquine manufacturing sites worldwide and is on track to quadruple production by the
14
15
16
summer of 2020.67
17
18
19 According to a Bloomberg article, India manufactures 47% of the world's supply of
20
21 hydroxychloroquine.68 The country initially banned its export on March 25, 2020, but reversed
22
23
24 this decision on April 8, 2020, after the U.S. requested supplies.69 Zydus Cadila is increasing
25
26 production from 3000 kg per month to 20,000-30,000 kg per month with the possibility of
27
28 further increasing to 50,000 kg if needed. Ipca Labs have a 20,000 kg capacity and can
29
30
31
manufacture 100 million tablets per month. They can increase capacity to 26,000 kg. Ipca
32
33 Labs has been barred from selling its products in the U.S. since 2015 for violation of GMP
34
35 regulations, but on March 20, 2020, the FDA made an exception for the import of
36
37 hydroxychloroquine.69 Dr. Reddy’s also manufactures hydroxychloroquine sulfate tablets.70a
38
39
40
41 Production in China was also curtailed due to a lack of raw materials but is expected to recover
42
43 by June, 2020. Manufacturers in China include Chongqing Kangle Pharmaceutical Co., Ltd.,
44
45
Nantong Jinghua Pharmaceutical Co., Ltd., and Chongqing Southwest No.2 Pharmaceutical
46
47
48 Factory Co., Ltd.70b
49
50
51
52
53
54
55
56
57
58 46
59
1
2
3
4 7. Favipiravir
5
6
7 Favipiravir (4.8) (sold under the tradename Avigan), a broad spectrum antiviral discovered by
8
9 Toyoma Chemical Company, Ltd (Fujifilm group), is a modified pyrazine analog that was
10
11 originally approved for influenza resistance in Japan.71 It inhibits replication of both influenza A
12
13
14 and B and has also shown activity versus avian influenza.72 Favipiravir (FPV) is a prodrug which
15
16 undergoes intracellular ribosylation and phosphorylation to become the active drug Favipiravir-
17
18 RTP (Scheme 8).71,73
19
20
21
22 Scheme 8. Metabolism of Favipiravir
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 47
59
1
2
3 O
4 F N
5 NH2
6
HO N O
7 H
8 Hydroxy-favipiravir
9
10
11 O O
12 O F N F N
NH2 NH2
13 F N
NH2
14 N O N O
15 N O
OH OH
H O O
16
17 Favipiravir 4.8
HO OH O O OH
18 P
19 HO OH
Favipiravir R
20 Favipiravir RMP
21 O
22 F N glucoronide
NH2
23 O
24 N O F N
25 H O NH2
26 Favipiravir glucoronide F N N O
NH2
27
28 O OH
N O
29
OH O
30 O O OH
31 P
OH
O OH O
32 O O
P P
33 OH OH
O O
34 O O
P P
35 HO
OH
HO
OH
36
Favipiravir RDP Favipiravir RTP
37
38
39
40
41
43
44 Favipiravir-RTP is an inhibitor of RNA dependent RNA polymerase which prevents viral
45
46 transcription and replication.74 Several hypotheses as to how favipiravir-RTP interacts with
47
48
49 RNA dependent RNA polymerase have been generated which show that it is incorporated into a
50
51 nascent RNA strand which prevent RNA strand elongation and viral proliferation.71 The presence
52
53 of purine nucleoside analogs can significantly reduce FPV’s antiviral activity suggesting there is
54
55
56 competition between both FPV and the purine nucleosides for the same binding site. Most of
57
58 48
59
1
2
3 favipiravir’s pre-clinical data is associated with its influenza and Ebola activity but the
4
5
6 compound has demonstrated broad activity versus other RNA viruses.71 The in vitro EC50 of
7
8 Favipiravir against SARS-CoV-2 is 61.88 uM/L in Vero E6 cells.20
9
10 The human bioavailability of FPV is very high at 98%, its metabolites are predominantly renally
11
12
13
cleared, and its half life is estimated to range from 2 to 5.5 hours.72
14
15 The most frequent adverse events of FPV reported during the development for influenza
16
17 treatment include mild to moderate diarrhea, asymptomatic increase of blood uric acid and
18
19
transaminases and decrease of neutrophil count.75
20
21
22 FPV has been approved for use in clinical trials of COVID-19 in China and it has also been
23
24 approved for experimental use in Italy, especially in the most affected regions.76
25
26
27
28
29 Limited and smaller patient number clinical trials are underway using a FPV starting dose of
30
31 1600 mg twice daily on day one followed by 600 mg twice daily for the next 9 days (Table 6).77
32
33 A few of these trials are comparing FPV with chloroquine, or in combination with other drugs
34
35
36
such as tocilizumab.
37
38
39
40 Thus far, there is little clinical evidence this drug has benefit for COVID-19 so further clinical
41
42
trials are warranted. A non-placebo open-label trial undertaken in in Shenzhen, China, compared
43
44
45 oral favipiravir (1600 mg twice daily for 1 day, then 600 mg twice daily) plus inhaled interferon
46
47 compared with a historical cohort of patients receiving lopinavir/ritonavir for 14 days. Those
48
49 treated with FPV and interferon had median shedding of virus of 4 days, compared with 11 days
50
51
52 in the lopinavir/ritonavir group.78 Radiographic improvement of chest CT scans was also seen in
53
54 91% of FPV-interferon treated patients compared with 62% of those on lopinavir/ritonavir.
55
56
57
58 49
59
1
2
3 Again, this was not a randomized, placebo controlled clinical trial so much caution is required in
4
5
6 interpreting these results. A prospective, randomized, controlled, open-label multi-center trial
7
8 based in China (China clinical trial CHiCTR20000302540 compared FPV (1600 mg twice daily
9
10 Day 1 followed by 600 mg twice daily Day 2-10) to Umifenovir (Arbidol) (200 mg thrice daily
11
12
13
for 10 days) involving adult COVID-19 patients. Favipiravir did not significantly improve
14
15 clinical recovery rate at Day 7 (61%) versus Umifenovir (52%).79
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 50
59
1
2
3 Table 6. Clinical trials underway with Favipiravir77
4
5
6
10 Dates
11 NCT04336904 A Multi-center, Randomized, 100 25Mar2020 – Day 1: FPV 1800mg, BID; Day 2 and thereafter: Placebo
12 Double-blind, Placebo-controlled, Jul2020 600mg, TID, for a maximum of 14 days.
13 Phase III Clinical Study Evaluating
14 the Efficacy and Safety of
15 Favipiravir in the Treatment of
16 Patients With COVID-19-Moderate
17 Type
18 NCT04349241 Efficacy and Safety of Favipiravir in 100 20Apr2020- FPV 3200 mg (1600 mg 12 hourly) loading dose on Standard of
19 Management of COVID-19 Oct2020 day-1 followed by 1200 mg maintenance dose (600 care
20 mg 12 hourly daily) on day 2-10
21 NCT04310228 Favipiravir Combined With 150 8Mar2020 – 1) FPV – Day 1 1600mg BID, then Day 2-7 600mg None
Tocilizumab in the Treatment of May2020 BID AND Tocilizumab 4-8mg/kg IV
22
Corona Virus Disease 2019-A 2) FPV – Day 1 1600mg BID, then Day 2-7 600mg
23
Multicenter, Randomized and BID
24
Controlled Clinical Trial Study 3) Tocilizumab 4-8 mg/kg IV
25
NCT04333589 The Mechanism, Clinical Outcome 210 1Apr2020 – FPV 1600mg BID Day 1, then Day 2-7 600mg BID Standard of
26 and Therapeutic Intervention of Sep2020 for 14 days care
27 Corona Virus Disease 2019 Patients
28 Whose Nucleic Acids Changed
29 From Negative to Positive
30 NCT04346628 A Phase 2 Randomized, Open Label 120 Apr2020 – FPV 1800 mg on the first dose (day 1) followed by Standard of
31 Study of Oral Favipiravir Compared Apr2021 800 mg BID for the next 9 days (days 2-10) care
32 to Standard Supportive Care in
33 Subjects With Mild COVID-19
34 NCT4319900 Clinical Trial of Favipiravir Tablets 900 5Mar2020 – 1) FPV (1600 mg BID, Day 1) then 600 mg Placebo
35 Combine With Chloroquine Apr2020 BID Day 2-10 AND chloroquine 500 mg
36 Phosphate in the Treatment of Novel BID Day 1, 500mg QD Day 2-3, 250 mg QD
37 Coronavirus Pneumonia Day 4-10
38 2) FPV (1600 mg BID, Day 1) then 600 mg
39 BID Day 2-10
40
41
42
43 51
44
46
47
1
2
3
4
7.2 Synthesis of Favipiravir
5
6 7.2.1 Routes from the Innovator Company, Toyama Chemicals
7
8 While favipiravir is a very small molecule, with a molecular weight of only 157, its synthesis is
9
10
not trivial. The first route to favipiravir reported by Toyama Chemicals (Scheme 9) required six
11
12
13 steps with an overall yield of about 2%.80 As is often the case with Medicinal Chemistry routes
14
15 which are designed to quickly generate analogs, several steps are unoptimized with low yields.
16
17 The synthesis starts with 3-amino-6-bromopyrazine-2-carboxylate (7.1), which was diazotized in
18
19
20 concentrated sulfuric acid with sodium nitrite, then added to MeOH to generate methoxy
21
22 compound 7.2 in 35% yield. Cross coupling of 7.2 with benzophenone imine with Pd catalysis
23
24 provided adduct 7.3 which was then hydrolyzed with 2M HCl to furnish amine 7.4 in 43% yield
25
26
27
for the two steps. The methyl ester 7.4 was converted to the primary amide 7.5 with methanolic
28
29 ammonia in 88% yield. Diazotization/fluorination was conducted using sodium nitrite in 70%
30
31 pyridinium hydrofluoride to afford fluoro compound 7.6 in 86% yield. In the final step,
32
33
conversion of the methoxy group to the hydroxyl group was accomplished with in-situ generated
34
35
36 TMS-I in acetonitrile to furnish favipiravir in 15% yield.80
37
38
39 Scheme 9. First Generation Route to Favipiravir
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 52
59
1
2
3 Ph2C=NH
4 Br N CO2Me Br N CO2Me (S)-BINAP
5 H2SO4, NaNO2
t-BuONa
6 N NH2 MeOH, reflux N OMe Pd2(dba)3
7 tol, 80 oC
7.1 35% 7.2
8
9
10 Ph Ph
11
2M HCl H 2N N CO2Me NH3
12 N N CO2Me
13 THF MeOH
14 43% (2-steps) N OMe 88%
N OMe
15 7.4
7.3
16
17
18 NaI
H 2N N CONH2 NaNO F N CONH2 F N CONH2
19 2 TMS-Cl
20 pyr-HF MeCN
N OMe N OMe N OH
21 86% 15%
22 7.5 7.6 favipiravir, 4.8
23
24
25
26
27
28 A second generation route was disclosed on a multigram to a few hundred grams by Toyama in
29
30 2001 (Scheme 10).81 The synthesis started with 3-hydroxy-2-pyrazinecarboxamide (7.7),82 which
31
32
33 was nitrated with sodium nitrate in concentrated sulfuric acid at 30-40 oC. Nitro compound 7.8
34
35 was crystallized by adding the reaction mixture to water, then slurried in dilute NaOH, resulting
36
37 in an isolated yield of 65%. Nitro pyrazine 7.8 was treated with phosphorus oxychloride and
38
39
40
pyridine with a ramp in temperature from 60 to 100 oC, resulting in displacement of both the
41
42 nitro and hydroxyl groups with chlorine and dehydration of the amide to the nitrile. After
43
44 aqueous workup and silica chromatography, 3,6-dichloropyrazinecarbonitrile (7.9) was isolated
45
46
in 77% yield. Displacement of both chlorines with fluorines was accomplished with KF and n-
47
48
49 Bu4NBr in DMSO at 90-100 oC for 6 h. After aqueous workup and silica chromatography, 7.10
50
51 was isolated in 79% yield. Hydrolysis of the nitrile to carboxamide was carried out with 12 M
52
53 HCl and THF at 30-35 oC to afford 3,6-difluoropyrazinecarboxamide (7.11) in 84% yield.
54
55
56 Selective hydrolysis of the 3-F group with sodium bicarbonate at 50 oC in dioxane/water
57
58 53
59
1
2
3 provided crystalline favipiravir in 52% yield. The overall yield for the five step synthesis was
4
5
6 17% with two purifications by column chromatography.
