Thesis Questions: introduction

Why do viruses have a high genetic variability?

resulting from the error-prone RNA-dependent RNA polymerase (RdRp) responsible for replication in most RNA viruses. This lack of proofreading capability leads to frequent mutations, enabling these viruses to evolve rapidly, adapt to new environments or hosts and develop resistance

DENV

Dengue virus, a member of the Flavivirus genus, causes dengue, one of the most widespread and severe viral diseases affecting humans. Symptoms range from mild fever and joint pain to severe liver, kidney, or brain damage, with approximately 400 million infections and around 22,000 deaths globally each year.

EBOV

Ebola virus disease (EVD), caused by viruses of the genus Ebolavirus, is frequently associated with severe or fatal clinical outcomes. The average case-fatality rate is approximately 50%, although mortality rates ranging from 25% to 90% have been reported depending on the outbreak and viral species. The largest outbreak to date occurred in West Africa between 2014 and 2016 and resulted in more than 11,000 deaths.

HCV

Hepatitis C virus (HCV) is a blood-transmitted pathogen that contributes significantly to chronic liver-related diseases and global morbidity and mortality. Despite control efforts, approximately one million new infections occur annually, with nearly 242,000 deaths reported in 2022 due to delayed diagnosis and limited access to treatment.^{6, 7}

COVID-19

Most recently, the coronavirus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), resulted in an estimated seven million deaths worldwide. Although the global emergency phase of the pandemic has passed, SARS-CoV-2 continues to evolve, demonstrating its adaptability and underscoring the importance of ongoing genomic surveillance and vaccine refinement.¹⁰

disadvantages DAAs

DAAs, which target viral enzymes or specific steps in the replication process,¹⁷ significantly complement vaccines. However, they often suffer from a narrow spectrum of activity¹⁸ and rapid emergence of drug-resistant viral strains due to frequent mutations.^{14, 19} In addition, challenges such as toxicity,²⁰ accessibility,²¹ and the need for rapid development during emerging outbreaks continue to limit their effectiveness.¹⁹

HDA + and -

Advantages

• broader-spectrum antiviral activity

• valuable in early stages of emerging infections when the virus is still unknown

• can enhance immune defence and restore cellular processes that are dysregulated during infection

• more resistant to viral mutations

challenges

• selecting target without causing toxicity

• knowledge about virus-host interations - can take time

• institutions such as NIAID have traditionally prioritized DAAs and monoclonal antibodies, which have more established development pathways and proven success.

HDA strategies

• block receptors used by viruses for entry

• interfere with host mechanisms essential for viral replication

• modulate immune responses to enhance antiviral defence

• correct dysregulated pathways that contribute to harmful inflammation

interferon

Interferons are a subclass of immunomodulators, and are a group of signaling proteins naturally produced by the immune system in response to viruses, parasites, and tumors.

immunoodulator

Immunomodulators are substances that alter the immune response to treat diseases

how does imiquimod help against human papilomavirus (HPV)

Imiquimod helps the body fight Human Papillomavirus Infection by “switching on” the immune system in the treated area. It causes the body to release interferons and other immune signals that help destroy HPV-infected cells.

what classes of kinases are there and what is GAK?

. Based on substrate specificity, they are classified as serine/threonine, tyrosine, or dual-specificity kinases which are the amino acids on the substrate where the phosphate is transfered to

Cyclin G-associated kinase (GAK) is primarily a serine/threonine protein kinase. This means it transfers a phosphate group from ATP mainly to the amino acids serine or threonine on target proteins.

General structure of kinases

tructurally, these enzymes adopt a conserved bilobal architecture consisting of a smaller N-terminal lobe, primarily composed of β-sheets and an α-helix, known as the C-helix, and a larger C-terminal lobe that is mainly α-helical. These two lobes are connected by a flexible hinge region that forms the ATP-binding pocket. The first residue of the hinge, commonly referred to as the gatekeeper residue, regulates access to a hydrophobic pocket adjacent to the ATP-binding site, which is inaccessible to ATP but can be exploited by small-molecule inhibitors.³⁷

glycine rich loop

In N-terminal lobe and stabilizes ATP phsophates during catalysis and coordinates to magnesium

catalytic loop

contains a conserved His-Arg-Asp (HRD) motif that participates in substrate positioning

activation segement

The activation segment typically spans 20–30 amino acids and extends from the conserved Asp-Phe-Gly (DFG) motif to the Ala-Pro-Glu (APE) motif. It contains the flexible activation loop (A-loop) (activation segment is not all loops), which plays a central role in substrate recognition and catalysis. Its correct positioning is required to form the substrate-binding cleft, and phosphorylation of a residue within this loop often stabilises the active conformation

DFG-in

In this active state, also called the DFG-in conformation and illustrated in Figure 2, the aspartate residue of the DFG motif is oriented towards the ATP-binding site and coordinates a Mg²⁺ ion that interacts with ATP, facilitating phosphate transfer.

