Medicinal Chemistry II

Manual for Practical Sessions

Level4 - PharmD

Fall Semester 2024/2025

Pharmaceutical Chemistry Department

 Table of Contents
Medicinal Chemistry 2: Course Specifications. ………………………………………………………………….
Course Structure, Assessment and Grading System ………………………………………………………….
Housekeeping Rules …………………………………………………………………………………………..….
(1) Introduction to Drug Pharmacokinetics (ADMET) ………………………………………………
Intended Learning Outcomes (ILOs) ………………………………………………………………………
Pre-session Reading ………………………………………………………………………………………………
Understanding where our course fits in the broader drug discovery development process
Physicochemical properties and Pharmacokinetics ………………………………………………
Learning Activity (1): Lipophilicity - Parameters & Binding ………………………………………………
Learning Activity (2): Lipophilicity - Lipinski’s rules & Balance ………………………………………………
Learning Activity (3): ionizability ………………………………………………………………………
Assignment (1) ………………………………………………………………………………………………
(2) Absorption ………………………………………………………………………………………………
Intended Learning Outcomes (ILOs) ………………………………………………………………………
Pre-session Reading ………………………………………………………………………………………………
Definition of Absorption and Bioavailability. Factors that influence bioavailability of a drug include:
Improving Solubility via structural Modifications ………………………………………………
Learning Activity (1): insertion of polar/ionisable group ………………………………………………
Learning Activity (2): disruption of hydrogen bonding ………………………………………………
Learning Activity (3): disruption of molecular planarity ………………………………………………
Learning Activity (4): bioisosterism ………………………………………………………………………
Learning Activity (5)٠ prodrug ………………………………………………………………………
Assignment (2) ………………………………………………………………………………………………
(3) Distribution ………………………………………………………………………………………………
Intended Learning Outcomes (ILOs) ………………………………………………………………………
Pre-session Reading ………………………………………………………………………
Around the Blood Supply, to Tissues, and to Cells. Physiochemical Properties Governing Volume of
Distribution. Blood Brain Barrier (BBB) penetration ………………………………………………
Plasma Protein Binding ………………………………………………………………………
Learning Activity (1): Improving Drug Distribution by Changing Acid/Base ………………………
Character. ………………………………………………………………………………………………
Learning Activity (2). Increase Volume of Distribution through Target Mediated ………………………
Drug Disposition (TMDD) ………………………………………………………………………
Learning Activity (3): Structural Modifications for Solving PPB Issues ………………………
Assignment (3) ………………………………………………………………………
(4) Metabolism ………………………………………………………………………
Intended Learning Outcomes (ILOs) ………………………………………………
Pre-session Reading ………………………………………………………………………
1. Functionalization Reactions ………………………………………………………………………
2. Conjugation Reactions ………………………………………………………………………
What is the significance of studying these types of metabolic reactions? ………………………
Learning Activity(1) ………………………………………………………………………
Learning Activity (2) ………………………………………………………………………
Assignment (4) ………………………………………………………………………
(5) Metabolic stability
Intended Learning Outcomes (ILOs) ………………………………………………
Advantages of Enhancing Metabolic Stability ………………………………………………
Strategies Used to Enhance Metabolic stability ………………………………………………
Learning Activity(1) ………………………………………………………………………
Learning Activity (2) ………………………………………………………………………
Assignment (5) ………………………………………………………………………
(6) Excretion …………………………………………………………………………………
Intended Learning Outcomes (ILOs) ………………………………………………
Pre-session Reading ………………………………………………………………………
Routes of Excretion ……………………………………………………………………………
Linking Chemistry to Excretion ………………………………………………
Implications for Drug Design ………………………………………………
Learning Activity ………………………………………………………………………
Assignment (6) ………………………………………………………………………
(7) Toxicity ………………………………………………………………………
Intended Learning Outcomes (ILOs) ………………………………………………
The Safety Window ………………………………………………………………………
Commonly Encountered Toxicophores ………………………………………………
Learning Activity (1) ………………………………………………………………………
Learning Activity (2) ………………………………………………………………………
 Assignment (7) ………………………………………………………………………
(8) Drug Design Case Studies ………………………………………………………………………
Intended Learning Outcomes (ILOs) ………………………………………………
Pre-session Reading ………………………………………………………………………
Learning Activity ………………………………………………………………………
Assignment (8) ………………………………………………………………………
Appendices ………………………………………………………………………
Appendix (1): An excerpt from the book “Medicinal chemistry for practitioners",
Li, Jie Jack (2020) ………………………………………………………………………
Appendix (2): An excerpt from the book "Organic chemistry”, Clayden,
Greeves, and Warren (2012) ………………………………………………………………………
Appendix (3): An excerpt from the paper “Acidic and basic drugs in medicinal
chemistry: A perspective”, Charifson & Walters, J Med Chern (2014) ………………………
Appendix (4): An excerpt from the book “An Introduction to Medicinal ………………………
Chemistry", Graham Patrick (2013) ………………………………………………
Appendix (5): An excerpt from the paper "In Vitro Metabolism and Covalent ………………………
Binding of Enol-Carboxamide Derivatives” (2008) ………………………………………………
Evaluation Sheet
Medicinal Chemistry 2: Course Specifications
A- Basic Information
Programme: Bachelor of Pharmacy (Pharm D)
Department responsible for offering the Department of Pharmaceutical Chemistry
course:
Department responsible for teaching the Department of Pharmaceutical Chemistry
course:
Academic year: Level 4 ' Fall Semester
Course title and code: Medicinal Chemistry 2; PC710
Prerequisite: Drug Design
Contact hours (credit hours): Lecture: 2 (2), Practical: 2 (1), Total: 4 (2+1)

