Org Prep Daily

March 11, 2007

Milkshake Manifesto

Filed under: procedures — milkshake @ 9:02 am


or The Twelve Theses For All Erudite Medicinal Men To Follow, To Purge The Sins Of Abominable Design And To Attain The Heavenly Joys of a Clinical Success

1. Do one change at a time. Keep your molecule small and simple. Cyclize. Fluorine is your friend.

If you make too many intuitive jumps in your structure without knowing the contribution of each change, you will not understand what did what. Your SAR table will become a jungle and you will miss important things.  If your compounds become hard to make early in the project, your progress will be slow. It is easy to gain potency by adding a big greasy piece like biphenyl or bromo-substituted benzyl but this back-fires in the longer run. Cyclic compounds have often better properties (cell permeation, PK) , the potency and selectivity can also dramatically improve by restricting the conformations. Replacing hydrogens with fluorines helps to fix the metabolism problem. One can gain additional potency with correctly placed fluorine with only a small penalty on the molecule lipophilicity and size. (Fluorine is also a wonderful NMR label that can be used to follow the chemistry and quickly determine the isomeric purity of compounds).

2. God created sharp SARs. 

Don’t despair if the permissible substitution pattern of your active compounds is very narrow – it is a good thing. Lousy and un-druggable molecules are usually from the muddy category (no matter what you change the molecules have always about the same potency). 

3. Lipinski rules and Polar surface area scores are only a shorthand. 

Lipinski rules and PSA scores are fairly crude but they point in the right direction – Don’t put too many hydrogen bond donors in your molecule if you want a cell permeable orally-active compound. More than 3 Amide/urea NH is bad news. Too many amide and heterocycle NHs together with good hydrogen bond acceptors (carbonyls, hetero-N) in the same flat polyaromatic molecule will likely result in lousy solubility – this is the typical problem with kinase inhibitors. (A small substituent like Cl, Me group next to NH can remedy the solubility/permeability problem). Be careful about sulfonamides and sulfones – these groups are quite polar and can cause problems with cell/brain penetration. Please note that the requirements are more stringent for brain-penetrable compounds. 

4. Acidic compounds have high plasma protein binding

If your molecule has acidic groups like carboxyl, tetrazole, isothiazolindione etc, your intrinsic activity has to be very high (single digit nM or better in cells) to ofset the extremely-high plasma protein binding of acidic compound. Greasy molecules are  problematic also – but acidic compounds are almost guaranteed to have 99+ % plasma protein binding and hence reduced relative potency in the whole blood assay. 

5. Basic molecules have typically improved solubility and cell potency - at a price

More basic amines (primary, secondary) have more pronounced accumulation within cell than less-basic amines (such as N-sunstituted morpholines). Adding amine side-chain to a molecule can also cause lowered selectivity and unexpected organ toxicity. Quartenary ammoniums tend to produce particularly nasty side-effects and should be avoided. Tertiary amines are metabolised by oxidative dealkylation quickly. (Putting fluorine or oxygen beta to amine can suppress the oxidative metabolism and at the same time lower the amine basicity.) Strongly basic molecules have typically very high volume of distribution – which means that due to partition of the compound deep into tissue the dosing has to be increased to achieve decent concentration in the blood – but at the same time the high volume distribution prolongs the half-life of the compound, even if the compound has rather high clearance due to rapid metabolism.

6. Amidines and guanidines are frustrating

It is easy to get an active compound with amidine but it is hard to get it orally active afterwards. Doing chemistry with amidine in the molecule is not fun either.  Other not-so-druggable functional groups are hydroxamic acids, aldehydes, thiols, reactive halogens. Nitro groups are a liability but they are not as bad as many people think - though you may want to try to replace a nitro group in the end. 

7. Test CYPs 

Basic heterocycles and polyaromatic compounds have problem with inhibition or induction of cytochomes. This is undesirable because of the drug-drug interaction liabilities. It is important to test for CYPs inhibition relatively early in the project, especially if the molecule has troublesome pieces like 4-monosubstituted pyridine, 5-azaindole, 1,2,4-triazole, N-monosubstituted imidazole, etc.