Small-molecule inhibitors that bind to the active DFG-in conformation are known as type I inhibitors and typically mimic the adenine ring of ATP, forming hydrogen bonds with the hinge region.

DFG-out

In contrast, in the inactive state, the DFG motif flips outward (DFG-out), causing the aspartate side chain to orient away from the ATP-binding site. This disrupts Mg²⁺ coordination, prevents substrate binding, while repositioning of the DFG phenylalanine exposes a hydrophobic backpocket.

The accessibility of this hydrophobic cavity is largely determined by the gatekeeper residue, which is located at the entrance of the pocket at the start of the hinge region. Kinases with a small gatekeeper residue possess a larger back pocket that can accommodate additional substituents on inhibitors, which may enhance both potency and selectivity. In contrast, kinases with larger gatekeeper residues restrict access to this cavity, limiting inhibitor binding in this region.

, type II inhibitors bind to the inactive DFG-out conformation and extend into the adjacent hydrophobic backpocket that becomes accessible in this state

type I1/2 inhibitors

More recently, type I½ inhibitors have been identified, which bind to the active conformation while also occupying part of this hydrophobic region

which residue is the gatekeeper residue in GAK

T123 as the gatekeeper residue side chian: (OH-)-CH2-(CH3)

The slightly more enclosed ATP pocket at the frant due to bulky Phe133 is compensated by the smaller, mor polar Thr123, compared o methionine or leucin in other NAKs. This residue contributes to the formation of a deeper backpocket, that can be occupied by eg. the morpholine of the isothiazolopyridine. or for our compounds the 3-position side chain

This is not the hydrophobic back pocket that becomes accessible in the DFG-out conformation but just a larger ATP binding pocket because the gatekeeper doesn’t take up as much space.

Which type of inhibitors are we trying to make

Cocrystalization experiments, supported by subsequent molecular modelling of compound 15 revealed that the isothiazolopyridine scaffold binds within the ATP pocket in a type I mode, meaning it targets the active (DFG-in) conformation of the kinase.

Our kinases are supposed to dock in the same type I mode.

The activation segment C-terminal helix (ASCH) largely eliminates the requirement for phosphorylation to adopt the active DFG-in conformation, rendering these kinases constitutively active

during screening we targeted DFG in:

targeting DFG-in conformation is generally relevant since the previously synthesised inhibitors also target DFG-in conformation

Other NAKs

• AAK1 (adaptor-associate kinase 1 )

• MPSK1

• BIKE

• GAK

ASCH

unique structural feature of NAKs which is an additional α-helical segment located C-terminally to the activation loop, known as the activation segment C-terminal helix (ASCH). This structural adaptation largely eliminates the requirement for phosphorylation to adopt the active DFG-in conformation, rendering these kinases constitutively active

domains of GAK

• N-terminal Kinase domain

• PTEN domain (recruitment of the portein to the vescile membrane by recognising the phospholipid head groups (new lipid environment))

  • the AA sequence is similar to that of the PTEN protein which is primarily a lipid phosphatase, which dephosphorylates phosphatidylinositol (3,4,5)-trisphosphatePIP3 (original lipid envrionment) to PIP2

  • the PTEN like domain in GAK does not have phosphatase activity but has high affinity for the new lipid environment formed after budding

  • so it is recruited by its affinity for clathrin and the lipid environment

• clathirn-binding domain

• J-domain: J-domain recruits the molecular chaperone Hsc70

Auxilin i has no kinase domain and restricted to neurons

Other functions GAK

• transcriptional coactivation

• mitotic chrmosome congression

• general cell growth

Steps chlathrin mediated endocytosis

• cargo binds to to receptors

• APs are recruited to the membrane and recognise the cytoplasmic sorting motifs of the bound receptors

  • GAK phosphorylates the µ-units increasing their affinity for the cargo receptors

• simultaneously APs recruit clathrin by binding to its heavy chain

• clathrin polymerises into a lattice coat

  • not a polymerization in the classic covalent sense, no actual bonds are formed its more of a reversible non-covalent self assembly process