B- Professional Information
The course aims and intended learning outcomes are based on that mentioned in the programme
specifications, with more course-related specific details.
1 - Overall Aims of Course.
Upon successful completion of this course the students will demonstrate:
 Knowledge of the basic concepts of pharmaceutical chemistry effectively.
 With the concentration on the molecular mechanism of the studied classes of compounds.
 Understand key drug properties and pharmacokinetics that are to be considered during the
process of drug design.
2 - Course learning outcomes:
Domain 1: Fundamental knowledge:
The students should be able to:
Program key elements Course learning outcomes
1-1-1-2- Display understanding of 1-1-1-2- Show understanding of knowledge of
organic, medicinal, analytical, and different classes of drugs.
biomedical studies of food & 1-1"1-2-a Show Understanding of knowledge of
pharmaceutical materials. antibacterial and antiviral agents.
1-1-1-2-0 Show Understanding of knowledge of
1-1-1-2-C Show Understanding of knowledge
of Antiprotozoal and anticancer agents.
1-1-1-2-d Show Understanding of knowledge
1-1-4-1-Explain the mechanism of 1-1-4-1- Describe the mechanism of action of
of hormones.
drugs, antibiotics 8 toxins action. drugs and how to optimize this action.
1-1-4-1-a Describe mechanism of action of
antibacterial and anticancer drugs.
1-1-4-1-b Describe mechanism of action of
antiviral, antimalarial, antifungal, antiprotozoal
agents, and hormones.
 1-1-4-2-Articulate information from 1-1-4-2- Gather information from basic
fundamental sciences to evaluate the sciences to determine the appropriateness,
appropriateness, and effectiveness of and effectiveness of drugs.
drugs and natural products in 1-1-4-2-a Gather information from fundamental
populations. sciences to determine the appropriateness,
and effectiveness of drugs including anticancer
drugs and hormones.
1-1-4-2-b Gather information from fundamental
sciences to determine the appropriateness,
and effectiveness of drugs including
Domain 2: Professional and Ethical practice antibacterial, antifungal, antimalarial 8
The student will be able to:
Program key elements Course learning outcomes
2-2-1-2- Design, synthesize 2-2-1-2- Design and improvement of proteins
and analyse pharmaceutical and drugs.
2-5-3- Design and perform
materials. 2-5-3- Design and apply research using
research using suitable appropriate methods.
methodologies.
Domain 3: Pharmaceutical Care
The student will be able to:
Program key elements Course learning outcomes
3-2-1- Connect mechanism of 3-2-1 - Combine mechanism of action &
action, uses, dosage, contraindications of different classes of drugs.
contraindications and adverse 3-2-1-a Combine mechanism of action &
reactions of drugs and drug contraindications of Antibacterial, anticancer
interactions. agents and hormones.
3-2-1-b Combine mechanism of action &
Domain 4: Personal Practice:
The students should be able to:
Program key elements Course learning outcomes
4-1-2 Analyse data, solve 4-1-2 Demonstrate responsibility for team
problems, and work efficiently Management skills.
in a team.
4-2-2- Utilize modern 4-2-2- illustrate understanding of modern
technologies & media to technologies to acquire good presentation skills.
acquire good presentation
skills.
3- Course Contents (Syllabus)

Week Practical
Date Topics Credit hrs. (1)

1 29-9 Drug Pharmacokinetics (ADMET) 1

2 13-10 Absorption 1

3 20-10 Distribution 1

4 27-10 Metabolism 1

5 3-11 Midterm Exam

6 10-11 Metabolic stability 1

7 17-11 Excretion 1

8 24-11 Toxicity ٩

9 1-12 Drug Design Case studies 1

10 8-12 Project 1

11 15-12 Practical exam 1

4- Teaching and Learning Methods

 Lectures (Tools: board, overhead projector, data show, online: Online
Meetings and Recorded Online Sessions).
 Practical Session (Tools: labs, boards, instruments, equipment, online:
Online Meetings, Recorded Online Sessions, and Online Tutorials)
 Assignments, seminars, research, and posters.
5- Student Assessment Methods

Written Midterm exam To assess The ability of students to follow-up the

course subjects.
Practical exam and To assess The ability of students to apply and
assessment of semester practice scientific knowledge.
work (class
Written finalactivities)
exam To assess The overall outcomes.
Oral exam To assess knowledge, understanding & intellectual
skills.
Assessments Schedule
Assessment 1 Periodic (Midterm) exams Week 5

Assessment 2 Formative assessment Week 8

Assessment 3 Practical exam Week 11

Assessment 4 Written exam Week 14

Assessment 5 Oral exam Week 14

Weighting of Assessments
Periodic examination 13.33 %
Final-term Examination 50.00 %
Oral Examination 10.00 %
Practical Examination 26.67 %
Total 100%
6- List of References
6.1- Course notes: Lecture and practical notes prepared by instructors.
6.2- Essential books (textbooks)
* Foye’s principles of medicinal chemistry, 8th edition 2019
* An introduction to medicinal chemistry, Patrick, Graham L. Oxford university
press, 2023.
6.3- Recommended books
* Medicinal chemistry for practitioners, Li, Jie Jack. John Wiley & Sons,2020.
* ADNIET for medicinal chemists', a practical guide, Tsaioun, Katya, and
Steven 4 Kates, eds. John Wiley 8 Sons, 2011
 Cont’d: List of References
6.4- Periodicals, Web sites, ... etc
- https://www.efmc.info/best-practices
- https://www.orqanic-chemistry.org
- https://pubchem.ncbi.nlm.nih.gov
- https://www.ebi.ac.uk/chembl
- https://drughunter.com

