Org Prep Daily

A friend had an unforgettable experience from hiking in Madagascar: a horror called terrestrial leeches. These slug-like creeps wait patiently in trees, bushes and tall plants. They can feel you coming from some distance and they converge and drop onto you from foliage, then bite through the shirt (pants, socks) and sneak in, migrating over your skin towards the juicier body regions where they attach themselves. This is painless - in fact you are not likely to notice the leeches munching on you under the shirt as the leeches exude a potent anesthetic in their saliva. They also inject a thrombin inhibitor that suppresses the blood clotting in the wound - which unfortunately causes you to bleed profusely (though painlesly) from the bite long after the leech has got her fill + dropped off to the ground, happy and slumberous; (this is a leech version of the Thanksgiving dinner - they can live of one good fill for many months). My friend said he knew nothing about these tree leeches until he returned from the hike and found himself covered with his own blood and with some dozen of leechey hitchhikers, attached to various parts…

The leech thrombin inhibitor hirudin is medicinally useful as an anticoagulant. There has been enormous amount of medicinal chemistry and biology done on the blood coagulation cascade. At the previous company we were working on factor VIIa inhibitors (that were supposed to be safer than the anti-thrombin or anti-Xa agents), for treatment of deep vein thrombosis. The thrombosis project got shelved eventually  - one reason was the difficulty with developing good orally-active compounds - but we made some pretty potent i.v. compounds. I have inadvertently found out how potent these compounds actually were: One night working in the lab, I noticed my jeans felt little heavy and sticky so I looked down and saw that the front of one trouser was completely soaked with blood, from the knee down to the sock and I could not find out where all this blood was oozing from! Eventually I noticed a tiny splinter of glass, barely 2 mm across, embedded in the knee and I realized that few hours before I was purifying some final compounds on prep-HPLC and I dropped an empty test tube (after active fraction) and I kneeled down to the floor in order to pick up the splinters - a tiny piece must have stuck to my knee then.   

Here is an example of the VIIa chemical monstrosity we were making  - a true embodiment of the god-awful drug design. But they sure had some long-lasting potency: 

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In US, the TV is now inundated with ads for Plavix. This inhibitor of platelet- aggregation is slightly more effective than aspirin in preventing heart attack (31% vs 23% risk reduction) and therefore widely prescribed as prophylactic over long periods of time. Plavix has more serious complications than aspirin - it causes prolonged bleeding. That makes the ER doctors quite unhappy as there is no available antidote for reversing the effect (like you get with the coumarine-based anticoagulants) and every mid-age weekend biker now seems to be on the drug whether they need it or not. With intracranial bleeding from head trauma, Plavix can provide a heroic and futile excersize for the entire surgery team.

Thanks to Abel Pharmboy for pointing out the story.

 credit:Zooillogix

There is an interview-exam that one takes when applying for US citizenship and one of the questions that came up was to name the benefits of becoming a US citizen. (One can vote, work for government, etc). The most obvious answer to this question was: “As a US citizen I cannot be harassed or deported for some arbitrary reason by government officials and I won’t have to deal with your bloody retarded immigration administration ever again.”  I kept that extended answer to myself.

Even as the citizenship application process was an unpleasant experience and took almost a year to complete (the immigration officials ”could not locate” my file - but only for 3 months), getting my green card ten years ago was far more nerve-wrecking business. I had a legal help since my employer sponsored me but I felt like naked and pushing through a scrub whenever I had to see the immigration officials: It was clear from their demeanor that I had no legal rights to get my visa/residency approved, that I could be removed from the country should they decide to do so - with or without a reason - and there are always some government regulations to provide such a reason in ways that even Franz Kafka could not have imagined.  I figured out that the actual role of the immigration officials is to keep out as many applicants as possible, and that it does not trouble them if few applicants are left hanging in limbo or pushed into the “out” group as a matter of a routine error of the immigration administration…

Getting up before the sunrise to stand in a line in front of the imigration office at 5:30 am (they opened at 9 am) to make it to the window already at 10:30am and turn the paperwork in - and then being told (incorrectly) that I needed to come back again with additional forms and documents - was just the initial experience. Being called for a hearing that was  canceled on the day of the appointment was another good one (my wife was doing internship in New York and she had to fly to this appointment to Arizona - twice because the nice people in Tucson office found it more convenient to re-schedule our hearing without sharing the news). Having friends who were removed from US did not attenuate my immigration-related paranoia either. (Husband and wife, both physicians, with 3 kids. He was a tenure-track assistant prof at university, she was a staff physician at a hospital. Their employers were sponsoring their H visa but the necessary J-to-H-visa waiver was denied by the immigration). Then another friend lost her job at Reuters in Boston and was doing an unpaid intern for 5 months because she had to re-new her J-visa-related work permit at the end of her first year in US. (But the immigration office did not care to process her renewal papers - which she turned in 3 months in advance). Also, practically every immigration official that I met came across as surly and tense, underpaid and overworked, and behaving like a postal worker about to turn serial.