  • APs therefor attacht the cargo containing receptor to the forming coat and also concentrate the recepters within forming CCPs

• when several of the hexagons of the clathrin coat contract to pentagons, the membrane pulls inward forming CCPs

  • molecular rearangements and exchange of clathrin allows formation of pentagons in the initially hexagon-rich lattice. these pentagon defects force the lattice to curve

• curvature increases until it buds of through action of dynamin

• now there’s a clathrin coated vesicle inside the cell

• PI(4,5)P2 is metabolised after budding, AP2 loses affinity for the vesicle

• GAKk is recruited via its PTEN like domain having high affinity for the new lipid environment AND the clathrin coat around it

  • meanwhile its clathrin binding domain attatches to the lattice

  • and its J-domain recruits Hsc70 to the lattice

• Hsc70 is then thought to bind to the clathrin lattice and pull it in a unfavourable postion, causing uncoating of the CCV and releasing a free vesicle.

  • The exact mechanism is complex, but Hsc70 is thought to bind to clathrin and use ATP hydrolysis to destabilise the clathrin lattice. This creates conformational strain or pulls parts of the clathrin triskelion into an unfavourable position, leading to disassembly of the coat.

24. Could GAK inhibition be toxic?

Yes, that is an important concern. Since GAK is involved in normal cellular trafficking and other cellular processes, complete or long-term inhibition could cause toxicity. The therapeutic idea would require a suitable balance: enough inhibition to disrupt viral replication or entry, but not so much that essential host-cell functions are severely impaired.

= optimal therapeutic window

Would inhibiting GAK block all endocytosis?

Not necessarily all endocytosis. Cells have multiple uptake pathways, including clathrin-independent routes. However, GAK inhibition can disturb clathrin-mediated trafficking and may therefore affect viruses or cellular processes that strongly rely on this pathway.

enveloped vs non-envoloped viruses

enveloped RNA viruses, with their genetic material enclosed in a protein capsid and surrounded by a lipid membrane. For enveloped viruses, attachment and entry are mediated by viral membrane proteins, whereas non-enveloped viruses use capsid proteins to interact with host-cell receptors.

viral entry

After clathrin uncoating, the endocytic vesicle fuses with early endosomes, which can subsequently mature into late endosomes. Along this pathway, progressive acidification can induce conformational changes in viral surface proteins,⁸² promoting membrane fusion or permeabilisation and enabling release of the viral genome into the cytosol.⁸³ After entry, many viruses utilise microtubule-based transport systems to reach their specific sites of replication,⁸⁴ where uncoating is completed and viral replication can start.

GAKs role in later stages of the viral life cycle

• transport to assembly sites through AP2 activated by GAK

• transport through the TGN using AP1: important for maturation of the particle

• GAK helps in the release of the viral particles (through clathrin/adaptor mediated processes)

• cell to cell spread

---

Transport to assembly sites: GAK helps regulate AP-2, which can support the transport of viral proteins, such as HCV core proteins, to sites where new virus particles are assembled.

• Virus maturation and transport: For viruses such as DENV and HCV, GAK supports AP-1-dependent trafficking through the Golgi/TGN, which is important for particle maturation and movement toward the cell surface.

• Viral release: GAK helps clathrin/adaptor-mediated vesicle transport, allowing mature virus particles to move toward the plasma membrane and be released from the cell.

• Cell-to-cell spread: In HCV, GAK and AAK1 also support adaptor-mediated trafficking that helps the virus spread directly from one cell to neighbouring cells.

gefitinib

less toxic alternative to erlotinib

inhibitor of the epidermal growth factor receptor (EGFR) for patients with lung cancer.

it has adverse effect like respiratory dysfunction suggesting it may have off-target inhibitory effects. later it was shown to inhibit GAK, and given its role in the CME it was proposed as a potential therapeutic target

gefitinib also inhibits GAK determined through a proteomics approach

Erlotinib in particular showed unexpectedly high affinity for GAK (K_d = 3.1 nM) and has been reported to inhibit viral infections, including those caused by DENV and EBOV, in both in vitro and in vivo studies

As this shows off-traget effects of inhibiting GAK can have bad adverse effects, why would inhibiting GAK be good as a therapeutic target?