8. Do not trust docking, do crystallography early.

Docking can provide some idea in absence of a real information (co-crystal X-ray structure) but most of time the docking amounts to a wishful thinking in front of the monitor. A co-crystal (or at least soaked-crystal) X-ray crystallographic data can be very illuminating. Use the crystallography as a source of ideas but don’t let it rule your design; X-ray data does not address electronic and entropic effects well, and it over-emphasizes hydrogen bonds and steric factors; you may easily get sidetracked by focusing on marginal interactions while overlooking some other important effects. Over-reliance on X-ray in finding additional interactions usually produces ugly compounds that have lots of floppy polar appendages and are hard to make. 

9. Get cell-based assay as early as possible to guide your project.

Biochemical data tels you about hidden potential of your compound but cell-based activity is the real thing. You don’t have a real lead compound until you can make it work in cell. Usualy everything that works in cell works also in biochemical assay but this is not true in the opposite direction - things will or won’t work in cell for mysterious reasons and only persistence and patience sometimes  helps to find the cell-active version of your molecule. Once you got a decent activity (say, 100nM in biochemical assay) it is important to have cell-based assay as the primary screen; it makes no sense to overoptimise on biochemical assay alone because without cell-based assay you will likely stray in wrong direction.

10. Ignore Caco-2 and do rodent PK tests instead, use human plasma and whole blood

Caco-2 permeability model is useless. Oral absorbtion/brain penetration tests in rodent should be done early in the project. Metabolic studies with liver microsomes can help to identify some metabolically weak spots (use LCMS to identify the metabolites) but microsome studies are not terribly helpful in prioritising the molecules - it is foolish to say “I have to have microsome half-time above 30 minutes.” (If all your molecules have a decent microsomal half-life and suddenly one has a very short one, maybe this one molecule can have a liability towards oxidative metabolism – but this suspicion cannot be taken as a substitute for doing a real PK in rat). Do plasma protein-binding studies with human plasma only – the calf-derived plasma is a poor substitute, often producing misleading results. Do a whole blood assay with human blood. If your advanced compounds are highly crystalline, measure the solubilities and prioritize based on solubility studies

11. Be nice but sceptical 

Be nice to your biologists but watch about the reliability of their data - if they got a weird curve or noisy assay they should be forthcoming about it. Don’t give the biologists the entire stock of your compound – you may not get it back.
Don’t force people to work on your ideas – let your lab colleagues choose what part of project they work on, which particular molecules to make. Make the project more enjoyable for them by scaling up the building blocks for the entire lab and by  ordering the labor-intensive advanced intermediates from a custom-synthesis company.

12. Patents are not scary 

Don’t worry over too much about patents – unless the exact compounds is in the patent experimental examples (with a good preparative procedure and NMR spectra) and is claimed there for the same protein. Very broad patent claims are easy to cast but they are also easy to challenge or skirt; it is almost always possible to get your own narrow patent on things that are within the scope of someone’s patent by describing “new and unexpected” properties of a class of compounds (that were claimed but not documented in the broad patent). 
Getting out of patent is much easier than many chemists think - look what Sepracor did, and how many closely-related me-too compounds are there on the market, from the competing companies.  Also, the patent-busting changes to the molecule can be quite minimal and can be made very late in the project.  It is good to get a broad patent coverage for your project – but if you cannot, all what one needs in the end to have patent-protected is just one compound, the one which eventually becomes a drug.

born5.jpg Credit: Adolf Born

Note: Totally Medicinal has a related discussion on Tricks of trade in medicinal chemistry.

Update: Derek Lowe has written many times on these subjects In the Pipeline and his take is always insightful. Although there is one idea on which we disagree – Derek claims that “nothing good ever came easy or cheap”. I would like to point out that my mother-in-law did pass away; and I did not have to hire anyone


  1. Interesting, although it’s pretty far from what I’m doing. Makes me want to do one for crystal engineering…

    Comment by Ψ*Ψ — March 11, 2007 @ 1:57 pm

  2. Words are like from another world. A lot to learn for me.

    Comment by liquidcarbon — March 11, 2007 @ 6:12 pm

  3. Liquidcarbon: If you independently ask several medicinal chemists to pick from a random list of chemicals the structures that they consider “nice-looking” or druggable, you will likely get similar selections. The rules – small, cyclic, not too polar, not too many problematic functional groups, etc – are easily learned by osmosis from working with more experienced chemists. (These rules originated from memory of project succes and failure). The learning curve is not too steep or long, just few months in decent medicinal chemistry program can give a good idea – the medchem stuff is just a small add-on, if one is a competent synthetic chemist.