My theory is that the original immigration rules were based perhaps on some common-sense reasoning but over the years the system got subverted by the changing agendas and got loaded with generous heaps bureacracy. It is now chaotic, inefficient and extremely slow and probably there is some political calculation in keeping it so. It works now like an obstacle course: the luckiest, the most persistant and informed applicants make it trough but others will not - and any idiotic reason is good for the purpose of keeping some immigrants out.

If you are US-born and if you had no personal experience with US immigration officials and if you think that I am exaggerating, please consider the experience that some people get at a DMV office or a courthouse. They could write about it in newspaper and complain to their senator,  go to another DMV office, and the worst misfortune that could befall them is a licence driving exam delayed for weeks. The US immigration is rarely held responsible for its mistakes, it gets away with screwing up lives, ruining careers and splitting families. It is certainly the most awful institution that I got to know since the end of communism.

I just learned that I have been approved and will have the oath-taking ceremony this Wednesday. It has been a huge relief for all the described reasons and also for being able to travel outside the US again.  And now that I won’t be deported I think I may try out some herbal remedies to celebrate.

Large pharmaceutical companies have poor research productivity record. In fact, they are hugely wasteful with the money they put into their research: One would think that the expertise and productivity improve with scale – yet it seems the corporate baloney, red tape and political infighting grow with size too. Some of the absurdities prevalent in the big  pharma companies today are possible only because these companies were able to afford them as they were living on profits of their past success.

The number of positive things that the management can do to improve the chances of a drug discovery project success is limited but there are limitless ways to ruin it.  It is hard to calculate what a company could have discovered - and didn’t because of some decisions going back ten years. How does one track the responsibility of managers who demoralized their best research staff and made alibistic decisions, did not provide the needed resources but by making unrealistic promises set others for failure? Bad pharma managers can keep awarding themselves massive bonuses for their “contributions” over many years  without ever having to verify that their management methods and ideas actually worked.  

Then there are unrealistic expectations of the stock-holders. They can bring in a vastly overpaid CEO star with a mandate to turn the company around. There is nothing that the new management can do quickly about the failing research - and even if they could somehow turn the research around  instantly there would be no apparent results for the next 3-5 years. So the new management will implement all the short-term fixes meanwhile, such as marketing the maximum out of their current drugs, licensing in few drug candidates from other companies or buying up a smaller company that has a promising drug on the market plus strong drug candidates in development. Firing the research staff usually follows, from the acquired company (the political consideration makes closing their own Central Research difficult - even if their own research produced nothing in a decade). When the management is paid in stock options, there is the incentive to raise the share price quickly- even at a huge long-term expense.

The new management is as likely as the old one to make cheery projections, promising the investors all the things that they want to hear. (“We will save two billions a year on post-merger synergies”). At the same time a new, formalized structure with teams, steering committees and frequent reports, meetings and evaluations is implemented – a re-organisation activity that hardly helps if the extent of the troubles and its true causes cannot be admitted - and with each new layer of bureaucracy  more people in research start updating their resumes . The struggling company is eventually acquired or merges, the correction takes place but it can take decades and the final money loses are staggering.  (In fact, none of the last decade big pharma mergers was profitable. It served well only the aggrandisment of the managemet who presided over them.)

Lunatic management ideas, superficial solutions, prevarication and dishonesty take hold in the industry because the consequences of bad management are not traced back to the managers. In some respect the situation in a big bad pharma company is analogous to Soviet Union - where the party nomenklatura steeped in ideology and propaganda but disconnected from the actual information and without a personal responsiblity for the true consequences of their actions were tasked with the over-fulfillment of the five-year plan quotas.