7- Facilities Required for Teaching and Learning

 Study halls
 Laboratories, equipment
 Online tools, books, audio-visual tools
 LMS System (Moodie)
Course structure, Assessment and Grading System
This course will cover 8 topics (1 topic/session) as outlined previously in the syllabus shown in the course
specifications. In each topic, we will be discussing a number of points that will be outlined in the Intended
Learning Outcomes (ILOs) section at the beginning of each chapter. These ILOs will be your guide for
studying each chapter. By the end of each chapter, measure your understanding of the topic by checking
that you have covered each point mentioned in the ILOs.
Next, you will find a Pre-session Reading section that will outline some resources that will help you get
ready before attending the session. These resources will offer a background for the topic that will be
discussed during the session and will prepare you to engage better in the learning process during the
session.
Following the Pre-session Reading, you will find the content of the topic that will constitute your learning
material in addition to any links or references that are referred within the text. Within each session, you
will find Learning Activities. These group activities will be used during the session for engaging the
students in an active learning process. Your active participation in these activities will be part of your
assessment for this course. In these activities, you will be asked to perform a task - in a group - aimed at
helping you understand the topic discussed in the session as a practical application.
At the end of each session, you will be asked to complete an assignment that will help assess your
understanding of the topic. This also is a part of your course assessment, and you will be expected to
hand in your assignment during the next session to receive a full mark. Late submission will result in a
50% deduction from your marks. For example, each assignment should receive 1.0 mark when submitted
on time (after 1 week), late submissions (submitted after 2-3 weeks) will receive a 0.5 mark. Much later
submissions (after more than 3 weeks) will not be eligible to receive any marks (0.0 mark for >3 weeks),
however, you will be welcome to discuss your answers with your demonstrator at any time.
The grading system used for this course is shown in the table below-
Grading Item Marks
In-session Activities 8 (1.0 mark X 8 sessions)
Assignments 8 (1.0 mark X 8 assignments)
Project 4
Practical Exam 20
Total Practical Marks 40

Your marks will be recorded by your TAs in the evaluation sheet at the end of this manual during each
session. You have to make sure that your marks are recorded after each session before you leave so as
not to lose any grades.
Maintaining and safekeeping the evaluation sheet is your responsibility. You will be asked to hand in your
evaluation sheet at the end of the course for recording.
Finally, we hope you will enjoy learning with US about the wonderful art of medicinal chemistry and drug
design throughout this course.

The Pharmaceutical Chemistry Department Staff

Fall Semester 2024-2025

Housekeeping Rules

We would love to hear your feedback on this course at

any time. Please follow the link to the course evaluation
form and share your thoughts with us!
https://forms.gle/SjoFvwM83Tpyv4gS8

Housekeeping Rules
To ensure that our practical sessions are running efficiently, the following rules are to be followed
at all times without exceptions:
 Lab Attendance: Sessions will always begin promptly. If you arrive more than 10 minutes late, you
will not be allowed into the lab without valid permission from a staff member. After 20 minutes, late
admittance will not be permitted under any circumstances.
 Food and Drink: Eating and drinking are strictly prohibited in the lab. Remember that the lab is
shared with organic chemistry courses, and chemistry experiments are conducted in the same
space, so please act accordingly.
 Lab Manual: You must have your lab manual with you at all times. Follow along during
demonstrations, fill in the blanks, and take relevant notes. You will also need your manual to receive
marks for activities and assignments.
 Completing Activities and Assignments: Make every effort to ensure you have the correct
answers for activities, assignments, and notes in your manual by paying attention during the session
 when answers are shared. Model answers will not be provided as a printout.
 Consulting with TAs: You are encouraged to discuss your answers with your TA’s during their
office hours, which will be shared during sessions.
 Group Participation: Active participation in group activities is expected, and each group member
should be prepared to answer questions about their group's results.
 Lab Conduct: Proper lab conduct must be observed at all times. If you are causing any disturbance,
you may be asked to leave the lab. If so, please follow your TA’s instructions. If you feel you have
been treated unfairly, you may speak with the supervising staff member on the same day.
 Feedback: You are encouraged to provide anonymous feedback after each session through the link
provided on the previous page.
 Mutual Respect: Remember, the lab is a place for learning. We are here to support your learning,
and mutual respect is essential.
Introduction to Drug Pharmacokinetics (ADMET)
Intended Learning Outcomes (ILOs)
1- Where our course falls in the drug discovery and development process.
2- What multifactorial optimisation means.
3- What property-based design and structure-property relationship mean
4- The relationship between physicochemical properties and drug pharmacokinetics, and which
physicochemical properties influence PK
5- Effects of lipophilicity on pharmacokinetics and pharmacodynamics, and how to manipulate it.
6- Effects of ionizability on pharmacokinetics and pharmacodynamics, and how to manipulate it.

Pre-session Reading
Read this paper: Veale, C. G. L. Into the Fray! A Beginner’s Guide to Medicinal
Chemistry. ChemMedChem 16, 1199-1225(2021).
Focusing on section 8, and then record your thoughts on the material you have
reviewed.
https://chemistry-europe.onlinelibrary.whiley.com/doi/10.1002/cmdc202000929
Notes from the pre-reading activity:
………………………………………………………………………………………………………
………………………………………………………………………………………………………
………………………………………………………………………………………………………
………………………………………………………………………………………………..
…………….
Understanding where our course fits in the broader drug discovery and development
process
At this point, you would have studied the drug design (PC 508) and medicinal chemistry l (PC 609)
courses where you would have explored the basis of rational drug design (hit finding, drug-target
interactions, bioisosterism, lead optimisation). You would have also ventured into the realm of
computational chemistry and the applications of molecular modelling in computer-aided drug design. By
now, you can understand the pharmacodynamics of drugs and how they achieve their intended biological
actions, and you are familiar with various drug design strategies aimed to improve and optimise drug
potency. But does a drug just need to be potent at engaging with its target? Is potency sufficient to
achieve clinical efficacy?
Remember the definition of pharmacodynamics?
…………………………………………………………………………………………………………………………
…………………………………………………………………………………………………………………………
……….
potent drug might be able to achieve the intended desirable effect in vitro, or even in some in vivo assays
under laboratory conditions. But for this molecule to progress into clinical trials and towards drug
approval, it will need to be able to reach its site of action and its target in vivo (remember bioavailability)
and translate this in vitro potency into a demonstrable in vivo effect, in other words, it needs to be
efficacious Additionally, it will have to be safe and stable enough for administration.
Remember the difference between in vivo and in vitro?
All these parameters and characteristics need to be carefully balanced in a molecule before it can
become a drug. As you can see in Figure 1, you might start with a hit compound that is highly potent, but
it is nowhere near a drug-like molecule. A multifactorial optimisation process must ensue where this hit
molecule is gradually modified to build in safety, stability and other desirable properties to achieve a safe
and efficacious drug. You can imagine how hard this process could be where we are simultaneously
optimising several factors - whilst maintaining potency - to obtain a molecule in that small intersection
(bullseye) that fulfils all these parameters required for a drug.
This hit-to-lead optimisation process is part of the role of medicinal chemists in the drug discovery
process. As you remember and as you can also see in Figure 2, medicinal chemiists are responsible for
the drug design process from the hit identification stage to the development of a drug (preclinical)
candidate. You are already familiar with the different approaches used for hit identification. Following hit
identification and validation, a medicinal chemist will start this journey of multifactorial optimisation to
achieve a safe and efficacious drug candidate. An efficacious drug needs to be potent at engaging its
target, able to reach its target in vivo, and safe for humans (or any other target organism). This ability of a
drug to reach its target in vivo and how a drug moves inside the body is known as pharmacokinetics.
Pharmacokinetics refers to the movement of the drug into, through, and out of the body which is also
known as ADMET that stands for Absorption, Distribution, Metabolism, Excretion, and Toxicity, which is
used to describe the various physiological processes in sequence that the drug substance encounters
from its initial input (dosing) to its final removal from the body.

