Org Prep Daily

April 6, 2019

Sodium Tetrachloro Bis-(1H-Indazole)-Ruthenate(III) dihydrate

Filed under: procedures — milkshake @ 9:30 am



Sodium sulfate 213.0 g (anhydrous, 3.0 mol of Na) was gradually added into a stirred slurry of Al2(SO4)3 .18H2O 1000g (3.0 mol of Al) in D.I. water 2L and the mixture was stirred to complete dissolution (about 30 min). The total volume was adjusted by addition of D.I. water to 2.7L and the solution was filtered through a fine porosity filter. The obtained 1.1M solution of NaAl(SO4)2 was combined with 226.9g of the Cs salt (350 mmol) in a large 4L beaker. Solid CsCl 6g was added to the stirred mixture, to seed the formation of cesium alum CsAl(SO4)2.12H2O crystals. The mixture was stirred in open Erlenmeyer flask at ambient temperature for 30 hours. During this time, the red-brown slurry of the cesium salt turned into coffee-brown black slurry of the sodium salt intermixed with fine white salt-like crystals of cesium alum. The solids were collected by filtration, rinsed thoroughly with saturated (=1.5M) aqueous sodium sulfate solution (in three portions 3 x 0.5L until the filtrates were colorless) and the obtained filter cake wet with sodium sulfate solution was dried by suction, and then in vacuo for 1 day, until completely dry free-flowing material was obtained. The solids were transferred into a 2L wide-mouth Erlenmeyer flask, 700mL of acetonitrile was added and the mixture was stirred mechanically for 15 min [Note 1]. The resulting deep orange slurry was filtered on a medium porosity Buchner funnel, the cake of insoluble sulfate salts was rinsed with additional acetonitrile (3x100mL until colorless) and was discarded. The combined orange filtrates in a 5L round flask were diluted with MTBE 4L (added in four 1L portions with gentle stirring), the flask was then set aside for 30 min to complete the precipitation. The precipitated crude sodium salt was collected by filtration, rinsed thoroughlt with MTBE (2×0.5L) and dried by suction and then in vacuo.

The crude product, 190.4g of a fluffy brown solid (retaining solvent residues and Cs, about 2500-4000 ppm Cs) was transferred into a dry 10L round flask. 191 g of powdered activated molecular sieves 4A [Aldrich 688363, sodium aluminosilicate, SYLOSIV A4 manufactured by Grace Davidson] was added to the flask followed by methyl ethyl ketone 4.2L. With stirring on high speed (800rpm), methanol 600mL was gradually added into the slurry over a 5 min period. The stirring was continued for 30 min, at this time nearly all dark brown lumps of the material dissolved. The resulting orange slurry was filtered through a fine porosity 3L large Buchner funnel [Note 1]. The spent molecular sieves were thorougly rinsed with additional MEK (2x200mL) and discarded. The combined filtrates were precipitated by a gradual addition of MTBE 10L with mechanical sitrring. After complete MTBE addition, the stirring was turned off and the material was allowed to precipitate for additional 30min. The solids were collected by filtration and rinsed thoroughly with MTBE (2×0.5L) and dried by suction. The purified product 184g, containing solvate-bound MTBE, was combined with 3.3L of wet MTBE (prepared by shaking 4L of MTBE with water 50mL in a closed flask, for 30min, and decanting water-saturated MTBE from the water droplets).  The mixture was stirred in open 5L wide mouth Erlenmenyer for 40 min. The brown solids were collected by filtration, rinsed with wet MTBE, dried by suction [Note 2] and then then in vacuo overnight (15h). The yield was 176.1g of a heavy dark brown granular solid (93.5% of theory), with HPLC purity 98.7% and a matching elemental analysis. The Cs content was below 100 ppm. There was no MTBE residual odor.

Note 1: The solvolysis with acetonitrile and also with methanol leads to a formation of detectable decomposition products on the timescale of hours. The filtration and precipitation needs to be performed immediately as the material is rather unstable in solution.

Note 2: The product has a tendency to retain organic solvent residues. A re-slurry with wet MTBE transforms the solvates into a more stable dihydrate. There is a second, metastable orange-red dihydrate polymorph that forms temporarily from MTBE-solvated product upon exposure to moist air. A sudden appearance of orange-red particles within the brown-black filter cake of the product during the final filtration is an indication that the material was not fully hydrated and retains still some MTBE solvate. The problem is fixed by repeating the re-slurry in wet MTBE.