7
8
9 An alternate sequence for conversion of 3,6-difluoropyrazinecarbonitrile (7.10) to favipiravir
10
11 was disclosed by Toyama in 2009 (Scheme 10).83 The 3-F position was hydrolyzed first using
12
13 KOAc in aqueous DMF and the resulting hydroxyl compound 7.12 was purified by
14
15
16
crystallization as its dicyclohexylamine salt in 82% yield. The crystallization of the salt was
17
18 carried out directly on the reaction mixture, thereby avoiding a difficult workup since 7.12 is a
19
20 water soluble compound. In the final step, the nitrile of 7.12 was hydrolyzed to the carboxamide
21
22
using aqueous hydrogen peroxide, affording favipiravir in 89% yield after crystallization from
23
24
25 the acidified solution.
26
27
28 Scheme 10. Second Generation Route Disclosed by Toyama
29
30
31 1) NaNO3
N CONH2 H2SO4 O 2N N CONH2 POCl3
32
33 2) Aq. NaOH pyr
34 N OH N OH
65% 77%
35 7.7 7.8
36
37 Cl N CN F N CN
38 KF, n-Bu4NBr
39 DMSO
N Cl N F
40 79%
41 7.9 7.10
12 M HCl
42 KOAc THF, 30-35 oC
43 DMF, H2O 84%
44 82%
45 F N CN F N CONH2
46
47 N OH N F
48 7.12 7.11
49 NaHCO3
50 H 2O 2, H 2O Dioxane, H2O
51 89% 50 oC, 52%
52 F N CONH2
53
54
55 N OH
56 favipiravir
57
58 54
59
1
2
3 An improved route to 3,6-dichloropyrazinecarbonitrile (7.9) was developed by Nippon Soda
4
5
6 (Scheme 11).84 The sodium salt of 7.7 is first prepared from 2-aminomalonic acid diamide (7.13)
7
8 and glyoxal in an alkaline aqueous solution with the product crystallized from the reaction
9
10 mixture in 92% yield. Bromination with molecular bromine was conducted in a mixture of
11
12
13
MeOH/acetonitrile. After reaction completion, water was added to crystallize product 7.14 in
14
15 76% yield. Chlorination of the 3- and 6-positions was accomplished using phosphorus
16
17 oxychloride and diisopropylethylamine in chlorobenzene at 90-100 oC. After aqueous workup
18
19
3,6-dichloropyrazinecarbonitrile (7.9) was generated as a solution in toluene that was used
20
21
22 directly in the next fluorination step under similar conditions as outlined in Scheme 10.
23
24
25 Scheme 11. Improved Route to 3,6-dichloropyrazinecarbonitrile (7.9)
26
27
28 CHO
NH2 CHO N CONH2
29
30 H2NOC CONH2 Aq. NaOH
31 N ONa
7.13 92%
32 7.7-Na
33
34 Br N CONH2 POCl3
35 Br2 i-Pr2NEt
36 PhCl
MeOH/MeCN N OH
37 76% 90-100 oC
7.14
38 83%
39
40 Cl N CN
41
42 N Cl
43 7.9
44
45
46 A third generation route from Toyama was disclosed in a 2013 patent application.85a,b This route
47
48
49
is longer (9 steps) but builds the core pyrazine ring system from simple and inexpensive starting
50
51 materials (Scheme 12). This route uses an isoxazole as a masking group for the ortho amide and
52
53 hydroxyl functional groups.85a,b The early steps are described on a kilogram scale, with gram
54
55
scale experimentals for the final steps. The synthesis starts with the saponification of ethyl
56
57
58 55
59
1
2
3 diethoxyacetate (7.15) to diethoxyacetic acid (7.16) in 95% yield followed by amide bond
4
5
6 formation with aminoacetonitrile mediated by CDI to afford 7.17 in 60% yield. Next, reaction
7
8 with dimethyl oxalate in THF mediated by t-BuOK generated 7.18, which was not isolated but
9
10 reacted with hydroxylamine hydrochloride with TFA in MeOH at reflux to furnish isoxazole
11
12
13
7.19 in 48% yield for the two steps. Formation of the pyrazine ring was accomplished by
14
15 hydrolysis of the acetal group with p-TsOH-H2O in HOAc at 77 oC to provide 7.20 in 58% yield.
16
17 Conversion of the hydroxyl group to chloride was conducted in POCl3 with triethylamine
18
19
hydrochloride at 85 oC to provide 7.21 in 89% yield. At this point in the synthesis, several
20
21
22 different esters were prepared, some of which gave somewhat better yields and ease of operation
23
24 in the next steps. Fluorination was carried out with KF in DMSO at 80 oC to afford 7.22 in 84%
25
26 yield. In some cases it was found that addition of 1-chloro-2,4-dinitrobenzene reduced the
27
28
29 amount of black tar generated in the reaction, perhaps as a sink for excess fluoride. Alternatively,
30
31 hydroxyl compound 7.20 could be directly converted to fluoro compound 7.22 with 2,2-difluoro-
32
33 1,3-dimethylimidazolidine (7.23);85c however, the yield with this expensive fluorinating agent
34
35
36
was modest at 44% after isolation by silica chromatography. Opening of the isoxazole with
37
38 aqueous NaOH in THF revealed the nitrile 7.12, which was purified by treatment with an ion-
39
40 exchange resin and then hydrolyzed to the amide (favipiravir) with basic hydrogen peroxide.
41
42
Favipiravir could be purified to 99.0% by crystallization of its dicyclohexylamine salt.85a,b
43
44
45
Scheme 12. Third Generation Toyama Route to Favipiravir
46
47
48
49
50
51
52
53
54
55
56
57
58 56
59
1
2
3 O
4 H 2N CN
EtO CO2Et Aq. NaOH EtO CO2H CDI, Et3N EtO
5 N CN
6 OEt 70 oC OEt CH3CN OEt H
7 7.15 95% 7.16 60% 7.17
8
9 CO2Me NH2OH-HCl H 2N
10 O CN O O
CO2Me TFA, MeOH N
11 EtO OK EtO
t-BuOK N reflux N
12 H
OEt CO2Me H CO2Me
13 THF 48%, 2 steps OEt
14 7.18 7.19
15
16
POCl3
17 N O N O
p-TsOH-H2O Et3N-HCl
18 N N
19 HOAc, 77 oC HO N 85 oC Cl N
20 58% CO2Me 89% CO2Me
7.20 7.21
21
22
23 Me N N Me
CH3CN
24 F F 44%
25 7.23
26
27 KF, DMSO N O NaOH N OH N OH
NaOH
28 N
80 oC THF/H2O F H 2O 2
29 F N N CN F N CONH2
30 84% CO2Me 90% 84%
7.22 7.12 favipiravir
31
32
33
An alternate four step route to intermediate 7.21P from maleic amide-ester 7.24 was also
34
35
36 disclosed by Toyama (Scheme 13).85 Michael addition of hydroxylamine to 7.24 in aqueous 2-
37
38 PrOH afforded ester-amide 7.25 in 59% yield after crystallization from the reaction mixture.
39
40 Condensation of 7.25 with an aqueous solution of glyoxal in 2-PrOH at 41 oC, and controlling
41
42
43 the pH at 9.0 by dropwise addition of sodium carbonate, provided pyrazine N-oxide 7.26 in 62%
44
45 yield after crystallization, which was converted to oxime 7.27 in 88% yield by treatment with
46
47 isoamyl nitrite and acetyl chloride in 2-PrOH and crystallization from the reaction mixture. Ring-
48
49
50 closure and chlorination upon treatment with phosphorus oxychloride in toluene/DMF at 70 oC
51
52 afforded the isopropyl ester 7.21P in 49% yield. While yields for this preparation are modest, all
53
54 compounds are crystalline and several directly crystallize from the reaction mixture.
55
56
57
58 57
59
1
2
3
4
5
6 Scheme 13. Alternate Route to Chloride 7.21P
7
8
9 H2NOC Aq. NH2OH HOHN CONH2
10
11 i-PrOH
CO2i-Pr
12 7.24 59% 7.25 CO2i-Pr
13
14
15 CHO
16 CHO N OH N
17 Aq. Na2CO3 O O
18 CO2i-Pr
19 i-PrOH, 41 oC N CH3COCl, i-PrOH
20 62% O 7.26 88%
21
22
23 N OH
POCl3 N
24 O
CO2i-Pr tol, DMF N
25 N
26 70 oC Cl N
O NOH
27 49% CO2i-Pr
28 7.27 7.21P
29
30
31 7.2.2 Summary of Innovator Routes to Favipiravir
32
33
The initial route from Toyama was intended solely as a way of generating small quantities of
34
35
36 material for initial testing and was not appropriate for scaling.
37
38
39 The second generation route, with contributions from both Toyama and Nippon Soda, provides a
40
41 scalable 6-step route to favipiravir in 33% yield. This includes the 3-step process from Nippon
42
43 Soda from the commercially available diamide 7.13 to 3,6-dichloropyrazinecarbonitrile (7.9)
44
45
46 (Scheme 11),84 followed by the improved Toyama route (sequence 7.9, 7.10, 7.12, favipiravir,
47
48 Scheme 10) that integrates purification of the penultimate intermediate 7.12 as a
49
50 dicyclohexylamine salt.83
51
52
53 The overall yield for the third generation route is 9% (nine steps, Scheme 12) or 10% (Scheme
54
55
56
12, 13). The length of the synthesis and lower yield makes this route less attractive than the
57
58 58
59
1
2
3 second generation route. In addition, the intermediates up to and including 7.19 are reported as
4
5
6 liquids that are isolated by concentrating to oils such that purification can only be carried out by
7
8 vacuum distillation. This route, however, has one important safety advantage. As noted in the
9
10 patent specification, 3,6-difluoro-2-pyrazinecarbonitrile (7.10), a key late stage intermediate in
11
12
13
the second generation route, has high skin irritancy and is volatile, thus requiring special
14
15 equipment for handling. On the other hand, compounds 7.20, 7.21, and 7.22 are solids with low
16
17 volatility and no skin irritancy that require no special handling.
18
19
20 7.2.3 Alternate Routes to Favipiravir from Academic Labs and Generic Companies
21
22
Several alternate routes to favipiravir have been disclosed either in journal publications or the
23
24
25 patent literature. The following routes are presented in chronological order. Routes which are
26
27 only minor variations on previously disclosed routes are not included here.
28
29
30 A short route to favipiravir was disclosed in 2012 by Zheng and coworkers at Shandong Qidu
31
32 Pharmaceutical Co. (Scheme 14).86 Nitro compound 7.8 was prepared via nitration of 7.7 as
33
34
35 outlined in Scheme 10 (75% yield). Reduction of the nitro group was carried out by
36
37 hydrogenation with Pd/C in HOAc to afford amine 7.28 in 65% yield after crystallization from
38
39 EtOAc. Amine 7.28 was dissolved in 70% pyridinium hydrofluoride and treated with sodium
40
41
42
nitrite at -20 oC. After aqueous workup favipiravir was isolated in 70% yield with no
43
44 purification. No details were provided on product purity. The three step route from 7.7 provides
45
46 favipiravir in 34% yield.86
47
48
49 Scheme 14. Alternate Route to Favipiravir from 3-Hydroxy-6-nitropyrazinecarboxamide
50
51
52
53
54
55
56
57
58 59
59
1
2
3 O 2N N CONH2 H 2N N CONH2
H2, 5% Pd/C
4
5 HOAc, 10 oC
6 N OH N OH
65%
7 7.8 7.28
8
9 NaNO2 F N CONH2
10 70% pyr-HF
11
12 -20 oC N OH
13 70%
favipiravir
14
15
16 A route published by Zhang and coworkers at Shandong Qidu Pharmaceutical Co. in 2013
17
18
19 (Scheme 15)87 intersects with intermediate 7.14 of the Nippon Soda synthesis (Scheme 11). The
20
21 synthesis starts with 3-amino-2-pyrazinecarboxylic acid (7.29). After ester formation,
22
23 bromination with NBS provided 7.31. Diazotization and hydrolysis followed by amide formation
24
25
afforded 7.14. The route appears scalable but longer than the two step synthesis of Nippon
26
27
28 Soda.84 Further optimization of this route was reported by Liu and coworkers, with the most
29
30 important contribution perhaps being the development of a one-pot process for the final three
31
32 steps of the process, thus avoiding isolating of volatile 3,6-difluoro-2-pyrazinecarbonitrile (7.10)
33
34
35 that has high skin irritancy (Scheme 16).88 According to the experimental provided on a 400 mg
36
37 scale, 3,6-dichloro-2-pyrazinecarboxamide (7.9) was treated with KF (6 equiv) and n-Bu4NBr
38
39 (0.4 equiv) in DMSO at 50 oC for 3 h to generate 7.10. Then potassium carbonate and aqueous
40
41
42 30% hydrogen peroxide were added at 0 oC, then stirred at 25 oC for 1.5 h to form amide 7.11.