Remember the definition of pharmacokinetics?

…………………………………………………………………………………………………………………………
…………………………………………………………………………………………………………………………
………
You will study the clinical applications of drug pharmacokinetics in detail in another course, however, in
this course we will focus on the medicinal chemistry perspective of drug pharmacokinetics. Meaning, we
will focus on how medicinal chemists modify chemical structures to influence and optimise the
pharmacokinetic profile of a hit compound. You are already familiar with the term 1‘structure-activity
relationships - SAR” where modifying the chemical structure of a compound affects the biological activity
of this compound. In this course, we
will learn about ‘Structure-property relationships', where the modification of the compound structure
affects its properties in terms of pharmacokinetics.1

Figure 1. Hit-to-lead optimisation can be a delicate balancing act of improving factors around your hit compound,
such as potency, solubility, safety, stability, etc. to ultimately achieve a safe and efficacious drug suitable for clinical
applications, (https://www.efmc.infofhit to lead)

The ultimate goal of lead optimisation into preclinical candidates is to achieve a drug that is …………….
And ………………………. And ……………………………………

1 A side note: in addition to the aforementioned medicinal chemistry courses, this course also builds on your
understanding of organic chemistry (drug structures) and physical chemistry (physicochemical properties).
Figure 2. A classic representation of the drug discovery pipeline, describing the various progressive steps in the
development of preclinical candidates. Typically, a medicinal chemist's most prominent role occurs from hit
identification to lead optimization. Reference: Veale, C. G. L. Into the Fray! A Beginner's Guide to Medicinal
Chemistry. ChemMedChem 16, 1199-1225 (2021).
Physicochemical properties and Pharmacokinetics
So, which drug properties do you think will influence its movement “kinetics” inside the body?
You have guessed correctly, that will be the physicochemical properties of the molecule. Logically, the
physical properties (that are directly related to and dependant on the chemical structure, hence
physicochemical) of the molecule will affect how it moves and interacts with the biological environment
inside the body. Imagine throwing a group of irregularly shaped small objects or trinkets in a bowl full of a
liquid that is being stirred, can you imagine the behaviour of the movement of these objects in the liquid
and what will influence this behaviour (Check Figure 3 for inspiration)? You might have guessed that their
sizes, their material (and consequently density), their shapes (if it is trapping air!) will affect their
behaviour. Similarly, but on a molecular scale, the behaviour of the drug molecule in the body will be
directly influenced by its physico-chemical properties.
 Figure 3. An illustration showing the trinkets falling into a bowl full of a
liquid that is being stirred with a magnetic stirrer at the bottom.
The figure was created using ChatGPT, so excuse the unrealistic
representation of a magnetic stirrer.

Course objectives:
In this course, you will learn which physicochemical properties influence each step of the ADMET
process, the relationship between the molecular structure and these properties (structure-property
relationships), and how medicinal chemists try to manipulate molecular structures to modify these
properties and optimise the pharmacokinetic profile of a drug (property-based design).

So, which physicochemical properties will directly influence drug pharmacokinetics?

With your group, you will be assigned either one of the following learning activities where you will explore
key physicochemical properties that influence pharmacokinetic profiles of drugs.

Learning Activity (1): Lipophilicity - Parameters & Binding

Lipophilicity is one of the most important physicochemical properties that influences the behaviour of
drugs both in vitro and in vivo and has a direct effect on each step of the ADME process. Lipophilicity is a
measure of how greasy a molecule is. It has a profound impact on a drug's ADME because it is closely
associated with drug’s solubility, plasma protein binding, metabolic clearance, volume of
distribution, biliary and renal clearance, enzyme/receptor binding ..

LogP and LogD are two of the most used quantitative measurements of
a molecule’s lipophilicity. With your group, discuss the difference
between LogP and LogD, and which one is more relevant to drug
design, and then answer the questions below.
You can use these links to help you find the answers for these
questions:
1) Bunally, s. B., Luscombe, c. N. & Young, R. J . Using
Physicochemical Measurements to Influence Better Compound
Design. SLAS Discovery 24, 791-801 (2019).
https://Journals.saoepub.com/doi/full/
10.1177/2472556219859845
2) Lipophilicity Descriptors: Understanding When to Use LogP
& LogD.
https://www.acdlabs.com/wp-contentzubioads/
download/app/physchem/logp vs logd.pdf
3) LogP—Making Sense of the Value.
httns.VfanMW.acds.com/WD-conte/uploads/
The definition of LogP,
download/app/physchem/making sense.pdf& the meaning of its values (scale
representation):
 The definition of LogD:

Which is more relevant for drug design?

“As the value of LogP increases, binding to targets such as receptors and enzymes
is increased.”, why would increased lipophilicity drive protein binding and
potency? (Hint: remember protein folding, and the hydrophobic effect, also, refer to
the pre-reading paper)

What is molecular obesity?