That is an important point. Gefitinib shows that off-target kinase inhibition can contribute to adverse effects, so GAK inhibition clearly needs to be approached carefully. However, gefitinib is not a selective GAK inhibitor; it primarily targets EGFR and has multiple additional activities. Therefore, its toxicity cannot be directly attributed to GAK alone.

Erlotinib

Erlotinib in particular showed unexpectedly high affinity for GAK (K_d = 3.1 nM) and has been reported to inhibit viral infections, including those caused by DENV and EBOV, in both in vitro and in vivo studies.⁹⁵ A common structural feature of these inhibitors is the 4-anilinoquin(az)oline chemotype, which has been shown to interact efficiently with the ATP-binding site of several kinases

antiviral activity of isothiazolopyridines

In addition, isothiazolo[4,3-b]pyridine-based compounds have emerged as a distinct chemotype with high selectivity and affinity for GAK, showing antiviral activity against DENV,¹⁰¹ HCV,¹⁰² ebola virus,¹⁰³ and CHIKV.¹⁰³

if isothiazolopyridines show antiviral activity, how has there not been a commerical medicine from them?

IC50 should be nM

not from the paper that discovered this compound but form the paper discussing 3,5 substitution instead of 3,6 —> wrongly cited

compound 16 showed antiviral activity against Dengue Fever, Hepatitis C, Ebola Virus Disease, and Chikungunya by inhibiting Cyclin G-associated kinase (GAK), a host protein needed for viral replication. However, it likely did not advance into a medicine because it had d no evidence of progression into clinical development.

Drugdevelopment also requires the optimization of other properties such as:

• cell toxicity and a safe therapeutic window,

• solubility

• selectivity over other kinases

• pharmacokinetics

Synthesis of 3,6-substitued isothiazolo[4,3-b]pyridines

3-nitro-5-bromopyridin2-carbonitirle’s nitro group is

• reduced to the amine,(first protonation and thenFe donates electrons forming radicals and then charges, it loses water twice to yield to amine)

• followed by coversion of the CN to a thioamide using P4S10.

• Then oxidative ringclosure is induced by H2O2 (although I couldn’t find the exact mechanism, it likely involves a sulfenyl type intermediate R-S-... like sulfenic acid: R-S-OH).

Then either the amine is kept as a side chain, and a 6-side chain is introduced via suzuki

or Sandmeyer reaction converts the amine into a bromine. The bromine is substituted with NaOMe or N-nucleophiles, followed by suzuki coupling under two conditions (likely depending on the solubility, stability or reactivity of the different arylboronic acids.

Why not first suzuki an then sandmeyer followed by substitution

• the amino group could interfere with the suzuki coupling

• more importantly the dibromo compounds is a better common intermediate

  • if they did suzuki first, they would need to perform and opitmize a sandmeyer reaction separately for every arylated analogue.

• otherwhise the 2 amino analogue is the common intermediate which still needs 3 steps, while otherwhise the dibromo analogue is the common intermediate which only needs 2 more steps

isothiazole interactions in pocket

Nitrogen

• H-bond with NH of Cys126 bakcbone

• weaker H-bond with SH of Cys126 side chain

sulfur

• weaker H-bond with OH of gatekeeper Thr123

• chalcogen bond with bacbone carbonyl of Glu124

  • glutamic acid

Chalcogen bond

A chalcogen bond is a non-covalent interaction involving a chalcogen atom, usually sulfur, selenium, or tellurium.

Although these atoms often look electron-rich, they can have a small electron-poor region along the extension of a covalent bond. This region is called a σ-hole. An electron-rich atom, such as oxygen, nitrogen, sulfur, or a halide, can interact with this σ-hole.

So in simple terms:

A chalcogen bond is an attractive interaction between an electron-poor region on a sulfur/selenium/tellurium atom and an electron-rich atom nearby.

For example, in a molecule containing a C–S bond, the sulfur can interact with a nearby electron donor such as a carbonyl oxygen or nitrogen lone pair:

C–S···O
C–S···N

Important features:

• It is directional, often aligned with the extension of the C–S bond.

• It is similar in concept to a halogen bond.

synthesis of 5-substitued derivatives

cis should have been more specific because two enantiomers that are cis, it should have beent he one with boht methyls pointing up

Synthesis starts from 3-nitro-6chloropridin-2-carbonitirle, now 6 substituent is introduced first via suzuki coupling, then the same steps of nitro reduction, nitrile to thioamide conversion, and oxidative ringclosure using H2O2 were applied yielding 5-aryl-3-amino isothiazolo[4,3-b]pyridines.