43
44 Water and sodium bicarbonate were then added and the mixture warmed to 50 oC for 8 h to
45
46 generate favipiravir. After aqueous workup, favipiravir was isolated in 60% yield after
47
48
crystallization from EtOH.88
49
50
51
52
53
54
Scheme 15. Alternate Preparation of 6-Bromo-2-hydroxy-pyrazinecarboxamide (7.14)
55
56
57
58 60
59
1
2
3 N CO2H
H2SO4 N CO2Me
4 NBS
5 MeOH CH3CN
6 N NH2 N NH2
7 7.29 7.30
8
9 Br N CO2Me NaNO2 Br N CO2Me
10
11 H2SO4
N OH
N NH2
12
13 7.31 7.32
14 Br N CONH2
15
16 Aq. NH3
N OH
17 7.14
18
19
20
21
Scheme 16. One-Pot Process for Conversion of 7.9 to Favipiravir
22
23
Cl N CN F N CN
24 KF, n-Bu4NBr K2CO3, H2O2
25
26 N Cl DMSO N F DMSO
27 7.9 50 oC 7.10 25 oC
28
29
30 F N CONH2 F N CONH2
NaHCO3
31
32 N F H2O, 50 oC N OH
33
7.11 60% (3 steps) favipiravir
34
35
36
37 Li at Zhengzhou University disclosed a two-step process for conversion of 2-pyrazinecarbonitrile
38
39 (7.33) to 3,6-chloro-pyrazinecarbonitrile (7.9) (Scheme 17).89 In the first step, both pyrazine
40
41
42
nitrogens were oxidized using aqueous hydrogen peroxide in HOAc at reflux to generate the bis-
43
44 N-oxide 7.34, which was isolated in 57% yield after crystallization from MeOH. While no
45
46 mention of safety issues were noted, compound 7.34 appears to be a highly energetic compound
47
48
that could be shock sensitive. This was followed with chlorination using POCl3 and Et3N at
49
50
51 reflux to afford 7.9 in 45% yield after isolation by silica chromatography. The final three steps
52
53 were also telescoped into a one-pot process using conditions similar to those in Scheme 14, with
54
55 a yield of 65% over the final three steps after crystallization of favipiravir from EtOH.89
56
57
58 61
59
1
2
3 Scheme 17. Two-step Route to 7.9 from 2-Pyrazinecarbonitrile
4
5
6 O
7
N CN 30% H2O2 N CONH2
8 HOAc
9
reflux
10 N N
57%
11
7.33 O
12
13 7.34
14
15 POCl3 Cl N CN
16 Et3N
17 reflux
18 N Cl
45%
19 7.9
20
21
22 An alternate four step synthesis of 7.9 from 2-aminopyrazine (7.35) in 48% yield was reported
23
24 by Xie and coworkers (Scheme 18).90 2-Aminopyrazine (7.35) was chlorinated in MeCN with N-
25
26
27
chloro-N-methoxy-4-toluenesulfonamide (TSA) in 80% yield followed by bromination with
28
29 NBS in dichloromethane to afford 7.37. Installation of the nitrile group was accomplished with
30
31 Pd-catalyzed cyanation with NaCN mediated by CuI to furnish 7.38 in 85% yield. Conversion of
32
33
the amine to chloride was carried out with t-butyl nitrite and titanium tetrachloride in
34
35
36 dichloromethane to provide 7.9 in 81% yield, and 48% yield overall for the four steps.
37
38
39 Scheme 18. Synthesis of 7.9 from 2-Aminopyrazine
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 62
59
1
2
3 NBS
4 N TSA Cl N CH2Cl2
5
6 MeCN 87%
N NH2 80% N NH2
7
7.35 7.36
8
9
10 NaCN, CuI
11
Cl N Br Pd(PPh3)4 Cl N CN
12 DMF
13 N NH2 85% N NH2
14 7.37 7.38
15
16
17 Cl N CN
t-butyl nitrite
18
19 CH2Cl2,TiCl4 N Cl
20 7.9
21 81%
22
23
24 7.2.4 Summary of Alternate Routes to Favipiravir
25
26 Several alternate routes to favipiravir have been disclosed starting from a variety of starting
27
28 materials, including 3-hydroxy-2-pyridinecarboxamide,86 3-amino-2-pyridinecarboxic acid,87,88
29
30
31 2-pyrazinecarbonitrile,89 and 2-aminopyrazine.90 Most of the routes are undeveloped, requiring
32
33 isolation by silica chromatography, none has been described on more than a few grams, and none
34
35 provides any insight on product purity and the impurity profile. Nonetheless, two of the most
36
37
38
notable advances in this collection of routes includes, (1) the three step synthesis of favipiravir
39
40 from 3-hydroxy-2-pyridinecarboxamide (7.7)(Schemes10, 14),76 a starting material that can be
41
42 synthesized in one step from 2-aminomalonic acid diamide (7.13); and the one-pot conversion of
43
44
3,6-dichloro-2-pyrazinecarbonitrile (7.9) to favipiravir that avoids isolation of the skin irritant
45
46
47 7.10.88,89
48
49
50 7.3 Final Forms and Formulation of Favipiravir
51
52
53 Two crystalline forms of neutral favipiravir are known, designated as Form A and Form B. Form
54
55 A is thermodynamically stable form and the form generated by the manufacturing process.91 A
56
57
58 63
59
1
2
3 crystal form of neutral favipiravir, crystallized from EtOH or 2-PrOH, was disclosed by Weihai
4
5
6 Guanbiao Information Technology Co.92 It is not known if this is the same crystal form that is
7
8 generated in the Fujifilm Toyama manufacturing process.
9
10
11 Toyama Chemical Co. has disclosed three crystal forms of the sodium salt of favipiravir, one
12
13 anhydrate and two hydrates.93 They have also disclosed a crystalline meglumine salt.94 Both salts
14
15
16
appear to have high water solubility, but the meglumine salt had better solubility properties when
17
18 formulated as a lyophilized cake.94
19
20
21 Favipiravir is currently available as a tablet containing 200 mg of the active ingredient. The
22
23 approved favipiravir dose for influenza in Japan is 1600 mg twice daily on day one followed by
24
25 600 mg twice daily for 4 days. The dose for Ebola virus infection is 6000 mg on day one and
26
27
28
2400 mg/day on days 2–9.91
29
30
31 7.4 Favipiravir – Outlook for Supply
32
33
34
Fujifilm Toyama announced on March 31, 2020, that it has increased production of Avigan
35
36 (favipiravir) at its domestic sites and with its international partners in order to supply the
37
38 Japanese government and other countries. Favipiravir is included in the national strategic
39
40 stockpile of medicines of Japan, with up to two million doses available.95 On April 15, 2020,
41
42
43 Fujifilm stated that it is freeing up capacity at its Wako Pure Chemical facility in Japan and is
44
45 exploring partnerships with external companies.96 By July, 2020, the company expects to be
46
47 able to manufacture 100,000 treatments per month, increasing to 300,000 per month by
48
49
50 September, 2020.96
51
52
53
The company noted that it is sourcing the starting material, diethyl malonate, from chemical
54
55 producer Denka who will re-open a facility that was shuttered in 2017 due to lack of demand.97
56
57
58 64
59
1
2
3 We note that diethyl malonate could be used to generate the 3-carbon starting material in Scheme
4
5
6 11 and indicates the commercial route does not start with a pyrazine derivative. On April 27,
7
8 2020, Fujifilm announced that it has teamed up with two Japanese companies. Ube Industries
9
10 will manufacture intermediates while Kaneka will supply the drug substance with the goal to
11
12
13
have initial supplies available by July 2020.98
14
15
16
In 2016 Fujifilm licensed the active ingredient of favipiravir to the Chinese company Hisun
17
18 Pharmaceutical that allowed them to develop, manufacture, and market the drug in China. In
19
20 February 2020, Hisun received approval to manufacture favipiravir in China.99
21
22
23 Lasa SuperGenerics announced on March 20, 2020, that it will work with the Institute of
24
25 Chemical Technology in India to develop favipiravir with the goal to begin manufacturing of the
26
27
28 drug within a few months.100
29
30
31 On April 30, 2020, Glenmark Pharmaceuticals announced it had completed development of the
32
33 API and formulation processes and had received approval from the Indian government to initiate
34
35 clinical trials of favipiravir in COVID-19 patients in India.101
36
37
38
39 8. Pirfenidone
40
41
42 Pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone, 4.13), marketed by Roche under the tradename
43
44 Esbriet, is an immunosuppressant approved in many regions for the treatment of idiopathic
45
46 pulmonary fibrosis.102
47
48
49
51
52
53
54
In vitro evidence shows that pirfenidone inhibits collagen synthesis, down-regulates profibrotic
55
56 cytokines and decreases fibroblast proliferation103-105 and this has translated well into animal
57
58 65
59
1
2
3 models of fibrosis. 106 Pirfenidone has also been shown to reduce the production of tumor
4
5
6 necrosis factor alpha (TNF-) and IL-1 in both cells and isolated human peripheral blood
7
8 mononuclear cells (PBMCs) which are consistent with both the antifibrotic and anti-
9
10
inflammatory activities seen in fibrosis animal models.107, 108
11
12
13 The absolute bioavailability of pirfenidone has not been determined in humans. Pirfenidone is
14
15
16 primarily metabolized in the liver by CYP1A2 to yield 5-carboxypirfenidone, its inactive
17
18 metabolite.109 The mean terminal half-life is approximately 3 hours in healthy volunteers and the
19
20 drug is excreted predominantly as 5-carboxypirfenidone in the urine (up to 80% of the dose).
21
22
23 Pirfenidone increases hepatic enzyme levels including aspartate and alanine transaminases and
24
25 gamma-glutamyl transpeptidase and is contraindicated in patients with severe hepatic
26
27 impairment.109
28
29
30 The FDA recently granted breakthrough therapy designation to Esbriet for unclassifiable
31
32
interstitial lung disease (uILD). The Phase II trial was shown to slow disease progression and
33
34
35 supported it efficacy on a number of lung function parameters including forced vital capacity
36
37 (FVC) in patients with uILD.110 Based on the encouraging results seen in the treatment of both
38
39 IPD and uILD, pirfenidone is being repurposed to treat patients with severe COVID-19 infection
40
41
42 (NCT04282902).111 This multi-center, randomized, placebo-controlled Ph III clinical study will
43
44 first assess the efficacy and safety of pirfenidone in 147 hospitalized adults patients diagnosed
45
46 with COVID-19 and will be compared to standard-of-care. Primary outcome measures include 1)
47
48
49
absolute changes in baseline lesion area, finger pulse oxygen, and blood gas from baseline at 4
50
51 weeks of chest CT images, 2) total score of King’s Interstitial Lung Disease Short Questionnaire
52
53 (K-BILD) and week 4 absolute change from baseline. Secondary measures include time to death
54
55
within 4 weeks due to respiratory causes and time to disease progression or death within 4 weeks
56
57
58 66
59
1
2
3 in addition to multiple other pulmonary fibrosis survival measures. Pirfenidone will be
4
5
6 administered orally 3 times a days, 2 tablets each time, for a period of 4 weeks. The estimated
7
8 primary completion date is April 30, 2020 and thus far the study data has not been reported yet.
9
10
11
12
13 8.2 Synthetic Routes to Pirfenidone
14
15 Pirfenidone is synthesized in two steps from 2-amino-5-methylpyridine (Scheme 19). Most of
16
17 the focus in the patent literature has been on optimization of the cross-coupling step to improve
18
19
20
yield and impurity profile.