‫ إ‬Learning Activity (2): Lipophilicity -- Lipinski’s rules & Balance
Before 1991, potency was the major thrust of medicinal chemistry. The medicinal chemist’s focus was to
make the drug as potent as possible and then handed it over to the “formulation people” to make it
bioavailable. Regrettably, in 1991,40% of drugs in clinical trials failed due to poor
pharmacokinetics/bioavailability. That untenable attrition rate sent the message home. The industry took
notice and started paying closer attention to drugs' pharmacokinetic profiles. Consequently, 10 years later
in 2000, the attrition rate from poor pharmacokinetics in clinical trials was reduced five-fold to merely 8%.
During that period, they conducted extensive research on the optimal physicochemical properties that can
allow molecules to become drugs, in other words, to have drug-like properties.
Among these studies, the most prominent one is by Lipinski et al. (1997)2 which later became known as
Lipinski’s Rule of 5.
With your group, discuss how these “Rule of 5" relate to
lipophilicity, and why would molecules that are following these
rules have good drug-like properties? Then answer the following:

Briefly, what is the Rule of 5?

1.
2.
3.
4.

How do you relate lipophilicity to these rules? Where can we see the influence of lipophilicity on
these drug-like properties?

2 Lipinski, C. A, et al. Experimental and computational approaches to estimate solubility and permeability in drug
discovery and development settings. Advanced Drug Delivery Reviews 23,
What about hydrophilicity? Do you see limits for hydrophilicity in
these rules? Why do you think there are limits for hydrophilicity?

Discuss the meaning of the following figure with your group.

How would increasing/decreasing lipophilicity affect potency? Solubility?

Permeability? Metabolism? And toxicity? [Check Appendix (1)]
Learning Activity (3): lonizability
"In addition to hydrophilicity and hydrophobicity, the acidic, basic or zwitterionic properties of small
molecules, and its relationship to ionization state has a significant influence on drug properties. For
example, basic compounds which are ionized at low pH environments, display good solubility but tend to
be poorly absorbed in the stomach, as well as being more frequently associated with mechanisms of
toxicity such as phospholipidosis and hERG binding. Acidic compound, on the other hand, display a
higher tendency to bind to plasma proteins, thus influencing bioavailability and volumes of distribution. In
addition, the ability of a small molecule to access a binding site (e. g., electrostatic steering) and form
favourable binding site interactions will in many cases be dependent on a ligand's ionization state at the
pH of the local environment. Therefore, the modulation of pKa through medicinal chemistry is an
important optimization consideration." - Veale, C. G. 1. Into the Fray! A Beginner’s Guide to Medicinal
Chemistry. ChemMedChem 16, 1199-1225 (2021).
Discuss with your group the previous passage, then answer the following questions. You can refer to the
following references for guidance:

1) Bunally, S. B., Luscombe, C. N. & Young, R.. Using Physicochemical Measurements to Influence
Better Compound Design. SIAS Discovery 24, 791-801 (2019).
2) Check Appendix (1) and (2) at the end of the manual.

What is pKa?
When will a compound be ionized in term of its pKa and the pH? Which compounds will be
ionized at pH 7.4?

Which is more favourable for a drug, to be acidic, basic, zwitterionic, or neutral in terms of
bioavailability and potency? And why?

The following figure illustrates the key properties that are influenced by the acid/base
character of drugs. [Reference: Manallack. et al. The significance of acid/base properties in
drug discovery. Chern. Soc. Rev. 42, 485-496 (2013)].
Discuss with your group how would ionizability influence these properties? (check Appendix
(3))
 Molecular interactions
ionic interaction
Dipolar interactions
ion dipole interactions
Hydrogen bending

LJrug design factors Drug Add/Base Formulation

Biological activity Equilibria Solubility
Receptor interactions Complexation
(lipophilic) ligand efficiency Partitioning
Drug-likeness Osmolarity
Off target activity Chemical stability
Biopharmaceutics
permeability
Absorption
Distribution
Metabolism
toxicity
A final note on the importance and impact of this course: as a pharmacist. your focus is the optimal
management of medication use, taking into consideration drug-drug interactions, patients’ conditions,
adverse effects,... etc.
So, it is important that you understand drug pharmacokinetics (PK), how to interpret PK profiles of drugs
in relation to their physicochemical properties, and how the body affect a given drug and vice versa, to be
able to make an informed decision on medication use.

Note-taking Space:
 Assignment (1)

In this paper:
Cotman, A. E. et al. Discovery and Hit-to-Lead Optimization of Benzothiazole Scaffold-Based DNA
Gyrase Inhibitors with Potent Activity against Acinetobacter baumannii and Pseudomonas aeruginosa.
Journal of Medicinal Chemistry 66, 1380- 1425 (2023)

https://pubs.acs.org/doi/full/10.1021/acs.jmedchem.2c01597
They wanted to develop Topoisomerase inhibitors as antibacterial agents against A. baumannii, which is
a type of resistant Gram-negative bacteria. They optimised the hit compound (1) into compound (27)
where they have managed to improve the enzyme inhibitory activity (on-target potency, Co against
topoisomerase), but they could not improve the cellular activity (MIC against A. baumannii). They tried to
further improve the physicochemical properties of (27) by reducing the lipophilicity which they have
managed to achieve in compound (66), however, the potency was severely compromised. Based on your
understanding of the topic, please answer the following questions:
1) Complete the following table using the data in the paper for the enzyme inhibition and MIC.
Use this online platform to calculate LogP and LogD? 4 and pKa (acid) for these compounds, and fill in
the table:
https://admetlab3.sobdd.com/
 Compound

A. baumannii
Topoisomerase IC50 (nM)

A baumanni, MIC (pg/mL)

Calculated LogP

Calculated LogD/7.4

pKa (acid)

2) Why do you think the on-target potency improved 10x fold from (1) to (27)? Why do you think this
improvement did not correlate with the cellular activity that remained the same in both compounds? (Hint:
Gram-negative bacteria favour more polar compounds; more polar compounds can penetrate Gram-
negative bacteria better).
3) What caused the changes in LogP from (1) to (27) to (66)? Can you identify the strategies used to
increase/decrease lipophilicity?

4) Why do you think there is a difference bet een the LogP and LogD 7.4 values of all three compounds?
5) Between compounds (1) and (27), which is more acidic? And why?