    As a chemist you will be unlikely in the position to decide what biological assays/animal studies are used for testing your compounds – but eventually you should understand how the drug discovery process works, so that you can figure out whether the people in charge of your program have a clue or if they are just wannabies (as it is unfortunately too common in academic medchem programs).

    We have been beneficiaries of companies like Asinex or Chembridge – they do make our life in medchem easier; it is much better to buy a somewhat costly building block rather than struggle for weeks trying to make it. We also had wonderful hit sets recently, coming from the high-throughput screening of commercialy-available chemical collections – and from looking at these nice compounds it is clear that they too were made in one-at-the-time fashion, by someone given a list of compounds to synthesize. Since we know that many of these compounds were made in Russia or Ukraina or China we have been sometimes joking about enslaved chemists in exotic countries far away. (I used to do some custom-synthesis work on side for money, when I was a student in Prague – but at that time we were not under pressure to take it in order to survive.)

    Comment by milkshake — March 11, 2007 @ 8:05 pm

  4. Neither was I under this sort of pressure, I did this myself only once. But some people were. It’s kinda fun – to play around with different stuff, when you don’t have total synthesis around. Some of these companies also provide decent money and experience for students, I could have worked there myself if I lived closer. Chembridge also runs annual organic chemistry olympiad and organizes things like ASCMC ’04 or ASMC ’07. Look at the speakers!

    I think they will slowly switch from custom synthesis to their own medchem projects, I only wonder when. As judged by our forum, they started to look for medicinal chemists out there, but at the moment they can’t offer a salary greater than $30K / year, – will a good chemist take the bait? Moscow is the most expensive city in the world.

    Comment by liquidcarbon — March 12, 2007 @ 12:59 am

  5. Medicinal men only?

    Comment by Ψ*Ψ — March 12, 2007 @ 1:56 am

  6. As you can see I was trying to emulate the tone of religious tractates from the reformation period. In those days many theologians believed that woman is an instrument of Devil, created to lead a righteous man astray from the path of salvation. One cannot argue with such a justifiable point of view.

    (But Luther himself was an early proponent of women liberation and affectionate husband – he organized a convent break and twelve nuns were smuggled out of a monastery in herring barrels. Luther then married one of these barrel-liberated nuns.)

    Comment by milkshake — March 12, 2007 @ 2:17 am

  7. Nice work Milkshake. To tie together two parts of your twelve – care should be taken when replacing a metabolically soft Ar-H with an Ar-F – you could turn a CYP metabolic liability into a CYP inhibition liability.

    Comment by TotallyMedicinal — March 12, 2007 @ 2:56 pm

  8. I did not know this – thank you. I heard of some take-bite-and-die modifications (i.e. cyclopropyl amine) but did not realize that fluorine can do this also

    Comment by milkshake — March 12, 2007 @ 3:14 pm

  9. This is your second greatest post ever. (The TLC stain post is still #1.)

    Comment by Paul — March 13, 2007 @ 1:01 am

  10. The stain post took no work – I just wrote down the recepies for stains that people were using in the group.

    The medchem suggestions are a common stuff, too. We have a project right now that is in early stages. At the start of a project there is always a number of directions one can take with the lead compound. We got fresh postdocs joining us and some of them were a bit nervous about not knowing which way to go about making new analogs. “Keep things small and simple, do one change at the time and don’t worry too much” was the usual advice. The point is that nobody really knows which compound will turn out active and all one can do is to inch forward and hope for the best. (Actually we had some cheers recently when we got a crystal structure that showed one of our molecules wrapped-around the protein in a pretty bizzare fashion and overlapping on one end with another, more conventional inhibitor that we co-crystalled before. But joyful moments like this happpen only few times a year)

    Comment by milkshake — March 13, 2007 @ 1:30 am

  11. Milkshake- to what extent do you consider tox studies from the literature? Is it verboten to even attempt to study certain classes of molecules with certain substructures that may cause tox problems? I could see this a cost-saving approach but it may also exclude desirable hits. I’m in academics so I don’t know how you approach these issues. I assume most researchers dont want to be confronted by a manager who says “You made what? The rats die within ten feet of that stuff!”.