21
22
23
Scheme 19. Route to Pirfenidone
24
25
N NH2 N OH
26 NaNO2
27
28 H 3C H2O, H2SO4 H C
3
29 8.1 61% 8.2
30
31
32 Ph
33 N O
PhX
34
35 Cu(I), 
36 H 3C
37
pirfenidone, 4.13
38
39
40
41 The route starts with readily available 2-amino-5-methylpyridine (8.1), which is diazotized with
42
43 NaNO2 in aqueous sulfuric acid to generate 2-hydroxy-5-methylpyridine (8.2). The published
44
45 procedures are based on the initial work in 1949 of Adams and Schrecker for the synthesis of 2-
46
47
48
hydroxy-6-methylpyridine.112 The primary issue with the synthesis is isolating the water-soluble
49
50 product from the aqueous reaction mixture. In the Adams procedure, the isolation consists of
51
52 neutralizing the reaction mixture, concentrating to dryness, grinding the resulting solids, and then
53
54
extensively extracting with boiling benzene. An improved isolation process was developed by a
55
56
57
58 67
59
1
2
3 team at Smith Kline French by neutralization of the reaction mixture to pH 6.5-7.0 and extraction
4
5
6 into EtOAc at 60 oC followed by crystallization from EtOAc.113 This procedure was applied to
7
8 the preparation of 2-hydroxy-5-methylpyridine (8.2) which was isolated in 61% yield.114
9
10
11 Conversion of 2-hydroxy-5-methylpyridine (8.2) to 5-methyl-1-phenyl-2-(1H)pyridone
12
13 (pirfenidone) was disclosed in 1974 by Affiliated Medical Research Inc.115 The process involved
14
15
16
reacting 8.2 with potassium carbonate and a catalytic amount of zinc precipitated copper powder
17
18 in refluxing iodobenzene (188 oC) for 18 h. At the end of the reaction benzene was added, the
19
20 mixture decolorized with charcoal, and then filtered and concentrated to an oil. The oil was
21
22
titurated in petroleum ether to afford pirfenidone in 85% yield.115
23
24
25 In the patent application from the innovator company, Intermune, pirfenidone is prepared from
26
27
28 8.2, bromobenzene (1.8 equiv), potassium carbonate (1.2 equiv), and cuprous oxide (5 mol %) in
29
30 DMF (2 vol) at 125 oC for 18-20 h. After aqueous workup pirfenidone was isolated in 85% yield
31
32 after crystallization from toluene/heptane. The material was further purified by recrystallization
33
34
35 from water (pH 11) to afford pirfenidone with the dimeric impurities less than 0.03% and all
36
37 other impurities less than 0.05%. The inventors note that the starting bromobenzene should be
38
39 highly pure and contain very low levels of dibromo impurities since these form dimers which are
40
41
42
difficult to remove. 116
43
44
Many other patent applications have been filed the conversion of 8.2 to pirfenidone that include
45
46
47 different conditions, stoichiometries and other copper catalysts. Of note, Procos has developed a
48
49 process using chlorobenzene as solvent at 175 oC, sodium carbonate, CuI, and the ligand N,N’-
50
51 dimethylethylene-1,2-diamine. The product is isolated by crystallization from EtOAc/hexane
52
53
54 followed by recrystallization from water with an overall yield of 72-84%.117
55
56
57
58 68
59
1
2
3
4
8.3 Pirfenidone Final Form and Formulation
5
6
7 Pirfenidone exists as a single crystalline form designated as Form A.102 The drug substance is
8
9
10
milled to less than 150 microns.116
11
12
The drug product is formulated as an immediate release hard capsule containing 266.7 mg of
13
14
15 pirfenidone drug substance. The recommended daily dose is three capsules three times daily with
16
17 food for a total of 2400 mg/day.102 An 801 mg tablet is also available.118
18
19
20
21 8.4 Outlook for Supply – Pirfenidone
22
23
24 Pirfenidone has orphan drug status in the US and EU suggesting that large supplies may not be
25
26
27 available. Roche has not provided an update on the supply situation for expanded us for
28
29 COVID- 19 treatment. The Indian manufacturer Cipla supplies generic pirfenidone but has also
30
31 not provided any update on supply through May 14, 2020.
32
33
34
35 9. Baricitinib
36
37
38 Baricitinib phosphate (sold under the tradename Olumiant by Lilly) was approved in the EU in
39
40 Feb 2017 and in the U.S. in May 2018 for the treatment of moderate-to-severe rheumatoid
41
42 arthritis in adult patients whose disease is not well controlled by other tumor necrosis factor
43
44
45
(TNF) antagonists.119
46
47
49
50
51
52 Baricitinib is a selective and reversible inhibitor of Janus Kinase 1 (JAK1) and 2 (JAK2). JAKs
53
54 belong to the protein tyrosine kinase family and play important roles in the proinflammatory
55
56
57
58 69
59
1
2
3 signaling pathways that are frequently over-activated in autoimmune disorders such as RA.120
4
5
6 Upon binding of extracellular cytokines and growth factors, JAKs are phosphorylated and
7
8 activate signal transducers and activators of transcription (STATs). Via these signaling cascades,
9
10 inflammatory cytokine and chemokine transcription is induced to form inflammatory mediators
11
12
13
including IL-2, IL-6, IL-12, IL-15, IL-23. The majority of viruses enter cells through receptor-
14
15 mediated endocytosis. The receptor that SARS-CoV-2 uses to infect lung cells is ACE2, a cell
16
17 surface protein on multiple cells including lung AT2 alveolar cells. AT2 cells are particularly
18
19
prone to viral infection.121 One of the regulators of endocytosis is the AP-2 associated protein
20
21
22 kinase 1 (AAK1). Inhibiting AAK1 might interrupt the passage of the SAR-CoV-2 virus into
23
24 cells and also the intra-cellular assembly of virus particles.122 In addition to Baricitinib’s potent
25
26 JAK1 and JAK2 activity, it also potently inhibits AAK1 and also binds cyclin G-associated
27
28
29 kinase, another regulator of endocytosis.123
30
31
32
33 It is postulated that the therapeutic dose of 2 or 4 mg daily is sufficient to inhibit AAK1, and
34
35
36
could be trialed in patients to treat COVID-19. The JAK1/2 activity may also inhibit the cytokine
37
38 release associated with SARS-CoV-2 virus and dampen the cytokine storm that frequently
39
40 manifests during Acute Respiratory Distress Syndrome (ARDS).124
41
42
43
44
45 In humans, Baricitinib is rapidly absorbed with an oral bioavailability of 79%. It takes
46
47 approximately 1 hr to reach peak plasma concentration. Baricitinib is metabolized by Cyp3A4
48
49 but <10% of the total dose is prone to this biotransformation. Baricitinib is excreted primarily as
50
51
52 unchanged active drug in the urine (69%) and feces (15%) with only minor oxidative metabolites
53
54 identified. The mean half life in patients with RA is 12.5 h.125
55
56
57
58 70
59
1
2
3
4
5
6 On May 8, 2020, the US National Institute of Allergy and Infectious Diseases announced it is
7
8 initiating a clinical trial to study baricitinib as a treatment for COVID-19 in combination with
9
10 remdesivir.126 The study is expected to enroll about 1000 hospitalized patients and will compare
11
12
13
remdesivir alone versus remdesivir plus baricitinib ( 4 mg oral daily).126
14
15
16 The majority of clinical trials using Baricitinib to date have been short duration (2 week), open
17
18 label trials to treat patients with mild to moderate COVID-19 infection (Table 7).127
19
20
21 Furthermore, as baricitinib is primarily eliminated by renal excretion, clinicians are also
22
23 combining with anti-virals such as lopinavir/ritonavir (NCT04320277).
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 71
59
1
2
3 Table 7. Clinical trials underway with Baricitinib127
4
5
6
10 Dates
11 NCT04340232 Safety and Efficacy of Baricitinib for 80 Apr2020 – 2mg oral dose of Baricitinib Placebo
12 COVID-19 Aug2020
13 NCT04362943 Clinical-epidemiological 576 20Apr2020- Arm 1: Baricitnib None
14 Characterization of COVID-19 Jul2020 Arm 2: Anakinra
15 Disease in Hospitalized Older Adults
16 NCT04346147 Prospective, Phase II, Randomized, 165 13Apr2020 – Arm 1: HCQ 200 mg BID oral Standard of
17 Open-label, Parallel Group Study to Sep2020 Arm 2: Lopinavir/Ritonavir 200/50 mg BID oral care
18 Evaluate the Efficacy of Arm 3: Imatinib 400 mg QD oral
19 Hydroxychloroquine Together With Arm 4: Baricitinib 4 mg QD oral
20 Baricitinib, Imatinib or Early
21 Lopinavir / Ritonavir in Patients
With SARS Cov2 Pneumonia
22
NCT04321993 Treatment of Moderate to Severe 1000 17Apr2020 – Arm 1: Lopinavir/ritonavir 200/50 mg tablets BID for Standard of
23
Coronavirus Disease (COVID-19) in Feb2021 10 days care
24
Hospitalized Patients Arm 2: HCQ 200 mg BID for 10 days
25
Arm 3: 2 mg PO daily for 10 days
26
NCT04320277 Baricitinib Combined With Antiviral 200 16May2020 – Baricitinib 4 mg/day/orally and lopinavir/ritonavir Standard of
27 Therapy in Symptomatic Patients Jun2020 tablets 200/50 mg BID care
28 Infected by COVID-19: an Open-
29 label, Pilot Study
30 NCT04345289 Efficacy and Safety of Novel 1500 20Apr2020 – Arm 1: Convalescent anti-SARS-CoV-2 plasma, IV Placebo
31 Treatment Options for Adults With Jun2021 infusion (2 x 300 mL)
32 COVID-19 Pneumonia. A Double- Arm 2: Sarilumab 200 mg s.c. injection
33 blinded, Randomized, Multi-stage, Arm 3: Baricitinib 4mg/day for 7 days
34 6-armed Placebo-controlled Trial in Arm 4: HCQ 600 mg/day for 7 days
35 the Framework of an Adaptive Trial
36 Platform
37
38
39
40
41
42
43 72
44
46
47
1
2
3
4
9.2 Synthetic Routes to Baricitinib
5
6
7 Two main routes to baricitinib have been disclosed. The original route from Incyte involves
8
9
10
coupling the lower and central fragments (disconnection B, Figure 6), followed by appending the
11
12 upper portion (disconnection A).128 Lilly has disclosed an alternate disconnection, with initial
13
14 coupling at point A followed by point B.129,130
15
16
17 Et
18
19 O S O
20 N
21 A
22 NC
23 N N
24
25 B
26 N
27
28 N N
H
29
30
31 Figure 6. Retrosynthetic Disconnections of Baricitinib
32
33
34
35
36 Both routes require synthesis of fragment 9.1. Several routes to 9.1 have been disclosed; three of
37
38 these are discussed below. The original Incyte route is outlined in Scheme 20.128
39
40
41 Scheme 20. Incyte Route to Sulfonamide 9.1
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 73
59
1
2
3 Ph Ph
4
5 HCl
O Ph2CHNH2 N Boc2O
6 Cl
7 MeOH H2, Pd/C
8 9.2 43% OH 91%
9
9.3
10
11 Boc Boc
12 N TEMPO N (EtO)2P(O)CH2CN
13
14 NaOCl KO-t-Bu, THF
15 OH KBr O 61%
16 9.4 97% 9.5
17
18
Et
19
20 Boc 3M Aq. HCl H O S O
21 N MeCN, rt N EtSO2Cl N
22
23 i-Pr2NEt
24 91%
25
CN CN CN
9.6 9.7 9.1
26
27
28
29 In the first step, described on a 15 mole scale, epichlorohydrin (9.2) was reacted with
30
31 benzhydrylamine in MeOH for three days at room temperature and three days at reflux, to afford
32
33 63, which was crystallized as its HCl salt in 43 % yield.131 After salt break, 9.3 was subjected to
34
35
36 hydrogenation using Pd/C in THF in the presence of Boc2O to afford Boc-azetidinol 9.4 in 91%
37
38 yield after purification by silica chromatography. Oxidation of 9.4 was carried out with sodium
39
40 hypochlorite, aqueous sodium bicarbonate, and catalytic amounts of KBr and TEMPO. After
41
42
43
workup, the product layer was concentrated to dryness to provide crude ketone 9.5 in 97% yield.
44
45 For the Horner-Emmons reaction, the anion of diethyl cyanomethylphosphonate was generated
46
47 with KOt-Bu in THF for 3 h at -10 oC, then ketone 9.5 was added and the reaction stirred
48
49
overnight at room temperature. After aqueous workup, 9.6 was isolated in 61% yield after
50
51
52 purification by silica chromatography. A solution of 9.6 in MeCN was treated with 3 N aqueous
53
54 HCl for 18 h at room temperature and then concentrated to dryness. The resulting crude 9.7 was
55
56
57
58 74
59
1
2
3 suspended in MeCN, then treated with diisopropylethylamine and ethanesulfonyl chloride at
4
5
6 room temperature overnight. After aqueous workup, sulfonamide 9.1 was isolated after silica
7
8 chromatography. Two reactions were described on a one kg scale for the final two steps under
9
10 similar reaction conditions with yields of 59% and 91%.