6) What would be the ionization state of these compounds at pH 7.4? Draw the major species. Which of
these compounds would have a favourable ionization pattern in terms of pharmacokinetics?
(2)Absorption
Intended Learning Outcomes (ILOs)
1- Understanding the process of drug absorption, the different routes of administration, and the
mechanism‫ ؛؛‬of absorption.
2- Understanding the main factors affecting absorption and bioavailability.
3- Explore strategies to improve solubility to increase drug bioavailability.

Pre session Reading

Watch this video on YouTube: "Pharmacokinetics: Drug absorption and distribution” by Osmosis from
Elsevier,
And then answer the following questions”

1 ) What are the different routes of drug administration

Routes of Administration

All the previous routes of administration undergo an absorption phase, except for the intravascular route,
which delivers the drug directly into the circulatory system without the need for absorption.
2) What are the different mechanisms of absorption (transport mechanisms)? And the nature of
the drugs that use each mechanism?

Mechanism Properties of drugs using this mechanism

Small, hydrophobic, unionized, mostly following

1
Lipinski’s rules.

2 Hydrophilic, polar.

4 large drugs (peptides).

3) Can you relate between the properties of drugs required for absorption and the physicochemical
properties that you studied last session? Can you see the influence of lipophilicity and ionizability on
absorption?
Definition of Absorption and Bioavailability
Absorption is the first step in pharmacokinetics, describing the drug's journey through the human body,
which consists of three stages: Absorption phase - where the absorption rate is higher than the
elimination rate. Post-absorption phase - where the elimination rate surpasses the absorption rate.
Elimination phase - where no significant absorption occurs, and only elimination takes place.
In simpler terms, it refers to the movement of the uncharged drug from the site of administration to the
bloodstream. This phase represents the time (/max) from drug administration until it reaches its maximum
concentration (Cmax) in the blood.

Drug absorption is quantified in terms of bioavailability (F%).

Remember the definition of Bioavailability?

………………………………………………………………………………………………………
……………..
………………………………………………………………………………………………………
……………………………………………………………………………………
In this session, we will focus on oral bioavailability and GIT absorption.

Factors that influence bioavailability of a drug include:

 Solubility of the drug;
 Permeability of the drug;
 Chemical stability;
  First-pass hepatic metabolism;
 and nature of drug formulation.
These factors affect the amount of drug reaching the bloodstream following oral administration in an
unchanged form. (Summarised in Figure 4)
For a drug to be absorbed, it has to be dissolved first from the solid form (the dosage form) into the GIT
fluids. Not surprisingly, aqueous solubility is a key factor to influence a drug’s bioavailability. Aqueous
solubility is directly influenced by lipophilicity (OgPogD) and ionizability (remember session 'I).
How do you think lipophilicity and ionizability influence solubility?
/ Lipophilicity   solubility
/ ionizability   solubility

The drug also must be permeable to permeate through the different membranes from the
GIT to the site of action.

Remember the different mechanisms of absorption from your pre-reading?

………………………………………………………………………………………………………
……………………………………………………………………………………………………

Drug permeation will depend on the physicochemical properties of the drug, if they are suitable for its
corresponding mode of absorption. You might have noticed that the properties required for good aqueous
solubility (hydrophilicity/ionizability) might hinder permeability (mainly by passive diffusion). The FDAs
Biopharmaceutical Classification System divides drugs into four classes as shown in figure below:
 Class I Class II
 High Solubility  Low Solubility
 High permeability  High permeability

Class III Class IV

 High Solubility  Low Solubility
 Low permeability  Low permeability

If we are aiming towards Class I drugs (high solubility and permeability), these properties (lipophilicity and
ionizability) will need to be carefully balanced to fulfil the requirements for the two aspects. This is where
Lipinski's rules come in (remember session 1). It was found that the majority of molecules that follow
these limits (of the Rule of 5) often have good oral absorption as they possess this balance of lipophilicity
required for both good solubility and permeability.
The third factor is the chemical stability of the drug molecule; it needs to be stable enough to endure oral
administration and passage through the Gif with all its enzymes and pH changes. For instance, drugs that
are chemically unstable in the stomach pH or susceptible to enzymatic degradation tend to disintegrate
within the Gif. A famous example of a drug that is chemically unstable - and hence cannot be
administered orally - is the miracle antibiotic "Penicillin G" which can only be administered by injection.
Penicillin G has a highly reactive and strained p-lactam ring that can be easily hydrolysed under acidic
conditions (the stomach). You will learn more about the reasons for this chemical instability and the
approaches that had been used by medicinal chemists to improve its stability in the lectures, however,
you can read more about this here in Appendix (4) at the end of the manual.
When a drug is absorbed from the GIT, it enters the portal circulation before entering the systemic
circulation. If the drug is rapidly metabolized in the liver or gut wall during this initial passage, the amount
of unchanged drug entering the systemic circulation is decreased. First-pass metabolism by the intestine
or liver limits the efficacy of many oral medications. For example, more than 90%% of
nitroglycerin is cieared during first-pass metabolism. Hence, it is primarily administered via the sublingual
or transdermal route. Drugs with high first-pass metabolism should be given in doses sufficient to ensure
that enough active drug reaches the desired site of action.

Figure 4. Route of absorption of orally administered drugs showing a summary of the factors affecting absorption.

Imorcvinu Solubility via structural Modifications

As previously mentioned, proper aqueous solubility is an essential property for drug-like molecules (to
possess good absorption and distribution), and aqueous solubility is directly influenced by lipophilicity and
ionizability. Therefore, attempts to improve the solubility of poorly soluble drugs will mainly involve
structural modifications that aim to increase hydrophilicity ( LogD7.4) as summarized below:
With your group, you will explore these different structural modification
strategies to improve solubility in one of the following learning activities:
You will need to use this online platform “ADMETIab 3.0” to calculate LogP and LogD/4 and pKa for some
compounds. You can obtain the SMILES for the marketed drugs from PubChem.
Learning Activity (1): insertion of polar/ionisable group
The addition of hydrophilic and ionizable groups are the most common and classical strategies to improve
solubility. It has been reported that the installation of hydrophilic and ionizable groups can enhance
solubility by reducing the lipophilicity (LogD7.4) of lead compounds. This is one of the impressive
techniques to improve the solubility of poorly soluble drugs even for large hydrophobic molecules. Below
are some examples that make use of this strategy to improve solubility.

