TRANSCRIBED FROM RECORD RECEIVED 2/24/58
Madison, Wisconsin

Dr. Joseph Lein
Bristol Laboratories Inc.
Syracuse 1, New York

Dear Joe:

Welcome back. Of course, I wonder if you were able to find your way back

since I understand that Syracuse is buried under 0 or 50 feet of snow. But
assiming that this gets to you, having penetrated not only through the snow

but through the pile on your desk, I just thought that you would be interested

in an account of a new anticancer drug or group of drugs that Charlie Heidelberger
gave at a seminar last night. He has apparently been collaborating with

Hoffman LaRoche on the synthesis of these materials and they are now in the
course of clinical trial,

The key compound in hia study has been 5«fluorouracil, analogous to the bromo-
uracil which various people have found to be an analogue of thymine in the
synthesis of DNA. I think that Charlie went after fluorouracil on the
supposition that this would be an even better analogue of thymine, but it

turns out that he got interesting results for entirely wrong reasons. For
example, I found myself that fluorouracil inhibited E. coli and that this
inhibition was reversed not at all by thymine or thymidine but very efficiently
by uracil. It thus appears that fluorouracil if anything corresponds to the
uracil in RNA rather than the thymine of DNA. Charlie and his collaborators
have done a number of studies with ascites tumors and other tissues and one

of my own students did some work with E. coli which lead to the conclusion
that a small amount of fluorouracil is actually incorporated into RNA and

none at all into DNA. Fluorouracil has in fact been found to have rather
definite anticancer activity, Charlie having even succeeded in curing a
substantial fraction of mice receiving certain transplantable tumors. The
response is quite different from one tumor to another as one might expect,

but the results were good enough to warrant clinical investigation and I
gather that fluorouracil may end up to be an agent of approximately the same
limited but definite sort of usefulness as mercaptopurine. Like the other
anticancer drugs, fluorouracil is extremely toxic, and as one of clinicians
put it last night, its application may be more a matter of prolonging the
"terminal state" than life itself, of course, as in any clinical investigation,
the trials with this new material have had to be confined to the most difficult
risks and the not entirely satisfactory results perhaps should not be taken
entirely at face value.

It looks as if fluorouracil has two pointe of action, or perhaps even three:

(1) A small amount is incorporated into RNA. Whether or not this is deleterious
to the function of the RNA is not known. (2) In some fashion, it inhibits

the incorporation of uracil into RNA so that one would assume that inhibitory
levels are in a synthesis at large will be blocked, Thia I suspect is the

main carcinostatic reaction. (3) A small amount of administered fluorouracil
does turn up as the fluoro analogue of dioxyuradylic acid. This fluoro
dioxyuradylic acid is apparently analogous to the natural nonfluore compound

and seems to funetion in preventing the methylation of the uracil residue
J. Lein 2 2/21/58

in the formation of thymidylic acid. There is, therefore, with fluorouracil
and especially with the dioxynucleotide itself, very substantial inhibition of
DNA synthesis. This, to my mind, may turn out to be the most interesting part
of the story. The report on the nucleotide was part fragmentary as they have
had to prepare it entirely by isolation and they have not had large enough
amounts to do very much with it. However, it is active in the inhibition of
tumors and of the biochemical mechani ams I have just mentioned at extremely
low dilutions of the order of 10°7molar for the inhibition of incorporation

of formate carbon into thymine, for example. So there may be indeed a very
promising approach on this last point. Whether it will result in a selective
toxin is of course another question that can be decided only by experiment,

These observations lead me back again to my hobbyhorse of substitutional
chemotherapy. I hope I have made it clear that I thought that the cancer
problem was the most intriguing point of application of these notions. For
obvious reasons, one would like to try it out on a simpler systen, for example,
bacterial antibiosis. However, you would want to make the executive decision
yourself whether to proceed step-wise or to Jump right in into the cancer field,
My explicit proposal at the present time is to use cancer cells themselves, in
fact, the sume tumor that you are using for test purposes later on as the source
of your natural metabolites, and to subject there crude materials to the range
of substitutional and modificational reactions that we have already discussed.
If you had included a reaction that would result in the fluoridation of uracil
residues, you yourself would have discovered the effects of fluorouracil that
we have just described, Actually, there is at least some possibility that
considerably greater selectivity might be discovered if you had substitutions

of small di~ or tri-nucleotides. We don't know very much about the penetration
of such materials into cells but Simply because there are a great many different
ones there is the opportunity of greater differential activity with such
materials. This is, of course, a half-way step to try to feed an entirely
fraudulent gene to the target cell.