The NH2 can the either be converted to bromide via sandmeyer reaction followed by suzuki, this gives the cis-2,6-dimethylmorpholine substituent.

Or it can be converted directly to the unsubstituted morpholine substituent using 2-bromoethylether, avoiding difficulties of the sandmeyer reaction.

problems:

For 5-phenyl moieties with one or two é donating substituents had low yields for the sandmeyer reaction, due to multiple brominatiens ath different positions of the 5-phenyl ring. to minimize these reactions milder reactions tBuONO, CuBr2 and dry acetonitrile) were used.

to circomvent the sandmeyr reaction completely a 3-N-morpholinyl moiety was directly synthesized from the corresponding 3-amino congeners. Reaction of compounds 12f, 12 g and 12i with 2-bromoethyl ether and potassium carbonate as a base generated the desired compounds 15f, 15 g and 15i in decent yields (71–77%).

Why first suzuki?

They actually first tried to make a common 3-bromo-5-chloro isothiazolopyridine intermediate, which would allow late stage diversification. But during nitro reduction the 6-chloro-2-cyano-3-nitropyridine, the cyano group was hydrolysed to the picolinic acid. as this nitrile is needed to form the thioamide, this route field;

therefore the aryl group at position 5 was introduced first via suzuki, which is less electron-withdrawing than the chlorine, reducess the acivation of the nitrile towards hydrolysis. through this approach the nitirle did survive the nitro reduction step.

sandmeyer reaction

1. formation of nitrous acid

2. this loses water and forms the nitrosonium ion (NO⁺)

3. diazotization of the aromatic amine

4. after several proton transfers and the loss of water forms a diazonium salt

5. SET by CuBr leads to N2 extrusion leaving an aryl radical to which one of the coordinated Br atoms can couple

why not focus on 4-anilinoquinazoline

optimisation began with modification of the aniline substituents. similar modifcations on the ring led to simlar trends in potency. however but showed a tenfold decrease in GAK potency and also reduced selectivity leading the authors to focus on the quinolines.

Both scaffolds share a common binding mode, in which the N₁ of the ligand forms a hydrogen bond with the third residue from the gatekeeper in the hinge region, Cys126. However, the quinoline is more potent because  the C3 hydrogen restricts the aniline torsion and pre-organises the molecule for GAK binding ( 60° torsinon angle between aniline and quinoline) , whereas the quinazoline remains nearly planar.¹⁰⁹

High affinity of aniline

Molecular modelling suggests that the favourable activity of the specific methoxy patterns arises from displacement of a high-energy water molecule in the GAK active site (Figure 13B).

A high-energy water molecule is a water molecule that is trapped in an unfavourable environment. For example, it may be:

• poorly hydrogen-bonded,

• located in a hydrophobic pocket,

• restricted in its movement,

• unable to form its normal water–water hydrogen-bond network.

When a ligand binds and displaces this high-energy water, the system becomes more favourable because that water molecule is released back into bulk solvent, where it can move freely and form better hydrogen bonds. This gives an energetic benefit and can increase binding affinity.

application of anilinoquinolines

With this highly selective inhibitor in hand, the role of GAK kinase activity in cells could be investigated, using prostate cancer as an illustrative example. Previous studies linked GAK to prostate cancer through knockdown of the entire protein, thereby eliminating all of its functions.¹¹¹ In contrast, selective inhibition of the GAK kinase domain alone was sufficient to suppress prostate cancer cell growth. This demonstrates that the catalytic activity of GAK is required for prostate cancer cell proliferation and confirms the inhibitor’s on-target activity.¹¹⁰

Lipinski’s rule of five

focuses on Mw, lipophilicity, H-bonds to predict affinity which do not account for three-dimensionality, so medicinal chemistry has favored relatively planar, aromatic molecules

Lovering’s discovery

Escape from flatland, much like natural products (rich source of drugs) have higher levels of saturation and molecular complexity—> increase saturation = increase likelihood of succes

quantification of lovering

fraction of sp3 hybridised carbon atoms (Fsp3) and amount of chiral centers as descriptors for three dimensionality and complexity.

these increase along the drug development timeline

explanation: greater 3D character leads to:

• may better complement binding site

• lower promiscuity (interacting with multiple targets)

• improved aq solubility

• lower Tm

• enhance oral absorption

example scaffold hopping

replaced benzothiazole oiety with a thiazolopiperidine increasing Fsp3, and yielding one of the most selective and potent Pi3K inhibiotrs

The thiazolopiperidine inhibitors themselves do not appear to have become an approved drug. They were part of the development of selective PI3Kγ inhibitor chemistry. A related selective PI3Kγ inhibitor, eganelisib/IPI-549, did progress into clinical trials for cancer immunotherapy, but it remains investigational rather than an approved medicine.

what is the Fsp3 of our structures

between 0.44 and 0.50 for our compounds submitted.

our average is about 0.46, which is very close to the often cited average for approved drugs, around 0.47, and above the commonly mentioned “drug-like” threshold of Fsp³ ≥ 0.42.