What is the difference between LogP and LogD7.4? and why do we use LogD/4 here to describe
lipophilicity?

During the discovery of the first tyrosine kinase inhibitor imatinib, a basic side chain containing piperazine
was attached to phenylaminopyrimidine to improve its solubility. The piperazine also enhanced ligand
binding to the BCR-ABL kinase which was a pleasant surprise. The excellent aqueous solubility of
imatinib achieves a remarkable bioavailability of 98% in humans.
Why would the attachment of this piperazine improve solubility? What is the effect of this piperazine on
the calculated LogD7.4and pKa)

Phenylaminopyrimidine Imatinib

logD

pKa

What do you think the ionization state of imatinib at pH 7.4 would be?

4-Aminoquinazoline is a potent kinase inhibitor, but it has poor solubility. In an effort to boost its aqueous
solubility, a basic piperidine ring was installed on the side chain replacing the triazole ring, which resulted
in better solubility with up to a 500-fold improvement at pH 7.4.

Why do you think this piperidine resulted in this remarkable improvement in solubility? (Think in terms of
its effect on LogD7.4and pKa)
The following compound, a potent, third generation HIV-1 attachment inhibitor displayed poor water
solubility at higher doses on oral exposure. Solubility enhancement was achieved by introducing polar
and ionizable amino moiety in the triazole side chain. The modified compound showed improved
solubility, but displayed poor antiviral potency and decreased membrane permeability in comparison to
the parent compound.

Why do you think this amino moiety impeded permeability and reduced potency?

Learning Activity (2): disruption of hydrogen bonding

For a molecule to dissolve in water, the existing intermolecular hydrogen bonding must be broken so that
the solvate molecule may form intermolecular hydrogen bonding with water molecules. Thus, installation
of hydrogen bond donors as well as hydrogen bond acceptors such as OH and NH2 groups can enhance
aqueous solubility by forming hydrogen bonds with water molecules. At the same time, it has been
reported that the removal of hydrogen bonds may also result in an increase in solubility possibly by the
reduction in melting point (melting point indicates the stability of the crystal lattice). Based on these
observations, we will cover some successful examples of improving solubility by addition/removal of
hydrogen bonding.
Why do you think the removal of hydrogen bonding can reduce the melting point and improve the
solubility?

Thalidomide is probably one of the most famous drugs because of its teratogenicity. It has low lipophilicity
and an abysmal solubility of 52.1 mg/L. N- methyl-thalidomide, with the hydrogen bond donor eliminated,
has an aqueous solubility of 275.9 mL, a more than 5-foki boost over that of thalidomide.

Why do you think the methylation of thalidomide improved its solubility despite the increase in its
lipophilicity? And why is the lipophilicity of the N-methyl-thalidomlde higher than that of thalidomide?

Pyramidinotriazine derivative was found to have cytoprotective activities against rotenone toxicity but
suffered poor aqueous solubility. Scientists tried to improve the solubility by incorporation of CF3-
substituted phenyl ring. Further
incorporation of a variety of alkyl groups resulted in no improvement in solubility. This points to the fact
that the observed improvement in solubility is due to the incorporation of CF3-substituted phenyl ring.

How would CF3 groups improve solubility despite their lipophilicity? What is the calculated LogP of these
compounds?

In another study on camphoryl khellactone derivatives, the starting compound was found to possess
moderate anti-HIV activity. The poor solubility and bioavailability of the compound were resolved by the
introduction of a hydroxymethyl side chain, and the resulting compound showed improved solubility and
anti-HIV activity.

Comment on this improvement:

Learning Activity (3): disruption of molecular planarity
A drug’s crystalline lattices must be broken first in order to be dissolved. The more crystalline the material
is. the more difficult for it to dissolve. As a compound progresses through the pipeline, one often finds that
its solubility keeps “diminishing". This.is the result of an artificial artifact. When the compound is prepared
for the first time, the purity requirement is not that stringent, greater than 95-98 ٥/ ٥purity is more than
sufficient for the purposes of biochemical and cellular assays. As the compound progresses from a hit to
a lead, then to a drug candidate, the criteria for its purity grow. Many of them are crystalline. In fact,
crystalline forms are vital intellectual properties associated with innovative medicines. Empirically, Π-Π
stacking enhances crystallinity of compounds containing aromatic rings. Therefore, disruption of planarity
thus aromaticity results in improvement of solubility. It is an alternative strategy to classical approaches
like the addition of hydrophilic groups or reducing hydrophobicity, which often leads to reduced bioactivity.

How does molecular stacking enhance crystallinity?

These are y-secretase inhibitors with similar potencies. Bicyclo [1.1.1) pentane (BCP) was employed as a
bioisostere for the fluorophenyl moiety. This disruption of aromaticity lead to a higher solubility, and
increased membrane permeability.

Why do you think the membrane permeability improved with this change?
This vanilloid receptor-"! antagonist, 4-oxopyrimidine, behaves like “brick dust” with no solubility to speak
of. Disruption of the aromaticity of the trifluorophenyl motif gave the resulting compound, which shows a
13-fold boost of aqueous solubility over the starting compound. The fact that the partially saturated
compound has a significantly lower melting point is a good indication that reducing the planarity disrupted
the crystal-stacking capacity.

What is the relationship between melting point, crystallinity and solubility?

The lead compound pyrazolopyridine inhibitor of B-Raf has a poor aqueous solubility because the
molecule is flat with extensive Π-Π stacking. Two maneuvers were taken to disrupt the planarity. One of
the two fluorine atoms was replaced with a chlorine atom, which might increase the energy barrier for the
phenyl-amide single bond rotation (which helps in disruption of its planarity by increasing the dihedral
angle of bi -aryl moiety). Secondly, the methoxy group was replaced with the bulkier cyclopropyl group to
afford the resulting analog, whose melting point is lower than the starting point. The solubility of the final
compound at pH 7.4 is 14 times higher than the starting.