April 5, 2019

Cesium Tetrachloro bis-(1H-indazole)-Ruthenate(III)

Filed under: procedures — milkshake @ 3:26 am



RuCl3.xH2O 100.0g (x~3, 382mmol) was combined with conc. HCl 0.6L and non-denatured ethanol 0.6 L. The mixture was stirred and distilled under air at normal pressure until the total volume of the mixture was below 400 mL. The distillates were discarded. The obtained dark brown solution remaining in the distillation flask was cooled, filtered through a medium porosity glass Buchner funnel and the filtrates were adjusted with conc. HCl to total volume about 0.5L.

In the meantime, indazole 300g (2.54 mol; 6.64eq.) was dissolved in a mixture of water 800 mL and conc. HCl 4.0 L (with 20 min stirring), the solution was filtered through a medium porosity glass Buchner funnel. This indazole solution was charged into a 15L glass-and-teflon jacketed reactor equipped with an efficient paddle-shaped stirrer, 0.5L addition funnel, a thermoprobe and air-cooled reflux condenser topped with a gas outlet tube for HCl gas release. An additional volume of conc. HCl 4.0L was then charged to the  reactor, the circulator-heating was set to 90C. The temperature in the reactor was let to stabilize for at least 30 min and then carefully maintained at 90 C throughout. The solution of ruthenium trichloride in HCl was added dropwise, at about 250rpm stirring, over a period of 5 hours, using an addition funnel with a stem extended with a piece of polyethylene tubing (to limit splashing). The addition funnel was washed down with a small volume of HCl (2x50mL). The obtained brownish slurry was then stirred at 90C for additional 10 hours. The reaction mixture was cooled down to 25C, with stirring, the slurry of the precipitated product was drained from the reactor through bottom valve into a 15L polyethylene bucket. The solids were collected by filtration on a large (3L) medium porosity glass Buchner funnel, the reactor was washed down with 2M aqueous HCl and the washings were added to the Buchner funnel. The product was rinsed with additional 2M HCl, about 2L, and partially dried by suction overnight. This provided a wet cake (moist with the residual 2M HCl) of the indazolium salt, 598g,  as a brown sticky solid. [Note 1]

The moist indazolium salt was transferred into a 10L wide-mouth flask equipped with an efficient mechanical stirrer with a teflon paddle. CsCl 180g (1.07mol, 2.8 eq., powdered briefly with a spatula to break any lumps) was added to the flask, followed by methyl ethyl ketone 2.0L and non-denatured ethanol (99%) 1.8L The mixture was stirred at 200 rpm for 5 min, the stirring was then turned to high speed and continued for 2 hours at 700 rpm at ambient temperature (22 C). The resulting bright orange slurry was collected by filtration (3 L medium porosity Buchner funnel), the solids were rinsed thoroughly with 99% non-denatured ethanol and partially dried by suction, for about 1 hour. The obtained bright orange cesium salt in the form of MEK-solvate intermixed with residual CsCl was transferred into a large 4 L beaker. 1 L of a 2:1 (v/v) ethanol-water mixture was added and the slurry was stirred in open beaker for 15 min at about 350 rpm. During this time the bright orange color of the MEK-solvated cesium salt slurry faded into cinnamon red-brown color of the hydrate. The solids were collected by filtration (using the same Buchner funnel),  washed thoroughly with 99% ethanol, about 1L. The material was dried by suction overnight  (14 h). The yield was 226.9 g (91.5% theory) of a red-brown heavy solid. The product is approximately monohydrate (it forms initially as dihydrate but loses a part of the solvated water upon drying). The material is bench stable.

HPLC purity 98.5-99% by HPLC (SB Zorbax C18, 3 micron, 4.6x150mm,  a 12 min 10% to 90% linear gradient of MeCN(+0.1%TFA) in water(+0.1%TFA) at 1.0mL/min), the product composition was confirmed by elemental analysis and X-ray crystallography

Note 1: The indazolium salt is a potent contact irritant. Indazole and ruthenium trichloride are caustic to skin. HCl is very corrosive. A full face shield and a protective apron are recommended when loading the reactor with large volumes of conc. HCl. The reactor needs to be completely disassembled and cleaned after the preparation, to prevent damage to seals and metal parts, and decontaminated from Ru residues (rinse with acetone, followed by methanol with added conc. ammonia, about 20:1 by volume, followed by water and acetone rinse)

Note 2: Since Ru(III) salts are paramagnetic, NMR is not helpful for purity determination. HPLC is useful but the resolution of the impurities is very specific to the particular type of reverse-phase HPLC column (SB-C18 Zorbax 3 micron). It is best to use single injections and prepare the individual HPLC samples just before the analysis because the material gradually decomposes in solution.