In a system where the market does not care about the astronomical cost of drug development, where the cost of entering into the market limits the competition, where the research-to drug cycle is very slow and patent-granted monopoly lasts 20 years and most customers do not directly pay the full price for the drug, huge inefficiencies can take ride along the drug development process. Promoters of the latest fads in management theory, gee-whiz consultants, corporate lawyers and hochstapler executives are among the many types who find a rich milieu in the pharma industry.  

A single failed reaction is a setback. 1 000 000 failed reactions is a library.

After two combichem industry jobs, these are the things I learned:

Testing cruds = sloppy 
On-resin-screening = crap
Mixtures of compounds = crap
One bead-one compound = crap
‘Libraries from libraries’ = crap squared
Million-compound library = we crap harder

What I ended up doing most of my time in combichem was making the precursors and building blocks, attaching them to the resin, deprotecting, loading the resin-bond compounds to the synthesizer or plates or syringes, then running one, maximum two combinatorial steps, then the cleavage, workup, purification. Lots of washing at every step. The preparatory and workup steps took more work then the actual combichem. (The purification was definitely the most tedious step, even with preparative HPLC.)  It was an experience that made me increasingly fond of the traditional medicinal chemistry.

One needs to be pretty cautious, about doing a medchem research in the combinatorial fashion. Even if most reactions could be adapted to combichem in principle, this usually means lots of development time. It is worth doing in limited number cases when:

1. The reactions are very clean
2. Lots of building blocks are available, preferably in a suitably-protected form
3. The set-up does not require a low temperature or strictly oxygen-free conditions 
4. The chemistry is insensitive to reagent excess, goes to completion and won’t ”over-react”when pushed hard (= turn into mess  because the optimum reacton time at given temperature was grossly exceeded).

So the best type of reactions for combichem is the robust kind “that a monkey can do”.  For example using a reaction that is highly moisture-sensitive or prone to overheating-induced decomposition (i.e. Mitsunobu) adds significant difficulties. Titrating the stoechiometry to 1.05 equivs of the reagent is not possible, just as optimizing the reaction conditions for every single building block. Adapting chemistry to combichem format grows exponentially harder if one is trying to synthesize a very large library at once. (Just preparing the reagent stocks for a large library is a chore. There are handling delays, etc).

The other problem with combichem is that it encourages sloppy techniques and wishfull thinking. One needs to be concerned about the building block combinations that do not work well. Failure is frequent if one tries to employ a wide-diversity set of building blocks - even straightforward  reactions like acylation and alkylation (with the excess of nucleophile) don’t progress or they produce mixtures unexpectedly in a substantial number of cases. To avoid bad surprises, one needs to experiment on smaller pilot libraries first - to test the suitability of the blocks, test the best solid-phase attachment, solvents, reaction conditions, cleaving method, purification sequence, etc.

Also one should not fool himself that an automated synthesizer will be a great time-saver - these systems are very impressive in showroom, pumping blue-colored water-  but keeping them up and running is a full time job, the gaskets can leak, the pipetting needle and the outlet chnanels get clogged, viscous solutions or stocks in highly-volatile solvents won’t get transferred in the correct amounts, the heating/cooling or nitrogen gas flow is uneven, and adjactent wells get cross-contaminated, moisture can condese in, etc. Turning on a big synthesizer can be unwieldy for a small-scale exploratory work, and it can take struggle to adapt the automated system to sensitive reactions that need lots of care and attention. Manual combichem experiments can be faster and easier to control. More than few times I saw a big $ 200k all-teflon automatic synthesizer system gathering dust in the corner - because the chemists got tired of the hassle. 

Over the years, combichem gradually evolved into parallel synthesis of small sets (tens or hundreds) of individual compounds. Nowadays the parallel-synthesis-produced compounds are purified - so there is no actuall difference from traditional medchem. The main impact that combichem had was popularizing the high-throughput solid-phase methods in the synthetic chemistry.

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The first combichem company that I joined (14 years ago) is still in business - they underwent a number of mergers and are now a part of Sanofi. Their new masters like them because they got good hits from their libraries with a reasonable frequency. (The chemists there have a pretty decent system for cranking out the compounds - it is too bad that they are discouraged from publishing.) It always remained a pretty small operation throughout the years but it survived.

I think the future is in outsourcing.  We have recently screened some commercially-available collections that were produced by a traditional chemistry abroad - and I was very surprised by the number and high quality of the resulting screening  hits. A chemist in Russia or China can make a set of compounds, $3 per compound, 90%+ purified and bottled.  A set of 1000 dissimilar compounds that were made individually, pure, is in my opinion more usefull than 10 combichem general libraries with 1000 compounds each.