11
12
13 The Lilly approach to 9.1 is three steps from with unprotected azetidin-3-ol (9.8) and requires no
14
15
16
protecting groups (Scheme 21).129, 132
17
18
19
Scheme 21. Lilly Route to Sulfonamide 9.1
20
21
Et 6% O2 Et
22 EtSO2Cl
H O S O in N2 O S O
23
24 N THF/H2O N TEMPO N
25 K3PO4 NaNO2
26
OH NaOH OH CH3CN O
27
28 9.8 95% 9.9 87% 9.10
29
30 Et
31
O S O
32
(EtO)2P(O)CH2CN N
33
34
i-Pr2NEt, 2-PrOH
35
36 83%
CN
37 9.1
38
39
40 In the first step, 9.8 was reacted with EtSO2Cl in aqueous THF to generate sulfonamide 9.9,
41
42
43 which is most efficiently isolated by continuous countercurrent extraction. In the first extraction
44
45 using water/toluene the product of reaction at both the amine and alcohol is extracted into the
46
47 toluene water leaving product 9.9 in the water layer. This is then extracted with EtOAc to bring
48
49
50 the product into the organic layer with a 95% yield for the step. Oxidation to the ketone is
51
52 described both in batch and flow mode. Batch processing using 6% oxygen in nitrogen, a
53
54 concentration of oxygen that will not support combustion, with sodium nitrite, HOAc and
55
56
57
58 75
59
1
2
3 TEMPO in acetonitrile, was carried out at 500 psi with the headspace being refreshed every
4
5
6 minute over 17 hours. For the reaction in flow, the reactants and reagents were supplied in four
7
8 feeds: (1) TEMPO in CH3CN, (2) NaNO2 in water, (3) alcohol 9.9 in 1:6 HOAc:CH3CN, and (4)
9
10 6% O2 in N2. The back pressure was set at 500 psi and residence time was 12 h, resulting in an
11
12
13
assay yield of 98%. At the end of the reaction in either batch or flow, a batchwise aqueous
14
15 workup was followed by solvent switch into 2-PrOH afforded ketone 9.10 with a step yield of
16
17 87-94%. The advantages of the flow route include (1) no need for a specialized large scale vessel
18
19
rated to >500 psi, (2) no need for continuous replacement of oxygen in the head space, and (3)
20
21
22 improved safety considering the smaller footprint for the high pressure reaction. Several other
23
24 oxidation procedures were also described. The 2-PrOH solution of 9.10 was combined with
25
26 diethyl cyanomethylphosphonate and diisopropylethylamine. Product 9.1 crystallized as the
27
28
29 reaction proceeded and was isolated in 83% yield after addition of heptane to increase
30
31 crystallization recovery.
32
33
34 A third route to 9.1 was disclosed by Hangzhou Cheminspire Technology Co. (Scheme 22).133
35
36 The HCl salt of 3-azetidinone (9.11) was reacted with ethanesulfonyl chloride mediated by
37
38
39
diisopropylethylamine in dichloromethane to afford sulfonamide 9.10 in 87% yield after silica
40
41 chromatography. Reaction of 9.10 with cyanoacetic acid in refluxing toluene mediated by
42
43 piperidine afforded vinyl nitrile 9.1 in 81% yield after isolation by silica chromatography. The
44
45
use of cyanoacetic acid instead of the high molecular weight phosphonate required for the
46
47
48 Horner-Emmons reaction (Schemes 20 and 21) reduces waste and product mass intensity for this
49
50 step.
51
52
53 Scheme 22. Hangzhou Route to Fragment 9.1
54
55
56
57
58 76
59
1
2
3 Et
Et
4
O S O
5 H EtSO2Cl O S O NC CO2H
6 HCl N N N
i-Pr2NEt piperidine
7
8 CH2Cl2 toluene, 
9 O 87% O 81%
CN
10 9.11 9.10 9.1
11
12
13 In the Incyte route, conversion of 9.1 to baricitinib was completed as shown in Scheme 23, with
14
15
16 completion of the “B” connection first followed by the “A” connection (Figure 3).128 First, 6-
17
18 chloro-7-deazapurine (9.12) was protected with SEM-Cl using NaH as base in N,N-
19
20 dimethylacetamide to furnish 9.13 in 89% yield after silica chromatography. Cross-coupling
21
22
23
with pyrazole boronate ester 9.14 was accomplished in with Pd catalysis in 1-BuOH/water at 90
24
oC to provide crude 9.15, which was deprotected with aqueous HCl in THF. The resulting
25
26
27 product 9.16 was crystallized from acetonitrile in 82% yield for the two steps. Michael addition
28
29
of imidazole 9.16 to vinyl nitrile 9.1was conducted in acetonitrile mediated by DBU. Addition
30
31
32 adduct 9.17 was isolated by addition of water to the reaction mixture and crystallization in 97%
33
34 yield. Deprotection of the SEM group was carried out in two steps. First, the silyl group was
35
36 removed using lithium tetrafluoroborate in aqueous acetonitrile at 80 oC overnight followed by
37
38
39 cooling to 10 oC, addition of ammonium hydroxide, then reaction overnight at room temperature
40
41 to remove the hydroxymethyl group. Isolation consisted of adding the reaction mixture to water
42
43 to crystallize baricitinib free base crude followed by purification via silica chromatography in
44
45
46
81% yield. The crystalline phosphate salt was generated in 90% yield by treatment with
47
48 phosphoric acid in acetonitrile/EtOH. The 6-step sequence to the phosphate salt with an overall
49
50 yield of 52% was described on the multi-kilogram to multi-hundred gram scale but required two
51
52
chromatographic purifications.
53
54
55 Scheme 23. Incyte’s Final Steps to Baricitinib
56
57
58 77
59
1
2
3 N
4 Cl N O
5 Cl B
Cl
6 O EtO O
SiMe3 9.14
7 N N
8 NaH, 0-5 oC N Pd(PPh3)4
N N N
9 CH3CONMe2 K2CO3, H2O
H 9.12 SEM 9.13
10 89%
11 1-BuOH, 
12
13
14 H NC O
15 EtO N N N S Et
N N
16 O
Aq. HCl 9.1
17
18 THF
N N MeCN, DBU, rt
19 82%
20 N N N N 97%
21 SEM SEM 9.16
9.15
22
23
24 Et Et
25 O S O 1) LiBF4 O S O
26 MeCN, H2O
N N
27 2) Aq, NH4OH H3PO4
28 NC 81% NC
N N N N
29
30 3) H3PO4
31 MeCN, EtOH
32 N 90% N
33 N N
N N
34
SEM H
35 9.17 baricitinib
36 phosphate
37
38
39
40
41
42 The alternate assembly of the building blocks via connection “A” followed by connection “B”
43
44
45 was disclosed by three independent groups in 2016 (Scheme 24). The conditions for nucleophilic
46
47 addition of pyrazine 9.18 to vinyl nitrile 9.1 are compiled in Table 8, all with comparable yields
48
49 of 83-86%, although purification by crystallization was only reported by the Lilly team.129 The
50
51
52
Lilly129 and Southeast University135 groups used DBU as base while the Sun group134 used
53
54 potassium carbonate in aqueous dioxane. For the palladium catalyzed cross coupling of boronate
55
56
57
58 78
59
1
2
3 ester 9.19 with 9.12, the reported yields ranged from 71% to 99% although again the Lilly group
4
5
6 was the only one to isolate and purify by crystallization (Table 9).129
7
8
9 Scheme 24. Alternate Final Steps to Baricitinib
10
11
HN N Et
12
13 O S O
14 N
15 B
Et O O NC
16
O S O N N
17
18 N 9.18
19
B
20 O O
21 CN
22 9.1
23 9.19
24
25 Et
26 Cl O S O
27 N
28 N
29 NC
30 N N N N
31 H 9.12
32
33 Pd catalysis N
34 N N
35
H
36
baricitinib
37
38
39
40 Table 8. Conditions for Synthesis of 9.19 from 9.1 and 9.18
41
42
43 Institution/Company (ref) Conditions Scale Yield
44 Lilly (129) DMF, DBU, rt, 22 h. Concentrate to 15 g 83%
45
46
dryness then crystallize from 2-PrOH
47
48
Sun (134) Dioxane/H2O, K2CO3, rt, 18 h. After 2g 86%
49
50 aqueous workup, crude 78 isolated after
51 concentration to dryness
52
53
54 Southeast University, MeCN, DBU, 60 oC, 4 h. Concentrate to 156 mg 84%
55
56 Nanjing, Chinaa (135) dryness, then silica chromatography
57
58 79
59
1
2
3
4
aInstitution of senior author
5
6
7
8
9
10
Table 9. Conditions for Conversion of 9.19 to Baricitinib
11
12
13 Institution/Company Conditions Scale Yield
14 (ref)
15
16
Lilly (129) THF, H2O, K3PO4, Dichloro[1,1'- 5g 71%
17 bis(dicyclohexylphosphino)ferrocene]palladium
18 (II), 90 oC, 19 h. Crystallization from 1-BuOH
19
20
21 Sun (134) Dioxane/H2O, K2CO3, Pd(PPh3)4, 80-85 oC, 5 h, 2g 99%
22
23 rt, 18 h. Workup and then concentrate to dryness
24 Southeast University, t-BuOH, H2O, toluene, CsF, Pd(PPh3)4, reflux, 48 870 84%
25
26 Nanjing, Chinaa (135) h, purification by silica chromatography mg
27 aInstitution of senior author
28
29
30
31
32
33 9.3 Potential Manufacturing Route to Baricitinib
34
35
36
37 The five step route outlined in Schemes 21 and 24 is a short and efficient route to baricitinib, but
38
39 is apparently not the commercial manufacturing route. One of the attractive features of the five
40
41
42 step route is that it requires no protecting groups. However, according to the European Public
43
44 Assessment Report (EPAR) for Olumiant, the final step of the process involves a deprotection
45
46 reaction.136 Some considerations for selecting a longer route that includes a protecting group:
47
48
49 (1) the purity of the final step from 79 may generate related substance impurities that are difficult
50
51
to control at acceptable levels;
52
53
54
(2) Pd may be difficult to remove from the final product;
55
56
57
58 80
59
1
2
3 (3) conversion of 79 to baricitinib is a relatively complex reaction that may be difficult to
4
5
6 reliably reproduce as a final step, whereas a straightforward deprotection may be more readily
7
8 reproducible;
9
10
11 (4) Lilly may not believe they have clear freedom to operate the protection-free route; and
12
13
14 (5) given the low dose of baricitinib (2 and 4 mg per day), the cost of the API per tablet will be
15
16 very low regardless of the efficiency of the synthesis, so freedom to operate, product quality, and
17
18
19
reproducibility will outweigh cost considerations.
20
21
The likely manufacturing route is disclosed in the same set of patent applications as the
22
23
24 protection-free route, with the final steps outlined in Scheme 25.129 Compound 9.12 was Boc-
25
26 protected using Boc2O and t-BuOK in THF at 55-60 oC. To the resulting cooled solution of 9.20
27
28 was added aqueous potassium phosphate, boronate ester 9.19, and PdCl2-XantPhos (0.25 mol %)
29
30
31 in THF, and the mixture was warmed to 55-60 oC for 4 h to afford a 97% assay yield of Boc-
32
33 baricitinib 9.21.137 The resulting solution was passed through a thiol-containing silica resin
34
35 column to remove Pd, then heated under pressure (300 psi) to 140 oC to effect removal of the
36
37
38
Boc group. The THF solution was then solvent switched to 1-BuOH and crystallized to afford
39
40 baricitinib in 90% yield.