March 31, 2019

Pearlman’s catalyst

Filed under: procedures — milkshake @ 9:00 pm

TL 1967(17), 1663-4

Pd hydroxide on charcoal, contains 20% Pd by weight

PdCl2 2.0g (11.3 mmol) and activated carbon 4.8g (HCl-washed grade) was combined with DI water 40mL in a 250mL round flask with a large egg-shaped stirbar. The slurry was stirred on a 80C oil bath under air-cooled condenser for 20 minutes. A solution of LiOH.H2O 1.0g in DI water 8 mL was then added in one portion with vigorous stirring, the heating was turned off and the mixture was stirred overnight (16 hours). The solids were collected by filtration, rinsed with DI water, then with a solution of acetic acid 0.2mL in water 40mL and then with DI water again. The filter cake was compressed with a spatula, the product was dried by suction and then on highvac overnight.

Y=5.74g of a black powder

December 23, 2018


Filed under: procedures — milkshake @ 8:31 am



Inorganic Syntheses Vol 35, p. 148

4.20g of RuCl3.xH2O (x~3) and 99% EtOH 100mL (non-denatured) was refluxed gently on a 95C oil bath under air-cooled condenser opened to air, for 3 hours. The obtained dark greenish solution was filtered through a medium porosity fritted funnel, to remove some insoluble particles, the filtrates were evaporated on rotovap in a 300mL flask. The dark honey-like residue was combined with DMSO 16mL. The mixture was stirred on a 150C oil bath under air-cooled condenser opened to air, for 2 hours. During this time the reaction mixture color changed to orange-red and then yellow precipitate formed. After 2 hours, the reaction mixture was allowed to cool to room temperature, the obtained slurry was gradually diluted with acetone 120mL and the mixture was allowed to crystallize overnight (14 hours). The precipitated yellow heavy crystalline solid was collected by filtration, rinsed thoroughly with acetone, dried by suction and then on highvac.

Y=6.58g (85% th) of a yellow crystalline solid.

1H(D2O, 400 MHz, a 10 minute-old sample): 3.46(s,6H), 3.44(s, 6H), 3.35(s, 6H), 2.69(s, 6H)

Note 1: This preparation works best when performed under air. Common Alihn reflux condenser unconnected to water source was used for the purpose.

Note 2: The oxygen-bound axial DMSO ligand solvolyzes readily, the D2O-NMR spectra  actually belongs to the aqua complex and free 1 equiv of DMSO formed in situ. The NMR sample in D2O is not stable, some new peaks start appearing after 20 minutes.



June 18, 2018

Season 2 – Breaking Bad In South Florida (1)

Filed under: Uncategorized — milkshake @ 12:40 am

This is a work of fiction. Names, characters, businesses, places, events and incidents are either the products of the author’s imagination or used in a fictitious manner. If you find any resemblance to actual persons, living or dead, or actual events, deadly or lively, or actual molecules, carbons or heteroatoms, it is purely coincidental.


Part 1

Five years in, the CEO of Timbermill* Pharmaceuticals, Inc. has run his company into a dead end. Slender but imposing with a mischievous smile, balding, polite and blindingly smart, he founded his little biotech on the pledge of quality, speed and capital-efficient development process. The year was now 2012 and not a single promise he made to the investors came through. The cash was running low, the latest news from the clinical studies were spelling doom and the reports from the CROs were downright disturbing. The original idea behind Timbermill Pharma was to create a virtual biotech company on the East Coast with a team of experts in cancer drug development and commercialization. It was supposed to work by in-licensing drug candidates from academia, setting up collaborations with academic groups to generate biology publications, and by doing the preclinical development at CROs – no labs in house so as to keep the expenses at minimum – the purpose was to get the compounds as quickly as possible into relatively inexpensive small open-label Phase 1 dose-escalation studies. (Cancer trials are ideal because the safety bar is so low and their dosing regimen is typically very short). The positive clinical result would be wrapped into a delicious little parcel and sold = profit!