    Comment by Clone Guy — March 13, 2007 @ 4:44 pm

  12. I would not worry too much about things like nitro group – it is in number of drugs – though one should at least try to replace it with something else.

    With putting stuff into animals – PK studies are relatively quick and can be done with 3 rats. But you still are going to make couple of people in DMPK, vivarium busy for days so you should have a justification for putting things into animals. I am not expert but my understanding is that tox studies are longer, take more animals and are more labor-intensive.

    I understand your concern – what if we missed something – but you have to decide on priorities in using the shared resources, to be considerate to other people. I think you would not like it if you urgently needed a tox study or PK of your preclinical compound but your compound would be repeatedly delayed for weeks – just because some doofus from another project put several awfull molecules into the queque before you.

    That said, some boses can be overly cautious and worried about things that may not even be a problem.

    Comment by milkshake — March 13, 2007 @ 5:13 pm

  13. Good rules, generically, all. Although all are subject to the willingness of the company you work for to take on risk. Your #12, for instance — my attorneys would vehemently disagree with you, arguing that “freedom operate” is very different from “ability to prevent infringement”. If we can’t get solid composition of matter, not only on the candidate but on lots of analogues, then we probably won’t take a molecule forward. You don’t want to provide a roadmap to follow-on molecules.

    The one I like best is your #2. Too many medicinal chemists get hung up on making lots of potent molecules. Well, you really aren’t learning anything if you do that, are you? The changes you’re making probably aren’t anywhere near the protein of the interest. Great for addressing PK and cross-reactivity, but how many molecules can you run through PK, anyway? If small change = big effect on activity, then you’re in a sweet, sweet spot.

    Comment by weirdo — March 13, 2007 @ 9:09 pm

  14. The opinions differ – I worked with people who were completely into me-too analoging of somebody-else’s compound (in hopes of finding a modification that the original patentholders did not think of) aiming at improving the PK properties or reducing some known side-effect potential. The advantage of this Sepracor-like approach is that by patent-busting you can get your own advanced compound rather quickly, within a year or so (depending on the number of chemists).

    I was also on early-stages projects where some chemists were very nervous, saying “we can’t make these analogues because so-and-so claims this class for an unrelated target.” I tried to explain that by the time we will need to get concerned about getting a patent, our compounds will most likely look very different from the early lead.

    Of course a patent lawyer will advise you to pick clean compound – it is a lot less work for him and a broader protection for you. But having been in the patent-busting business before I can tell you that it is impossible to protect a broad IP estate thoroughly; some of the patent-busting tricks are trivial. I would encourage people to worry about chemistry and biology first – and if you get something good you can probably find a way to protect it, in the end.

    Comment by milkshake — March 13, 2007 @ 9:49 pm

  15. Truer words were never spoken! Working for a company that uses Caco-2 and Xliver microsomes in place of PK studies to prioritize what moves forward, because of a lack of PK resources, I sometimes want to leave meetings through the plate glass window. Get the resources for PK and use LM and caco as it is meant to be, an adjunct to the screening cascade, and NOT as a filter for promising compounds.

    Comment by industry guy — March 13, 2007 @ 9:54 pm

  16. The irony is that having a small rodent vivarium and three people taking care of the animals and doing the PK work is actualy not too expensive in the relative terms. They need a decent LC/MS, but overall expenses are lot less than for example buying, setting up and maintaining one 400MHz instrument. I think nobody sane would suggest that chemists should save money by running TLCs in place of NMR and send out samples for their most important compounds to a contract NMR lab .

    Comment by milkshake — March 13, 2007 @ 10:06 pm

  17. I’m guessing you didn’t much care for your mother-in-law?

    Comment by Ψ*Ψ — March 14, 2007 @ 1:08 am

  18. Some relatives are good people, deep down.

    Comment by milkshake — March 14, 2007 @ 1:34 am

  19. good commment. Could you please give me your E-mail address?


    Comment by wei — February 24, 2009 @ 2:06 am

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