41
42
43 Scheme 25. Potential Final Steps of the Manufacturing Route for Baricitinib
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 81
59
1
2
3 Cl
4
5 N
6
7 N N
8 H 9.12
9 Boc2O
10 t-BuOK
11
Et THF Et Et
12
13 O S O Cl O S O O S O
14 N N N
15 N
16 NC NC NC
N N N N N N N N
17
18 Boc 9.20 140 oC
19 K3PO4 90%
20 B N N
O O PdCl2-XantPhos
21 THF, H2O N N
N N
22
55-60 oC Boc H
23
9.19 97% 9.21 baricitinib
24
25
26
27
28
9.4 Final Form and Formulation of Baricitinib
29
30
31 According to the Olumiant EPAR, several crystalline forms of baricitinib free base were
32
33
identified. Crystalline form I, the thermodynamically stable anhydrous form, was selected for
34
35
36 Phase 3 clinical studies and commercial development. 136
37
38
39 The finished product is an immediate release film-coated tablet in dose strengths of 2 mg and 4
40
41 mg. The bioavailability after oral administration of baricitinib from the commercial tablet was
42
43 79%. Early clinical studies were conducted with the phosphate salt. A comparative
44
45
46 bioavailability study on the 8 mg Phase 2 capsule of the phosphate salt versus the 8 mg Phase 2
47
48 tablet of the free base showed no statistical difference in AUC or Cmax in fasted volunteers.136
49
50
51
52
53
54
55
9.5 Outlook for Supply of Baricitinib
56
57
58 82
59
1
2
3 In an April 10, 2020 press release, Lilly indicated that they do not anticipate shortages of
4
5
6 baricitinib, which is widely available in countries where it is approved.138
7
8
9
10
11
12 10. Camostat
13
14
15 Camostat (4.9) was first reported in a patent by Ono Pharmaceutical Co., Ltd in 1977.139 The
16
17 mesylate salt of camostat was approved in Japan in 1985 as a tablet formulation (Foipan) for the
18
19
20 treatment of reflux esophagitis and chronic pancreatitis.140 The drug is both well-tolerated and
21
22 has a relatively low in vivo human half-life with a three times daily dose recommendation of
23
24 100-300 mg.141 Camostat mesylate has additionally been investigated for the treatment a wider
25
26
27
range of indications including oral squamous cell carcinoma142, dystrophic epidermolysis143,
28
29 exocrine pancreatic enzyme inhibition144, and chronic pancreatitis.140,145
30
31
32
33
10.1 History, Mechanism of Action, and Status of Clinical Trials
34
35
36 Camostat is an inhibitor of the enzyme transmembrane protease serine 2 (TMPRSS2). As
37
38
described earlier in the introduction, SARS-CoV-2 uses the SAR-CoV receptor ACE2 for entry
39
40
41 and the serine protease TMPRSS2 for S protein priming. TMPRSS2, cleaves the S protein,
42
43 allowing the virus to fuse with cell and start to replicate inside it.
44
45 A study in 2012 demonstrated that inhibition of TMPRSS2 by camostat partially blocked
46
47
48 infection by SARS- CoV and human coronavirus NL63 in HeLa.146 Further recent in vitro
49
50 studies have shown that camostat significantly reduces the infection of Calu-3 lung cells by
51
52 SARS-CoV-2 and inhibited entry into primary human lung cells.147 In mice, camostat dosed at
53
54
55
56
57
58 83
59
1
2
3 concentrations similar to the clinically achievable concentration in humans reduced mortality
4
5
6 following SARS-CoV infection from 100% to 30-35%.
7
8 There are only 4 clinical trials underway to explore the efficacy of Camostat in COVID-19
9
10 patients (Table 10).148 A randomized, placebo-controlled Phase IIa Danish trial is now underway
11
12
13
(NCT4321096) in 180 patients. Patients will receive 2 x 100 mg of camostat mesylate or placebo
14
15 3 times a day for 5 days, the maximum dose given to patients with pancreatitis in Japan.
16
17 Estimated primary completion date is Dec. 31, 2020 and the estimated study completion date is
18
19
May 1, 2021.148
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 84
59
1
2
3 Table 10. Clinical trials underway with Camostat148
4
5
6
10 Dates
11 NCT04321096 The Impact of Camostat Mesilate on 180 31Mar2020 – Camostat Mesylate 2x100 mg pills 3 times daily for 5 Placebo
12 COVID-19 Infection: An Dec2020 days
13 Investigator-initiated Randomized,
14 Placebo-controlled, Phase IIa Trial
15 NCT04353284 The Effect of Camostat Mesylate on 114 30Apr2020- Camostat mesylate 200mg taken 7 days. Placebo
16 COVID-19 Infection in Ambulatory Mayt2021
17 Patients: An Investigator-Initiated
18 Randomized, Placebo-Controlled,
19 Phase IIa Trial
20 NCT-4338906 Evaluation of the Efficacy and 334 1Jun2020 – Arm 1) Camostat (400 mg TID) + None
21 Safety of Camostat Mesilate + Jun2021 hydroxychloroquine (400 mg BID day 1, 200 mg BID
Hydroxychloroquine Combination D2-D7)
22
Therapy in Hospitalized Patients Arm 2) Placebo (TID) + hydroxychloroquine (400 mg
23
With Moderate COVID-19 Infection BID day1, 200 mg BID D2-D7
24
NCT04355053 An Open-Label Study to Compare 250 11Apr2020 – Arm 1) Hydroxychloroquine 400 mg BID on day 1 Placebo
25
the Efficacy, Safety, and Tolerability Oct2020 then 200 mg BID on days 2-5 + Azithromycin 500 mg
26 of Hydroxychloroquine Combined QD on day 1 and 250 mg QD on days 2-5
27 With Azithromycin Compared to Arm 2) Hydroxychloroquine 400 mg BID on day 1
28 Hydroxychloroquine Combined then 200 mg BID on days 2-5 + Camostat mesylate
29 With Camostat Mesylate and to "no 200 mg TID for 10 days
30 Treatment" in Hospitalized Patients
31 Suffering From a Mild or Moderate
32 SARS CoV 2 Virus
33
34
35
36
37
38
39
40
41
42
43 85
44
46
47
1
2
3
4
10.2 Synthesis of Camostat
5
6 The first synthesis of Camostat mesylate, originally known as FOY-305, was reported on a gram
7
8 scale in the initial 1977 disclosure by Oni Pharmaceutical Co., Ltd.139 The synthesis of Camostat
9 is relatively simple and constitutes two consecutive esterification reactions as outlined in Scheme
10
11 26. 4-Hydroxyphenylacetic acid (10.1) was alkylated with N,N-dimethyl-2-bromoacetamide in
12
13 refluxing acetonitrile to afford the O-alkylated intermediate 10.2 that precipitated in 58%
14
15
isolated yield upon removal of acetonitrile under reduced pressure followed by water addition. In
16 the subsequent esterification, the crude acyl chloride 10.3 prepared from p-guandinobenzoic acid
17
18 was used to synthesize camostat followed by a precipitation of camostat mesylate from a
19
20 methanol/diethyl ether mixture. A Chinese patent published in 2015 claims higher yields on a
21 low-gram scale by modifying the exact esterification reaction conditions147 but application to any
22
23 production scale syntheses of camostat mesylate remain unpublished.
24
25
26
27
Scheme 26. Original Synthesis of Camostat Mesylate Disclosed in 1977
28
29
30 Br CONMe2 O CONMe2
31 CO2H
32 Et3N, MeCN O
HO HO
33 reflux
34 10.1 10.2
58%
35
36 COCl O CONMe2
37 NH O
1)
38 O
H 2N N NH O
39 H
40 10.3
pyridine H 2N N
41 H camostat mesylate
42 2) MeSO3H MeSO3H
43 MeOH, Et2O
44
34%
45
46
47
48 10.2 Formulations and Salt Forms of Camostat
49
50 Camostat has been demonstrated to be readily hydrolyzed by human liver esterase and other
51
52 esterases.150 In an effort to alleviate extensive gut hydrolysis and thereby increasing the resulting
53
54
55 camostat in vivo half-life, Ono Pharmaceutical Co., Inc. patented a variety of soft gel capsules
56
57
58 86
59
1
2
3 containing mixtures of camostat mesylate and middle-chain triglycerides.151 Such soft gel
4
5
6 capsules displayed significantly improved camostat bioavailability compared to intravenous
7
8 administration of a saline solution of camostat mesylate when tested in rat intestine. In a later
9
10 development, Novartis described the formation of a number of additional crystalline camostat
11
12
13
salt forms in good to excellent yields: hydrogen succinate (88%), succinate (88%), phosphate
14
15 (75%), acetate (90%), hydrogen tartrate hemihydrate (99%), glycolate (96%), xinafoate (99%),
16
17 hippurate (83%), adipate (98%) and glutarate (98%).152 These salt forms as well as a novel
18
19
polymorph of camostat free base (designated as form II) were evaluated for their potential use in
20
21
22 inhaled formulations of camostat.
23
24
25 10.3 Outlook for Supply
26
27
28
29 Camostat mesilate (or mesylate) is supplied for the Japanese market by Ono Pharmaceuticals
30
31 under the tradename Foipan.140 A number of generic suppliers in Japan and South Korea also
32
33
34 manufacture the drug. Little information is available on the expected future worldwide supply of
35
36 camostat mesylate other than Ono Pharmaceuticals Co., Inc. announcing that shipment of the
37
38 drug would be limited due to an increased demand based on initial reports that it might be
39
40
41 effective in the treatment for COVID-19.153 Given the short synthesis of camostat from readily
42
43 available raw materials, supply could likely be ramped up quickly if needed.
44
45
46
47
48 11. Lopinavir/Ritonavir
49
50
52
53 The treatment of HIV was revolutionized in the mid-1990s when a number of HIV protease
54
55
56
inhibitors emerged, resulting in a dramatically reduced death rate for AIDS.154 The chemical
57
58 87
59
1
2
3 structures of these successful HIV protease inhibitors generally resemble small peptides and
4
5
6 typically incorporate highly modified synthetic amino acids.155, 156 The first HIV protease
7
8 inhibitor to reach the market was saquinavir that was developed by Hoffmann-La Roche and
9
10 approved in December 1995. Ritonavir (5) is a contemporary HIV protease inhibitor developed
11
12
13
by Abbott Laboratories that received FDA approval in March 1996. Soon thereafter, Abbott
14
15 Laboratories reported an improved second generation HIV protease inhibitor ABT-738 that later
16
17 became known as lopinavir (4).157 As described in more detail below, a dramatically improved
18
19
bioavailability of lopinavir is observed in the presence of ritonavir. These findings led to the
20
21
22 development of Kaltera, an FDA approved fixed dose combination of lopinavir and ritanovir for
23
24 the treatment of HIV/AIDS in several countries, in which lopinavir is the predominant active
25
26 drug and ritanovir functions primarily as a P450 CYP3A4 inhibitor.158
27
28
29
30 LPV is an inhibitor of the HIV-1 protease enzyme. The HIV-1 protease enzyme, a dimeric
31
32 aspartate protease, is the enzyme responsible for cleaving the Gag polyprotein, which functions
33
34
as the major structural protein of virus, and plays a vital role in the HIV viral life-cycle.159 LPV
35
36
37 is a peptidomimetic drug wherein the molecule contains a hydroxyethlyene scaffold which
38
39 mimics the normal peptide linkage (cleaved by HIV protease) but which itself cannot be
40
41 cleaved.154 By inhibiting HIV-1 protease activity and subsequent proteolysis of the Gag
42
43
44 polyprotein, this results in the production of immature, non-infectious HIV-1 virus particles.
45
46
47 LPV, when administered alone has a very low human bioavailability of ~25%, primarily due to
48
49 extensive oxidative metabolism by P450 CYP3A4 enzymes.157 It is exclusively co-administered
50
51
52 with ritonavir which reduces drug metabolism and significantly improves LPVs bioavailability.
53
54 In healthy human volunteers, co-dosing of 400 mg of LPV and 50 mg RTV significantly
55
56
57
58 88
59
1
2
3 increases the area under the concentration curve of LPV in plasma by 77-fold over that observed
4
5
6 after dosing with LPV alone. This results in concentrations of LPV which exceeded the anti-viral
7
8 50% effective concentration (EC50) for >24 hours.157
9
10
11
To date, there is no in vitro SARS-CoV-2 data reported for lopinavir/ritonavir, nonetheless
12
13
14 lopinavir was found to weakly inhibit both MERS-CoV and SARS-CoV replication in cell
15
16 cultures with EC50’s of 8 and 17 M, respectively.160 Most clinical studies have investigated the
17
18
use of lopinavir/ritonavir in SARS-CoV but these studies have been mostly retrospective and
19
20
21 observational in nature.161 One such retrospective matched cohort study of SARS patients
22
23 showed that treating SARS-CoV patients with LPV (400 mg)/RTV (100 mg) orally every day for
24
25 14 days significantly reduced ARDS. Patients also appeared to run a milder disease course in
26
27
28 recurrence of fever, reduction of viral load and worsening of chest radiographs.162 It was also
29
30 noted that timing of administration was critical during the early viral replication phase (initial 7-
31
32 10 days) because delaying therapy further had no effect on clinical outcomes.