A suitable value is considered to be Fsp3 0.42, and 84% of marketed drugs meet this criterion

indeed though it is mostly due to the aliphatic substituent, BUT if you are asked you can always tell that even though tetralons might have only 2 additional sp3 carbons that are still restrained by the overall fusion, the whole planarity of the molecule is gone and it is more flexible and more likely to accommodate the geometry of the bindinng pocket. Plus indeed we have chosen for aliphatic sp3 rich substituents to engage that hydrophobic pocket of kinase and have higher Fsp3

what does this image mean?

This figure shows a PMI analysis(principle moments of intertia), which is a way to describe the overall 3D shape of drug molecules. The x-axis is the 3D score, calculated from the principal moments of inertia, and the y-axis is the molecular weight. A low 3D score, close to 1, means the molecule is more linear or planar, while a score closer to 2 means the molecule is more spherical or three-dimensional.

What is important here is that most DrugBank molecules fall in region I, with a 3D score between 1.0 and 1.2. In this dataset, 79.7% of the molecules are in this region, while only a very small fraction reach the more 3D regions IV and V. This means that many approved or experimental drugs are still relatively flat or elongated rather than highly three-dimensional.

For my compounds, I used Fsp³ as a simpler descriptor of saturation and potential 3D character. My Fsp³ values, between 0.44 and 0.50, suggest a reasonable degree of sp³ character. However, Fsp³ and PMI are not exactly the same: Fsp³ tells how many carbons are sp³-hybridised, while PMI/3D score describes the actual molecular shape in 3D space. So this figure mainly supports the general idea that increasing molecular three-dimensionality is relevant in drug design, but to place my compounds directly in this plot, I would need to calculate their PMI/3D scores.

what is clogP, and why is it beneficial for 35 to have alower value?

cLogP means how a compound distributes between a lipophilic phase and an aqueous phase, usually expresed as the partitionaing between octanol and water.

a lower value means the compound is less lipophilic and usally more water-compatible

in this context the authors described as a benefit, since the drugs had better drug-like properties like aqeous solubility.

However, you should be careful: lower is not always better. If cLogP becomes too low, the compound may have reduced membrane permeability or weaker interactions with a hydrophobic binding pocket.

commercial drugs with isothiazole motifs

• denotivir, (herpes)

• ziprasidone ( schizophrenia and bipolar disorder)

also applications in agriculture, ligands, catalysis, pesticide development

how are c and d-fused analogues prepared

The [d]-fused analogues are usually prepared by oxidative cyclisation of appropriately functionalised aryl precursors. The [c]-fused analogues are more challenging and much less reported, with only a few examples in the literature. That is why developing access to [c]-fused isothiazoles is synthetically interesting.

c-fused example, see isothiazolo[4,3-b]pyridine examples

mechanisms

Takeshima:

Takehsima: Cyclopentanone first forms an enamine with ammonia. This enamine reacts with carbon disulfide to give an aminodithiocarboxylate intermediate. Elemental sulfur then promotes oxidative intramolecular N–S bond formation, closing the isothiazole ring. Finally, under the basic ammoniacal conditions, the thione is present as the ammonium thiolate salt.

mechanism Dieter and chang

First, compound 38 reacts with hydroxylamine. The nitrogen of NH2OHNH_2OHNH2​OH attacks the ketone carbonyl, giving a carbinolamine intermediate. After proton transfers and loss of water, the oxime 39 is formed. This is the normal condensation of a ketone with hydroxylamine.

The second step is the key ring-forming step. In the presence of SOCl₂ and pyridine, the oxime OH is activated. The oxime oxygen attacks SOCl₂, forming an O-sulfinyl chloride / chlorosulfite-type intermediate. This converts the oxime hydroxyl group into a much better leaving group. Pyridine traps the HCl that is formed.