I am still not sure but that the bacterial system may not still be the more

appropriate for pilot studies. ‘There is every reason to pursue the analogy

between bacterial and cancer growth in the first stages of which one cannot

hope to find a greai deal of selectivity. You can, however, hope to find a

class of compounds and ways of producing them which can then be applied in a
iarger scope to your experinents with tumor cells,

I am, of course, anxious to hear how far the seeds that I am trying to drop are
germinating,

To avert to a slightly different theme, I would like to know whether you do —
have any concrete plans with regard to the production of analogues of neuraminic
acid. I have in mind particularly the nitrophenol derivative which I had
proposed to you earlier as a likely good homogenic substrate for the
neuraminidase activity of influenza and other viruses. The production of such
& material would be a tremendously valuable tool in fundamental virus studies
and in addition might have a rather wide application for diagnostic purposes.

I think I need not enlarge on this muen further than pointing out how desirable
it would be to have a good sensitive chemical or enzymatic test for virus
activity. Such a test could be made specilic by virtue of the specific
inhibition of the virus by antibody, Whether the diagnostic applications of
J. Lein 3 2/2h,/28

such a material would have a sufficient market to justify commercial interest,

I do not know, but I have no doubt about the extent of scientific interest.

If, therefore, you do not have tangible plans for the exploration, I hope you
will let me know about it so that I can try to encourage other people to get
inte it. There are some possibilities, some of them being, in fact, commercial
ones. Since this particular area is,as I have indicated, of marginal commercial
interest, it is a little hard to predict what your reaction to that would be,

One of my colleagues here, Steve Kubie, has told me that he made a reasonably
good preparation of nitrophenolgalactacide by the straight fusion reaction,
simply fusing the two reactants without very much more fuss or bother. I
suspect that the unreacted nitrophenol could then be extracted by ether or
other measures that your chemical friends will know better about. The same
procedure might just happen to work reasonably well with colominic acid as a
source of the neuraminic acid residue.

Another trick I'd like to try out of my ow curiosity sometine, would be to

see if one can make nitrophenol conjugates of cell walls or even intact cells
by similar methods with a view again to using such preparations as specific
substrates for wall splitting enzymes. The only reason I bring this up is that
you may sometime happen to have some nitrophenolated cells as a by»product of
some other reactions along this line, and if any such come up I'd be delighted
to see whether bacteria phage had any specific effect on such a substrate.

Joe, I wonder if it happens that you are a member of the New York Academy of
Soiences? If not, I would strongly recommend it to you as the Academy puts on
quite a number of symposia each year which deal with topics within your
professional interest. I will be very happy to Sponsor your membership, if
you would be interested. The dues are $15 a year and you would certainly cet
a remarkable series of publications for it.

For example, I just had a notice that there is going to be a conference on
"Screening Procedures for Kxperimental Cancer Chemotherapy” next March 1315

in New York. Looking at the program, I would judge that this is one of the
sort of meetings that you would be quite interested to attend yourself. I
believe that in general these meetings are not open to the public cxeept by
invitations As a sort of postscript, Joe, I wonder if you can add $30 to my
next check on account of expenses for servicing the dictating machine, supplies,
and a case.

With all the best.

Yours,

Joshua
s/ Joshua Lederberg

JLtac

P.S. I hope you have been following the publications fram art fornberg at
Washington University, St. Louis, and hie group very closely. The last word
is that under certain conditions, some of his enzyme preparations are able to
J. Lein 4 2/24/58

incorporate unnatural nucleotides into DNA. As I think most people will agree
this may ultimately prove to be one of the most promising approaches towards
antiviral and anticancer chemotherapy. I would doubt that any part of it is
quite ready for detailed commercial exploitation, unless you were in a position
to do some very elaborate enzyme work indeed, But I strongly advise you to
keep your eyes open.

That's it again.

Josh.