33
34
35
36 Initial reports of lopinavir/ritonavir for the treatment of COVID-19 involve small retrospective,
37
38 non-randomized cohort studies. A recent randomized, controlled open-label trial involving 199
39
40 hospitalized adults patients with SARS-CoV-2 in Wuhan/China compared the efficacy of
41
42
43 lopinavir/ritonavir vs standard of care.163 Treatment with lopinavir/ritonavir was not associated
44
45 with a difference from standard care in the time to clinical improvement. In addition, mortality at
46
47 28 days was similar in the lopinavir-ritonavir group and the standard-care group (19.2% versus
48
49
50
25%) and the percentages of patients with detectable viral RNA were similar at various time
51
52 points.164 Further clinical trials are underway to assess both the efficacy and safety of
53
54 lopinavir/ritonavir (Table 11).164 It has been reported that many clinicians are abandoning the use
55
56
57
58 89
59
1
2
3 of lopinavir/ritonavir for the treatment of COVID-19 after the negative findings from the above
4
5
6 study. A recent report sent to the editors of NEJM suggest this action is premature. They argue
7
8 the mortality rate lowering is significant, in addition to the lowering of ARDS. They advocate
9
10 that therapeutic guidelines retain this combination as a treatment option against COVID-19,
11
12
13
awaiting completion of the WHO SOLIDARITY trial.165a
14
15
16 A recent report in Lancet suggests that the combination of lopinavir, ritonavir, ribavirin, and
17
18 interferon could be effective in treating COVID-19 in the early stages of the disease.165b Patients
19
20
21 were randomly assigned (2:1) to a 14-day combination of lopinavir 400 mg and ritonavir 100 mg
22
23 every 12 h, ribavirin 400 mg every 12 h, and three doses of 8 million international units of
24
25 interferon beta-1b on alternate days (combination group) or to 14 days of lopinavir 400 mg and
26
27
28
ritonavir 100 mg every 12 h (control group). Between Feb 10 and March 20, 2020, 127 patients
29
30 were recruited; 86 were randomly assigned to the combination group and 41 were assigned to the
31
32 control group. The combination group had a significantly shorter median time from start of study
33
34
treatment to negative nasopharyngeal swab (7 days [IQR 5–11]) than the control group (12 days
35
36
37 [8–15]; hazard ratio 4·37 [95% CI 1·86–10·24], p=0·0010).
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 90
59
1
2
3 Table 11. Clinical trials underway with Lopinavir/Ritonavir164
4
5
6
10 Dates
11 NCT04307693 Randomized Controlled Clinical 150 11Mar2020 – Arm 1) Lopinavir/ritonavir 200mg/100mg 2 tablets by Placebo
12 Trials of Lopinavir/Ritonavir or May2020 mouth, every 12 hours for 7-10 days
13 Hydroxychloroquine in Patients Arm 2) Hydroxychloroquine 200mg 2 tablets by
14 With Mild Coronavirus Disease mouth, every 12 hours for 7-10 days
15 (COVID-19)
16 NCT04359095 Effectiveness and Safety of Medical 1600 11May2020- Arm 1) Hydroxychloroquine PO 400 mg every 12 Standard-
17 Treatment for SARS-CoV-2 Oct2020 hours for 24 hours, then 200 mg every 12 hours for 10 of-care
18 (COVID-19) in Colombia: A days.
19 Pragmatic Randomized Controlled Arm 2) Hydroxychloroquine 400 mg PO every 12
20 Trial hours for 24 hours, then 200 mg every 12 hours for 10
days + Lopinavir / Ritonavir PO 400 mg/100 mg
21
every 12 hours for 10 days
22
Arm 3) Hydroxychloroquine PO 400 mg every 12
23
hours for 24 hours, then 200 mg every 12 hours for 10
24 days + Azithromycin PO 500 mg once daily for 5 days
25 NCT04328285 Chemoprophylaxis of SARS-CoV-2 1200 14Apr2020 – Arm 1) HCQ 200 mg : 2 tablets on the evening at Day Placebo
26 Infection (COVID-19) in Exposed Nov2020 1 and 2 tablets on the morning at Day 2 and 1 tablet
27 Healthcare Workers : A Randomized once daily afterwards
28 Double-blind Placebo-controlled Arm 2) LPV/RTV 200/50 mg, 2 tablets twice daily
29 Clinical Trial
30 NCT04328012 Comparison Of Therapeutics for 4000 6Apr2020 – Arm 1) lopinavir/ritonavir 400mg/200mg mg PO BID Placebo
31 Hospitalized Patients Infected With Jan2021 x 5-14 days depending on availability
32 SARS-CoV-2 In a Pragmatic Arm 2) hydroxychloroquine sulfate 400 mg BID on
33 aDaptive randoMizED Clinical Trial Day 0 200 mg BID Days 1-4, days 1-13 if available
34 During the COVID-19 Pandemic Arm 3) losartan 25 mg PO QD x 5-14 days depending
35 (COVID MED Trial) on availability
36 NCT04330690 A Multi-centre, Adaptive, 440 18Mar2020- Arm 1) Lopinavir/ritonavir will be administered 400 Standard-
37 Randomized, Open-label, Controlled Mar2022 mg/100 mg orally for a 14-day course, or until of-care
38 Clinical Trial of the Safety and discharge from hospital, whichever occurs first
39 Efficacy of Investigational Arm 2) Hydroxychloroquine 800mg BID for 1 day
40 Therapeutics for the Treatment of then 400mg BID for 10 days plus optimized
41 COVID-19 in Hospitalized Patients supportive care
42
43 91
44
46
47
1
2
3 (CATCO: Canadian Treatments for Arm 3) Remdesivir 200mg IV on day 1, followed by
4 COVID-19), in Conjunction With 100 mg IV daily infusion for 9 days plus optimized
5 the Public Health Emergency supportive care
6 SOLIDARITY Trial (World Health
7 Organization)
8 NCT04372628 Trial of Early Therapies During 900 1May2020- Arm 1) Hydroxychloroquine 400 mg PO BID for two Placebo
9 Non-hospitalized Outpatient Dec2020 doses (Day 1), then 200 mg BID for subsequent eight
10 Window (TREAT NOW) for doses (Day 2-5), then placebo twice daily for
11 COVID-19 subsequent eighteen doses (Days 6-14)
12 Arm 2) Lopinavir/ritonavir 400 mg/100 mg PO BID
13 for twenty-eight doses (Days 1-14)
14 NCT04364022 Efficacy of Pragmatic Same-day 420 Apr2020- Single dose HCQ 800 mg PO and Lopinavir/ritonavir Placebo
15 Ring COVID-19 Prophylaxis for Oct2020 2 x 200mg/50mg PO BID for 5 days
16 Adult Individuals Exposed to SARS-
17 CoV-2 in Switzerland: an Open-
18 label Cluster Randomized Trial
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43 92
44
46
47
1
2
3
4
11.2 Synthesis of Lopinavir
5
6 A scalable synthesis of lopinavir (4.4) was described in two publications166, 167 and a patent168 by
7
8 a team at the innovator company, Abbott Labs (now Abbvie) in 1999 and 2000.169 The
9
10
retrosynthesis of lopinavir is outlined in Scheme 27.
11
12
13
Scheme 27. Retrosynthetic Approach to Lopinavir
14
15
16 Ph
17 O OH
18 H
N
19 N O
20 H
O N O
21 Ph
22 HN Lopinavir, 4.4
23
24
25
26
27
28 CO2H Ph
OH
29
O N + NBn2
30 H 2N +
HO2C O
31
32 HN Ph
33 11.1 11.2 11.3
34
35
36
The central core diamine, 11.2, containing three chiral centers, is the same for both ritonavir
37
38
39 (4.5) and lopinavir (4.4), and is described in earlier publications from Abbott.170 The first chiral
40
41 center is derived from the chiral pool (L-phenylalanine) and this center is then used to direct
42
43 installation of the other two chiral centers, as outlined in Scheme 28. The process chemistry
44
45
46 route to 11.2, described on 14 to 122 kg substrate scale, begins with the tribenzylation of
47
48 phenylalanine (11.4) with benzyl chloride to afford ester 11.5, which is isolated as a crude oil
49
50 after aqueous workup. The next two steps, addition of the acetonitrile anion to the benzyl ester,
51
52
53
and reaction of the resulting nitrile with benzylmagnesium chloride, were carried out as a one-
54
55 pot reaction in MTBE. Sodium amide was the base used to generate the acetonitrile anion. This
56
57
58 93
59
1
2
3 is not a common base used at scale due to concerns with handling and flammability, but a survey
4
5
6 of other bases, including KOH, KOtBu, NaH, KHMDS, and LiHMDS, led to incomplete
7
8 reactions, slow conversions, or racemization.170 MTBE was found to be a preferred solvent for
9
10 the reaction, with no racemization occurring when the reaction was carried out at 0-5 oC. In an
11
12
13
earlier procedure using THF as the solvent, the reaction was conducted at -45 oC to prevent
14
15 racemization.170 Upon formation of ketonitrile 11.6, vacuum was applied to remove ammonia,
16
17 then the benzylmagensium chloride was added at 25 oC and stirred for 2 h. After aqueous
18
19
workup, enaminone 11.7 was crystallized from EtOH with >99% ee in overall 79% yield for the
20
21
22 three steps.
23
24
25 The diastereoselective reduction of enaminone 11.7 to aminoalcohol 11.2 required extensive
26
27 development. Use of a single reagent to effect reduction at both centers afforded mixtures of
28
29 diastereomers. Sodium borohydride-TFA complex provided the best result with 72% of the
30
31
32 desired diastereomer 11.2 and 28% of a mixture of the other three diastereomers. Reductions
33
34 using NaBH4 with mineral acids led to reduction of only the enamine double bond to generate
35
36 11.8 with a dr of approximately 95:5. This suggested a stepwise process could be feasible –
37
38
39
reduction first of the enamine double bond followed by ketone reduction. The initial reduction
40
41 using NaBH4 with sulfuric acid followed by the ketone reduction by addition of NaBH2(TFA)2 in
42
43 THF afforded 11.2 with a dr of 95:5. While this reaction worked well in the laboratory, scale up
44
45
resulted in thick mixtures due to ring-opening polymerization of THF. Addition of water helped
46
47
48 minimize THF decomposition and, unexpectedly, also improved the diastereoselectvity.
49
50 However, a better solution was to exchange sulfuric acid with MeSO3H and carry out the
51
52 reaction in the alternate solvent, dimethoxyethane (DME). While water improved selectivity, it
53
54
55
56
57
58 94
59
1
2
3 also slowed the reaction so the final conditions used 2-PrOH as additive instead. The role of the
4
5
6 alcohol in improving diastereoselectivity remains unknown.
7
8
9 The resistance of the ketonitrile to reduction was presumed to be due to complexation of the
10
11 ketone, perhaps as a boron enolate. To enable the second reduction, a boron complexing agent
12
13 was added. A survey of aminoalcohols found triethanolamine to be optimal. Final conditions for
14
15
16
reduction of 11.8 to 11.2 were treatment of the solution of 11.7 in DME with triethanolamine
17
18 followed by addition of NaBH4 in dimethylacetamide and stirring for 2 h at 15 oC. Aqueous
19
20 workup provided 11.2 as a solution in EtOAc, containing 89-93% of the desired diastereomer
21
22
with the remainder a mixture of the other diastereomers.166-168 This solution was used without
23
24
25 further purification for downstream conversion to lopinavir (Scheme 30). The reduction process
26
27 (11.7 to 11.2) was described on a 400 kg scale.168
28
29
30
31
32
33 Scheme 28. Abbott Synthesis of Central Chiral Core 11.2 of Lopinavir
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58 95
59
1
2
3 Ph PhCH2Cl Ph CH3CN
4
K2CO3, KOH NaNH2
5
6 H 2N CO2H H O, reflux Bn2N CO2Bn MTBE, 0-5 oC
2
7
8 11.4 11.5
9
10
11 Ph Ph
NaBH4
12 PhCH2MgCl MeSO3H
13 NH2
Bn2N CN Bn2N
14 THF, MTBE, rt DME, 2-PrOH
O 79% (3 steps) O
15 Ph 5 oC
16 11.6 11.7
17 ee >99%
18
19 Ph Ph
NaBH4
20
21 NH2 (HOCH2CH2)3N
NH2
Bn2N Bn2N
22
O MeCONMe2 OH
23 Ph 15 oC Ph
24 11.8 11.2
25
dr 95:5
26
27
28
29 The Abbott synthesis of fragment 11.1 is outlined in Scheme 29.166 In the first step, L-valine
30
31 (11.9) was converted to carbonate 11.10 using phenyl chloroformate. The reaction required
32
33 significant development to ensure operability at scale. Lithium chloride was added to reduce the
34
35
freezing point, allowing the reaction to be carried out at -10 oC. Control of the pH between 9.5-
36
37
38 10.2 was critical to minimize formation of valine dipeptide and acylated derivatives. Neutral
39
40 alumina prevented gumming and emulsion formation that was essential for mixing and
41
42 maintaining pH control. After aqueous workup, carbonate 11.10 was isolated in 92% yield after
43
44
45 crystallization from toluene/heptane. Treatment of 11.10 with 3-chloropropylamine
46
47 hydrochloride and solid NaOH in THF at 10 oC for 2 h furnished alkylated product 11.11, which
48
49 was cyclized to 11.1 by addition of KO-t-Bu in THF at 20 oC. After aqueous workup, 11.1 was
50
51
52 isolated in 77% yield and >99% ee after crystallization from EtOAc.