Then, one of the neighbouring methylthio groups attacks intramolecularly at the activated oxime nitrogen, forming a new N–S bond. This closes the five-membered isothiazole ring. Since that sulfur initially bears a methyl group, the cyclised intermediate is a sulfonium species. Loss of the methyl group, for example as MeCl, restores a neutral sulfur atom and gives the aromatic fused isothiazole 40. The second methylthio group remains as the substituent on the isothiazole ring.

shibashaki

1. Allyl cyanide acts as the pronucleophile. The base deprotonates the carbon next to the nitrile group, because this proton is relatively acidic due to stabilization by the nitrile. The carbanion is captured by the Cu catalyst.

2. The Cu(I) catalyst, with a chiral ligand, coordinates to the soft sulfur atom of the thioamide. This activates the conjugated thioamide system toward Michael-type attack and places it in a chiral environment.

3. The deprotonated allyl cyanide then adds via the allyl group to the β-position of the α,β-unsaturated thioamide. Because the electrophile is bound in the chiral Cu complex, one face is favoured, giving the product with high enantioselectivity.

4. Copper then helps the cyclisation by coordinating to the thioamide sulfur and also activating the nitrile. This makes the nitrile more electrophilic and brings the reacting groups into the correct orientation.

5. next the postiion alpha to the thioamide is deprotonated which attacks on the nitrile

6. the cu complexes between the imine and sulfur, this is oxidized under influence of air cu(II) and the thione isomerizes to the thioenol

7. the Cu complex can then undergo reductive elimination to form the isothiazole

singh

The first step involves the selective nucleophilic attack of ammonia (generated in situ from NH₄OAc) at the carbonyl carbon of 1 to give imine intermediate A, which undergoes intramolecular nucleophilic attack of sulfur on the imine nitrogen, forming the S–N bond to generate cyclic intermediate B. Subsequent aerial oxidation of intermediate B gives the desired isothiazole 2.

postfunctionalization singh

Due to the immense importance of alkylsulfonyl derivatives of heterocycles in the medicinal field, (17) we thought to functionalize the 5-thioalkyl group to alkylsulfonyl derivatives. The alkylsulfonyl compounds can improve pharmacological properties compared to their nonsulfonyl analogues. Subsequently, we treated compounds 2d and 2p separately with 3 equiv of m-CPBA in CH₂Cl₂ at room temperature. The reaction proceeded well, affording the corresponding sulfonyl derivatives 3a and 3b in good yields (

In contrast, post-functionalisation strategies, where the core scaffold is modified after its formation, are highly advantageous in medicinal chemistry. They enable late-stage diversification from a common intermediate, facilitating the rapid generation of analogues and more efficient SAR studies.¹⁴³

xiao and deng

Initially, nucleophilic attack of enaminoesters 2 with S8 generates polysulfide A and protonated B. At the same time, 2-methylquinoline (1a) could generate radical intermediate C in the presence of elemental sulfur. Intermediate C could couple with A to afford intermediate D via single-electron transfer with elemental sulfur. Then enamine isomerization of intermediate D produces intermediate E, which undergoes intramolecular nucleophilic addition to give intermediate F. Finally, F was oxidized to product 3.

The key coupling step then occurs between radical C and intermediate A. The quinolylmethyl radical adds to the electron-poor double bond of A, forming a new C–C bond. This radical addition gives a new carbon-centred radical intermediate. Elemental sulfur can then accept one electron from this radical intermediate in a single-electron transfer step. In other words, sulfur acts as a mild oxidant:

organic radical intermediate+S8→oxidised organic intermediate+S8∙−/Sx2−\text{organic radical intermediate} + S_8 \rightarrow \text{oxidised organic intermediate} + S_8^{\bullet -}/S_x^{2-}organic radical intermediate+S8​→oxidised organic intermediate+S8∙−​/Sx2−​

After this SET oxidation, loss of a proton gives intermediate D.