Except that by the end of 2012 there were no serious takers left for Timbermill and its projects – one by one, they reconsidered after doing their due diligence. Nobody wanted to invest; It was necessary to unload the Timbermill Pharma even on unfavorable terms, with a generous commission. And then, few months later, the impossible actually happened: Our own struggling little company bought them! The acquisition of Timbermill Pharma in 2013 turned out to be one big unmitigated calamity for us; its impact is still felt today (with the same magnitude as the fallout from our CEO cooking Ecstasy on the university campus). As our research director later put it in a moment of weakness: “Only the go-betweens who cashed their finder’s fee benefited from the deal, they took the rest of us for a ride”.

The history of the Timbermill Pharma clinical projects illustrates how virtual biotech companies do a disservice to their research projects, to their shareholders and buyers. If someone tries to sell you a “nimble virtual company composed of seasoned industry veterans” who can do it better, faster and cheaper by contracting stuff out to CROs without performing any research in house, it helps to be extra cautious. Even if the formula could somehow work, there is a moral hazard with this setup. You could buy into a mirage of an impressive drug project where everything later turns out to be sketchy and done on the cheap while the problems are skillfully papered over. This is how the faster-cheaper-CRO works: Doing things for appearances and leaving the intractable problems for the buyer to discover later on.

Timbermill Pharma licensed its two clinical candidates from an academic research group in Austria, located about hundred miles from where I was born. (I also met the CEO of Timbermill about a decade earlier when we were both at Pharmacia-Pfizer; it’s a small world). The university research group in Austria had no real experience with drug development and they produced some irreproducible half-baked procedures in their process patents, to create a semblance of a manufacturing method… The clinical compounds  from Austria were two inorganic coordination complexes, intended for the treatment of cancer. And both were also ugly-looking metal complex small molecules with a murky mechanism of action. These two compounds had low cytotoxicity on their own and were supposed to work somehow as potency-improving and resistance-reversing add-ons to the established chemotherapy drugs, by indirect mechanism that is synergistic with DNA-targeting cytotoxic drugs, etc.  These clinical compounds were nearly impossible to follow in vivo with analytical methods because they were markedly unstable in plasma so there was no PK to speak off; a cherry blossom of a project from the clinical development standpoint. (It is true that cis-platinum and related platinum drugs also have stability issues in solution but they have at least a reasonable PK and a well-defined activated metabolites/degradation products and direct DNA-dependent mechanism of action. None of this was true for the clinical candidates from Austria.)

One compound that Timbermill Pharma put into clinic was a complex of gallium. Unfortunately they could not get a composition of matter patent on it; the compound was known for more than a century. Why they did not bother to perform a simple ligand  study to get few close analog complexes not yet described in the literature? – The compounds that they could then patent for themselves before putting millions into a clinical trial. The normal medicinal chemistry mode of thinking was foreign to the Timbermill expert team and at any rate they did not have the lab nor the chemist in house for the job. Going forward just with the help of CROs, they were not able to solve even the relatively straightforward formulation task of making shelf-stable pills that would release the compound at a predictable rate in the gut before going into the clinic with it. The clinical study was a bust.

The gallium compound did not have much biology research to back it up either: lots of hand-waving about remodeling the Golgi apparatus and endoplasmic reticulum and the synergy with cytotoxic DNA active agents but no real insight. The older known gallium compounds that previously failed in the clinic supposedly acted on transferrin, by gumming it when binding gallium in place of iron – it was not clear how this clinical candidate was any different as it was falling apart in the plasma so rapidly that the parent compound could not be detected even right after injection into the animal. The only advantageous thing that the clinical candidate had was its oral availability: previously gallium was tested in cancer therapy mostly in the form of injection, although another orally available compound was also in the clinic – and failed.

Gallium has cumulative kidney toxicity, if you put a small amount of gallium into bloodstream (for example in radioisotope-based gallium scan diagnostic procedure), the kidney damage risk is negligible. Not so with the repeated treatment or with a massive dose… Obviously, the main goal in this project should have been to demonstrate new and unexpected properties of the therapeutic agent. For example, to show the oral compound does not damage kidneys nearly as much as injectable gallium salt at a comparable dose. Unfortunately, Timbermill Pharma failed to show this and the animal studies provided no real PK insight: all that could be observed in plasma was just inorganic gallium and the free ligand. If anything, the early problems with the unreliable release from the pill in humans demonstrated that gallium oral therapy was more problematic than the old injectable forms of gallium.
It was merciful that my colleagues put this clinical candidate from Timbermill Pharma on the shelf shelf just after few cursory PK and biology experiments: The gallium project soon disappeared from our company website and the management treated it with an embarrassed silence.