[This post was inspired by Derek Lowe’s In the Pipeline]

 Credit Jiri Sliva

Levitra is a close Viagra knock-off and there is little difference between the two for the patient. “Money wasted on developing yet another version of the erection pill could have been put into malaria research instead.” And so on.

I am not a fan of Big Pharma but I would like to point out that the misery of the Third World was not caused by the lack of drugs or by their high prices - the root problems have to do more with awful governments and wars. But if most patients there cannot pay for the new antimalarials / HIV inhibitors / tuberculostatics, the research interest will get re-directed elsewhere and one can hardly blame the industry for that.  (Another complication is that even if the drugs are provided for free, it is hard to get these patients to take their medications in a disciplined manner for the prescribed period of time - and hence the multidrug resistance emerges soon)

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Sepracor business plan used to be entirely based on patent-busting; Sepracor was scorned for it by many people in the industry. Yet this kind of ”bottom-feeding” research saved some good drugs from oblivion - drugs that would not be introduced into US otherwise. As a result, a next generation of drugs appeared in previously stagnant fields. (Antihistaminics, sleep meds.) 

A competition from me-too drugs produces pressure that pushes the innovation - without it, companies selling popular drugs are in no hurry developing their more advanced follow-on candidates if their established drug is doing fine. 

A late-comer drug needs to offer some demonstrable advantage over the more established ones. The me-too drug can for example have a different pharmacology from the original drug - whether this was a part of the design or just a coincidence. (The drug distribution, metabolism, drug-drug interactions and side-effect profile are notoriously hard to predict: a small change in the structure can have an important effect which might not be apparent until the testing is done in patients). The main reasons for doing the me-too medchem research is that it is a lot easier: Developing a new structural class from a low-potency screening hit can take years and there is no guarantee that this optimization will ever produce a drug candidate. Starting with a proven competitor’s compound and modifying it in few places greatly shortens the development time and improves the chances of success. 

It is just impossible to exhaust all possibilities inherent in a class of molecules. The chemists and biologists in the team that discovered the original drug must have made choices at numerous points of the project- when they picked the directions they thought were worth pursuing. Their project had deadlines, it was influenced by personal experience and bias.  Independent re-visiting the original data and premises with the hindsight knowledge of the clinical performance and with a competitive mindset can lead to surprises, and eventually to an improved drug candidate.  

There is also a natural tendency of compounds coming out from different groups to converge:  It is a common practice in the industry to re-synthesize and test the published compounds. The added insight from their testing (especially when combined with the X-ray crystallography) can quickly close the gap between the competing groups. This can also produce a resemblance of a me-too approach.

 Credit: Jiri Sliva

I have been doing Arndt-Eistert homologation (example: OrgSyn 79, p.154, 2002) so I needed pure anhydrous diazomethane solution. I made diazomethane from nitrosomethyl urea (NMU) and to avoid the distillation, I transfered diazomethane from my KOH-dried diazomethane solution in toluene by passing a stream of Ar through it (at R.T. for 30 min) and condensing the liberated dry Ar-diluted diazomethane gas directly into the reaction mixture cooled to -78C. (I heard of this alternative of diazomethane solution distillation from Rapoport group - and it worked, though I cannot recomend doing this kind of gas transfer on large scale because diazomethane is so nasty and toxic.)

I have only a limited experimental experience with diazomethane. There is plenty literature on the subject. But I would like to ask you about your personal perspective: What is you favorite method of making high-purity dry diazomethane solutions (that can be used for sensitive applications such as Arndt-Eistert)? 

One reason why I am asking this is that it apears to me that all the commonly used precursors of diazomethane have some problems associated with their use: Diazald needs a presence of alcohol (preferably a high-boiling one) or a phase-transfer catalyst to work with aqueous hydroxide and the produced diazomethane solution should be distilled to remove the sideproducts. MNNG is highly toxic and difficult to buy nowadays. NMU is expensive, explosive, very carcinogenic and the NMU-produced diazomethane solution contains trace of methylamine. So I would be delighted to learn what you have tried and liked.

The other reason is that I have one rather simple-minded idea - that I would like to try, to see if it works -  for a method of preparing contaminant-free anhydrous diazomethane solution without the risky distillation step. I would like to know if you think it is a worthwhile use of lab time, if you think a new method is even needed - a method that would be convenient and work with benign commercial reagents.