Thummel

This is an interesting reaction because you would expect ring fusion at the 2 and 3 postition and one at the 4 and 5 position (metafused) but instead you get fusion at the 2 and 3 position and at the 5 and 6 position (para fused), this indicates that the amine the beta amino alfa beta unsat ketone is not the nitrogen source of the pyridine

This was explained by the fact that under these conditions, the enamine of the enaminone can equilibrate to the imine, than become hydrolysed expelling ammonio forming an enol which tautomerizes to an aldehyde, this aldehyde can be attacked by the enamine formed of the reaction of the other ketone with NH3, which cyclises to the product

Not a Friedlander condensation:

Ikeno

Bohlmann–Rahtz reaction

when the reaction iscarried out using cyclic ketones as substrates, kinetic controlis expected. After the Michael reaction between the enaminederived from cyclic ketones and propargyl ketones, the resultingdouble bonds adopt the Z-form, which is favorable for cycliza-tion (intermediate D). In particular, the double bond on thecyclohexane ring, formed by nucleophilic attack from the highlyreactive cyclic enamine, can only adopt a Z-form. Therefore,the Bohlmann–Rahtz reaction with cyclic ketones is expected toproceed rapidly at room temperature, at higher temperatures th E isomer would form

through allene intermediate

gagan

that aniline derivatives react readily with β-chlorovinyl aldehydes 56 forming imino-enamine salts 57, which cyclise to quinolines fused to a saturated ring 58 when heated in acetic acid (Scheme 6).¹⁵⁵

goswami

, it was assumed that the reaction proceeds via enamine, which undergoes addition−elimination followed by cyclization and condensation with β-bromo-α,β-unsaturated aldehyde to form the pyridine products (Scheme 1, path B).

However, the expected pyridine product was not observed in the crystal structure. On the basis of the crystal structure, a plausible reaction mechanism is proposed in Scheme 1. The in situ generated “ammonia” from ammonium acetate reacts with β-bromo-α,β-unsaturated aldehyde 1 to form aminoaldehyde I, which reacts with 1,3-diketone to form intermediate II. Enamine intermediate II then undergoes cyclization to form the pyridine products.

= Friedlander type condensation

Hantzsch pyrdine synthesis

• 1 equivalent of an aldehyde (e.g., formaldehyde or benzaldehyde)

• 2 equivalents of a \(\beta \)-keto ester (e.g., ethyl acetoacetate)

• 1 equivalent of a nitrogen donor (typically ammonia or ammonium acetate) [1, 2]

The Result: The reaction natively produces a 1,4-dihydropyridine (1,4-DHP), often called a "Hantzsch ester". In a second step, this intermediate can be oxidized (dehydrogenated) to yield the corresponding pyridine. [1, 2]

transition metal catalysed for fused pyridines

Ni or Pd catalysis of alkynes with alkyne nitriles

optimized Goswami reacton

difficult mechanism but reacts through a xanthene dione intermediate by reaction with a second diketone which allows eco friendly conditions of ammonium acetate in ethanol water solvent

b-chloro-a,b-unsaturated aldehydes, 1,3-diketones, and ammonium acetate in ethanol : water (1 : 1) solvent under eco-friendly conditions.A plausible mechanistic pathway was proposed to explain the base-catalyzed formation of dihydro-6H-quinoline-5-ones from b-chloro-a,b-unsaturated aldehydes (Fig. 3). The b-chloroa,b-unsaturated aldehyde initially went through a Knoevenagel condensation with the 1,3-diketone in the presence of a nonnucleophilic base. Subsequently the presence of a vinylic chlorine atom facilitated the incorporation of the second molecule of 1,3- diketone to produce the xanthene-dione intermediate (8). The in situ product 8 immediately reacted with ammonia, generated from ammonium acetate under the reaction conditions, to form the b-enaminone through ring opening. This b-enaminone ultimately formed the dihydro-6H-quinoline-5-one through internal rearrangement involving N–C bond formation and C–C bond cleavage losing one molecule of the 1,3-diketon

why p4S10Py4

combination of P4S10 1 and pyridine, as a successful reagent for thionation, but lawesson’s reagent more commonly used

• it has a higher thermostability than LR (LR decomposes above 110 °C)

• no chromatographic purification

• alows use of ‘neutral solvents’ like ACN

• he salt 11a is readily soluble in water, easily removed during workup

• easier to prepare

• odorless (when sufficiently pure)

• pure thionated products (less side reactions)

disadvantages LR

• Additional drawbacks with the reagent 2a are its low solubility, the coformation of foul-smelling side products that are difficult to separate from the desired molecules28-30 (column chromatography is often required), and that HMPA (a solvent that is prohibited in Sweden and many other countries) quite often has to be used.

Why does it react fasterthan P2S5

• P4S10Py4 is more soluble in contrast to p2S5 which is poorly soluble

• thermostability

• contains negative charge compared to P4S10

mechanism thionation

It is at present not clear if the reagent 3 thionates as such or via dissociation to species such as 15 or 16. Formation of 16 has previously been formulated as part of the dissociation of P4S10. 6