But the second project we got from them was a real bummer: Timbermill Pharma put its second clinical candidate through the Phase 1 dose escalation open label study – that is to say, by hiding the GMP manufacturing and formulation problems from the FDA – and we unfortunately took it from there. Our company obtained Orphan Designation for this drug candidate based on their study – by submitting the study data to the FDA. Our research director did it while knowing quite well that the dose-escalation Phase 1 trial was invalidated by the Timbermill misconduct and that it should have been re-done a long time ago.

*A long and unrelentingly depressing tale about a fictional company. So I added at least pictures of kittens.


September 9, 2017

Breaking Bad in South Florida – The Aftermath

Filed under: Uncategorized — milkshake @ 7:15 pm

This is a work of fiction. Names, characters, businesses, places, events and incidents are either the products of the author’s imagination or used in a fictitious manner. If you find any resemblance to actual persons, living or dead, or actual events, deadly or lively, or actual molecules, carbons or heteroatoms, it is purely coincidental.

The Aftermath

(here is Part 1Part 2Part 3Part 4Part 5, Part 6, Part 7, Part 8, Part 9, Part 10)

The company recently raised millions from an investment fund for the clinical trials. They are initiating the phase 1 trial with a polymer-based i.v. formulation of an old chemotherapy drug. It is the same problematic nanoparticle formulation over which my biologist friend was fired.

It has been difficult for me to find a job since I went against the company. The pattern has been the same: a successful phone interview followed (after few weeks) by a brief communication that the position got already filled. The company also sent the campus police after me last October. I was suddenly called by the university police and asked to immediately report to the campus where they served me a trespass notice and a permanent campus ban – supposedly for previous loitering outside the incubator building. (I did walk by the building once, curious if the labs were still in use – they are on the ground floor, with floor-to-ceiling glass wall so one can easily look into them from the distance).

The purpose of this theatrics was to paint me as an unstable disgruntled loner who is about to bomb the campus. I think it was the idea of our research director – and the university obliged: three police cars and four armed overweight campus police officers puffed up making the campus safe for the drug kingpin in their biotech incubator…

At this time my father became critically ill, I had to go to Prague to look after him so I did not have the energy to fight this baloney trespass. By the time I returned to Florida, I pretty much gave up – I was rather depressed and tired of the affair.

It probably looks petty – what helped me to pick up the fight again were the recent company research publications: The company has been publishing our old research results to impress the potential investors and buyers. I saw enough of my work published without my name on it and I was getting resigned about it. In one case I still caught a publication in the galley proofs because the co-authors alerted me and after my protests the research director reluctantly added my name back to the author list.

But then in summer 2017, the company published a summary overview of its proprietary polymer chemistry – showcasing the remarkable ease of manufacture of new version of our elaborate polymers and the simple-yet-effective drug encapsulation with the self-assembled nanoparticles – and my name was left out again. The scheme was my proposal, and about 30% of the man-hours spent on it was also mine. The research director actually asked me to write the official history of the project for the company in 2015 because I am the main inventor on the patent claiming these polymers. But when I contacted the authors on the paper and asked them to submit an addition/correction to put my name back on the author list, they told me to get lost; some of them in a rude way.

Lucky for us, this is an ACS journal – they have the COPE guidelines for cases just like this one: The journal editors already opened an investigation. I can’t predict the outcome but there is an overwhelming evidence that it was my work and I was left out on purpose – I hope this will give me an opportunity to tell my story in C&EN.

Another issue is the clinical candidate currently in the Phase 1. The company was under the obligation to provide all available safety and efficacy animal data in the IND submission to obtain the approval for the Phase 1 clinical trials. I am not sure they submitted everything: The research director despised the animal data that were showing a poor stability of the drug-loaded nanoparticles in mouse blood – it is the research study for which my biologist friend was fired after presenting his results. I believe that the FDA probably found out about the problems with the IND data submission recently, together with the company background story. They never comment on the ongoing investigations so it is hard to confirm; the only people who will know with certainty are employed by the company, or at CDER.

And then there are the shareholders who saw the value of their investment plummet over the years – who got diluted by the acquisition of a virtual biotech company (because of a poorly-done due diligence) and recently they were diluted yet again by the investment fund. If the company is acquired or goes public, the early investors who are getting hurt will probably question why the management involved itself in cooking MDMA and other drugs in 2013-14. The continuing presence of our research director will predictably turn into a magnet for the shareholder lawsuits. When it happens I will be there to help.


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