53
54
55
56
57
58 96
59
1
2
3 Scheme 29. Abbott Synthesis of Fragment 11.1
4
5
6
7
8
9
LiCl, LiOH
10 CO2H
11 CO2H PhOCOCl Cl NH3Cl
12 HN OPh
13 NH2 H2O, Al2O3 NaOH, THF
14 pH 9.5-10.2 O
15 11.9 92% 11.10
16
17
18 CO2H
19 CO2H
KOt-Bu
20 H O N
HN N Cl
21 77%
22 O HN
23
11.11 11.1
24
25
26
27 The final steps to lopinavir are shown in Scheme 30.166 Amine 11.2 was coupled to acid chloride
28
29 11.12, generated from acid 11.1 with thionyl chloride, using imidazole (3.0 equiv) in
30
31
32
EtOAc/DMF at 30 oC. After aqueous workup and concentrating to an oil, the crude amide 11.13
33
34 was debenzylated in MeOH with Pd/C and ammonium formate in MeOH at 50 oC to furnish
35
36 amine 11.14. Upgrade of the diastereomeric purity was accomplished by crystallization of the
37
38
salt with L-pyroglutamic acid in 1,4-dioxane to generate 11.14 PGA salt in 74% and >98.5%
39
40
41 purity. The final amide coupling of 11.14 PGA with acid chloride 11.16 (generated from 11.3
42
43 with thionyl chloride) was conducted under Schotten-Baumann conditions (EtOAc/aq. NaHCO3)
44
45 at room temperature. Crystallization of lopinavir was originally carried out from
46
47
48 EtOAc/heptanes, but removal of residual solvents proved problematic so an alternate
49
50 crystallization from EtOH/water was developed. Overall yield from 11.2 was 58%.
51
52
53 Scheme 30. Final Steps to Lopinavir
54
55
56
57
58 97
59
1
2
3
Ph Pd/C
4 Ph Imidazole O OH
5 OH HCO2NH4
6 DMF, EtOAc NBn2
NBn2 N
7 H 2N H MeOH, H2O
8 O N 50 oC
9 COCl Ph
Ph
10 HN
11 11.2 O N 11.13
12
13 HN
14
15
11.12
16
17
18
Ph
O OH Ph
19
NH2 O OH
20 CO2
21 N Dioxane NH3
H N
22 O N H
23 Ph O N NH
24 HN O CO2H Ph
N
25 11.14 H HN 11.14 PGA O
26
11.15
27
28
29
30
31 Ph
32 O OH
33 ClOC O H
N
34 11.16 N O
35 H
O N O
36 EtOAc, water Ph
37
NaHCO3, rt HN Lopinavir, 4.4
38
39 58% (4 steps)
40
41
42
43
44
45 Abbott chose to upgrade the diastereomeric purity by formation of the L-pyroglutamic acid salt
46
47
of amine 11.14. In the experimental procedure provided, the yield for the crystallization of the
48
49
50 purified 11.14 PGA salt was 74%. This is a significant yield loss for the penultimate
51
52 intermediate in a lengthy synthesis. Laurus Labs have recently disclosed a process wherein the
53
54 diastereomeric purity upgrade is carried out at an earlier stage, amine 11.2, by formation of the
55
56
57
58 98
59
1
2
3 L-tartrate salt.170 The two-step reduction from 11.7 to 11.2 was carried out similarly to the
4
5
6 Abbott process (Scheme 28), then the L-tartrate salt of 11.2 was crystallized from MTBE in 79 %
7
8 isolated yield with a purity of 99.0%. The levels of the three undesired diastereomers were
9
10 0.16%, 0.24%, and 0.38%.171
11
12
13 In the same patent application from Laurus, the inventors note that the Abbott process produces
14
15
16
up to 5% over-acylated byproduct 11.17 in the final step, an impurity that Laurus claims is
17
18 difficult to remove by crystallization.171 The Laurus team has found that this impurity can be
19
20 hydrolyzed to lopinavir by treatment with base. In one example, lopinavir was prepared starting
21
22
with 0.95 equiv of acid 11.3 and 1.00 equiv of 11.14 PGA salt. The crude material contained
23
24
25 0.8% of impurity 11.17. This material was treated with methanolic ammonia, then crystallized
26
27 from acetone to afford lopinavir in 90% yield and 99.9% purity with an undetectable level of
28
29 impurity 11.17. In the comparative example in the Laurus patent application, lopinavir was
30
31
32 prepared starting with 1.00 equiv each of acid 11.3 and 11.14 PGA salt. The crude from this
33
34 reaction was crystallized from EtOAc/heptane to provide lopinavir with 94% purity containing
35
36 3% of impurity 11.17. We note that in the Abbott publication a 20% excess of acid 11.3 is used
37
38
39
for this reaction and, after crystallization from EtOAc/heptane a purity of 99% is obtained.166
40
41
42 Ph
43 O OH
H
44 N
45 N O
46 H
O N O
47 Ph
48 N
49 O 11.17
50
51
O
52
53
Figure 6. Structure of Lopinavir Impurity 11.17
54
55
56
57
58 99
59
1
2
3
4
11.3 Salt Forms and Formulation of Lopinavir and Ritonavir
5
6
7 Given the formulation of Kaletra is a fixed dose combination of ritonavir and lopinavir, the form
8
9
10
and physical characteristics of both drug substances must be considered for formulation
11
12 development.
13
14
15 Abbott has disclosed four crystal forms of lopinavir designated as Type I, a hydrated form
16
17
18 containing 0.5 to 2.0 moles of water; Type II, a solvated form containing either 2-PrOH, EtOAc,
19
20 or chloroform; Type III, a solvated form containing either EtOAc or MeCN and a desolvated
21
22 form; and Type IV, a non-solvated form. Other solvates within type III were mentioned but not
23
24
exemplified.172 According to the European Public Assessment Report (EPAR) for Kaletra, the
25
26
27 commercial form is crystallized from EtOH and water and is a mixture of the amorphous form
28
29 and crystalline type I.173
30
31
32 A cyclohexane solvate of lopinavir and its desolvated form were subsequently disclosed by
33
34 Hetero.174
35
36
37 Two crystal forms of ritonavir have been identified, Forms I and II.175 At the time of the initial
38
39
40 approval of ritonavir (tradename Norvir) in 1996, only Form I had been encountered in
41
42 development and launch. Given the low bioavailability of crystalline ritonavir, the two
43
44 formulations that were marketed, an oral solution and a capsule, contained ritonavir as a solution
45
46
47
in EtOH/water. In 1998, several lots of formulated drug undergoing release testing failed the
48
49 dissolution specification. An investigation revealed that a new polymorph (designated as Form
50
51 II) was crystallizing in the capsules. Soon, the new polymorph was showing up in other lots of
52
53
formulated drug and the API. Form II was found to be the thermodynamically most stable form
54
55
56 that was approximately 5-fold less soluble than Form I in EtOH/water mixtures. This form was
57
58 100
59
1
2
3 no longer fully soluble in the solution used for the capsule formulation nor under the refrigerated
4
5
6 storage conditions for the oral solution. As such, both formulations of the product were
7
8 withdrawn and new formulations were investigated. A soft gelatin capsule formulation was
9
10 developed with a solvent composition of EtOH, oleic acid, and water to ensure complete
11
12
13
solubility of Form II. Polyoxyl 35 castor oil was added to improve the bioavailability.176
14
15 Subsequently, a film-coated tablet (100 mg) was developed and marketed.177 The oral solution
16
17 was reformulated in a mixed solvent system of water, EtOH, propylene glycol, and polyoxyl 35
18
19
castor oil.176
20
21
22
23
24
25 Both ritonavir and lopinavir drug substances have very low water solubility and the crystalline
26
27
28 forms have minimal bioavailability. This led to an initial marketed formulation (U.S approval
29
30 September 2000) of a soft gelatin capsule containing133.3 mg of lopinavir and 33.3 mg of
31
32 ritonavir as a solution in a mixture of oleic acid, propylene glycol and water, with a storage
33
34
35 condition of 2-8 oC.178 To reduce the pill burden and avoid cold storage, a tablet formulation was
36
37 subsequently developed using hot melt extrusion technology to render each drug substance
38
39 amorphous in the formulation. Film-coated tablets containing 200 mg lopinavir and 50 mg
40
41
42
ritonavir were approved in the U.S. in October 2005 with room temperature storage
43
44 conditions.179
45
46
47
48
49
50 11.4 Lopinavir/Ritonavir Outlook for Supply
51
52
53
54
55
56
57
58 101
59
1
2
3 On January 28, 2020, the Indian generic company Cipla announced that they have raw materials
4
5
6 available to manufacture 10-12 million doses of lopinavir/ritonavir. They noted, however, that
7
8 current demand, and therefore supply, for HIV is low given the introduction of better drugs over
9
10 the past 20 years.180
11
12
13 On March 9, 2020, AbbVie stated that is does not anticipate any disruption to its supply chain
14
15
16
but continues to monitor its resources.181
17
18
19
On March 19, 2020, Israel approved the licensing of a generic version of ritonavir/lopinavir to
20
21 treat patients infected with the coronavirus.182
22
23
24 On March 23, 2020, AbbVie announced it would no longer be enforcing patents on the drug
25
26 combination.183 The composition of matter patent expired in 2016 but the tablet formulation
27
28 patent would have been in effect in the U.S. until 2028.184
29
30
31 On March 25, 2020, Mylan announced that it would waive its exclusive rights to market generic
32
33
34 lopinavir/ritonavir in the U.S. to allow other generic companies to manufacture the drugs.185
35
36
37 On April 7, 2020, the Philadelphia-based company Lannett announced it was poised to increase
38
39 production of lopinavir/ritonavir if demand increased.186 Lannett currently markets the generic
40
41 version of lopinavir/ritonavir oral solution.187
42
43
44 Other manufacturers of the drug combination include the Indian-based companies Aurobindo,
45
46
Cipla, Hetero, and Macleods. The Indian government has asked these companies to increase
47
48
49 production in anticipation of a greater need to treat COVID-19.188
50
51
52
53
54
55
56
57
58 102
59
1
2
3
4
5
6 12. Summary and Conclusions
7
8
9
10 Designing, developing, and testing a new drug for the treatment of COVID-19 is likely a multi-
11
12 year endeavor even with accelerated development, as a drug candidate must first be shown to be
13
14 safe in animals even before clinical trials can begin. Then, a series of three phases of clinical
15
16
17 trials must be completed: Phase I to demonstrate safety and tolerability in healthy volunteers,
18
19 Phase II to show efficacy in COVID-19 patients, and Phase III to confirm efficacy and safety in a
20
21 larger cohort of patients. A shorter pathway to treatment would be to repurpose older drugs –
22
23
those that have been shown to be safe and efficacious against other diseases, and may have
24
25
26 efficacy against SARS-CoV-2. A global collaboration of scientific groups, spearheaded by the
27
28 University of California at San Francisco, has identified 29 approved drugs that may have
29
30 activity against SARS-CoV-2.189 In this article, we have reviewed seven promising repurposed
31
32
33 drugs that are currently being studied in large clinical trials in COVID-19 patients. One such
34
35 drug, remdesivir, an antiviral drug originally developed for the treatment of Ebola virus, has
36
37 shown efficacy in a well-controlled clinical trial and has now been granted approval for
38
39
40 emergency use in the U.S. and Japan. An extraordinary effort has been underway since February
41
42 2020 at Gilead to ramp up production, with the goal of having one million doses available by the
43
44 end of the year. In the next few months we expect trial data for many other drugs, with the hope
45
46
that a cocktail of repurposed drugs can provide a meaningful treatment for the majority of
47
48
49 COVID-19 patients.
50
51
52
53
54
55
56
57
58 103
59
1
2
3
4 13. References
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6
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8 1) Geller, C.; Varbanov, M.; Duval, R. E. Human Coronaviruses: Insights into Environmental
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3 22) World Health Organization. WHO R&D blueprint: ad-hoc expert consultation on clinical
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