How did these changes disrupt molecular planarity?

Learning Activity (4): bioisosterism

Bioisosteric replacement is one of the most used classical approaches to improve solubility, potency,
selectivity, and other desired pharmacokinetics of the problematic molecules Bioisosteres can be
classified into two types i.e. classical bioisosteres (monovalent, divalent, trivalent atom or groups, tetra
substituted, and ring equivalents) and non-classic bioisosteres (cyclic & noncyclic functional groups).
Bioisosteric replacement of phenyl group of a lead molecule with pyridine, thiophene, or any other
heterocyclic may improve solubility.
CK-2-68 is a new molecule having good potency towards Plasmodium falciparum but it suffered from
pharmacokinetic limitations due to high LogP value and lack of aqueous solubility. CK-2-68 had a
quinolone core with a phenyl side chain. In order to reduce the logP and improve aqueous solubility,
several bioisosteric replacements of the phenyl side chain were carried out using various heterocyclic ring
systems, which ultimately resulted in a new molecule called SL-2-25 which had a pyridine side chain
instead of a phenyl. The pyridyl quinolone derivative helped in improving the solubility and activity against
p. falciparum (IC50 = 54 nM), and reduced LogP from.6.4 to 4.2.

Why did this bioisosteric replacement improve solubility?

2-Aryl quinolone derivative was found to have good inhibitory activity against mitochondrial cytochrome
complex in malaria. 2-Phenyl-quinolone derivative had very low aqueous solubility and LogP value of 5.6.
To enhance the aqueous solubility of the lead compound, the phenyl ring was substituted with a pyrazole
ring. The 2-pyrazolyl quinolone derivative showed improved aqueous solubility and lower lipophilicity
(LogP 3.7).
Why did this bioisosteric replacement improve solubility?

This novel benzodiazepine derivative is a T-type calcium channel blocker with poor aqueous solubility.
The optimization studies resulted in the bioisosteric replacement of the benzene ring of benzodiazepine
with pyridine which resulted in improved aqueous solubility and activity (IC.50 = 55 nM).

Why did this bioisosteric replacement improve solubility?

Learning Activity (5) prodrug
Prodrug strategy is one of the alternative approaches to overcome various obstacles in drug discovery,
including poor solubility. A prodrug is an inactive compound metabolized either chemically or
enzymatically in a controlled or predictable manner to the parent active form inside the body. Two major
classes of prodrugs are carrier linked prodrugs and bio-precursors.
Carrier-linked prodrugs is a convenient method to design drugs with major solubility issues, which are
connected to a nontoxic carrier or moiety through covalent linkage to change or get rid of their
undesirable physicochemical properties. These prodrugs undergo enzymatic or nonenzymatic cleavage to
release the active drug moiety. Carrier-linked prodrugs can be a bipartite prodrug (carrier is directly
connected to parent dugs) or tripartite prodrugs (spacer connects carrier to the parent drug). Various
types of carriers have been used to improve the solubility of poorly soluble compounds, such as amide
prodrug, phosphate prodrug, imine prodrug, carbonate prodrugs, and ether prodrugs.
Bio-precursor prodrugs are the type of prodrugs produced by chemical modification of the active
molecule. Different enzymes transform them into the active form. Bio-precursors do not have a temporary
linkage between a carrier group and the active moiety.

Draw a diagrammatic representation of the two types of prodrugs highlighting the differences between
them:
Ampicillin is currently a widely used antibiotic due to its broad-spectrum bactericidal activity and reduced
toxicity However, its oral bioavailability is only 30% to 50% due to its low absorption due to the presence
of two polar groups (NH2, COOH). An improvement has been achieved by esterification of the carboxylic
group which leads to increased lipid solubility and improved absorption. Bacampicillin is an example of a
classic ampicillin prodrug with bioavailability greater than 95%.

Calculate the LogD7.4 and pKa of both compounds, then demonstrate the effects of this carrier on
solubility, ionizability and permeability.

Ampicillin Bacampicillin

LogD7.4

pKa

Solubility

lonizability

Permeability
The prodrug approach was used for anthelmintic drug mebendazole to improve its solubility. A
prodrug containing a phosphoryl-based moiety showed almost 10,000-fold improvement in
aqueous solubility and improved oral bioavailability and metabolic stability.

Why did this side chain lead to that dramatic improvement of solubility?

Tedizolid phosphate is an FDA approved prodrug of tedizolid, used for the treatment of acute
bacterial skin infections. This drug is more effective than its parent drug tedizolid due to its higher
aqueous solubility with lesser adverse effects.

How is tedizolid phosphate converted into tedizolid?

 Assignment (2)
1) Summarise the strategies of structural modification used to improve solubility that were discussed
during the session.

strategy Physicochemical rationale

1

5
2) Using the online platform of SwissADME (httowww swissadme.chindex.Dlo) to predict solubility,
suggest 3 structural modifications to Improve the solubility of the following compound (and
calculate their solubilities):

Suggested Modification strategy Calculated solubility

(3) Distribution
Intended Learning Outcomes (UOs)
1- Understanding •drug distribution, and its parameters.
2- Understanding the physicochemical properties affecting distribution.
3- Understanding the limitations of distribution caused by the blood brain barrier.
4- Understanding the effect of plasma protein binding on distribution.
5- Explore strategies to manipulate drug distribution.

Pre-session Reading
Watch this video on YouTube: “Pharmacokinetics: Drug absorption and distribution” by Osmosis from
Elsevier,
https://youtub.be/a0-8a0zrzi4?si=2xZDMc5UBzerrla
(you can start the minute 8:00) and then answer the following questions:
1) What are the factors governing the extend of drug distribution? And the physicochemical
properties affecting distribution?
1

3
2) What is the apparent volume of distribution (Vd)?

Volume of Distribution (L) = ----------------------------------------------

Around the Blood Supply, to Tissues, arid to Cells

Once a drug is absorbed, it is subsequently distributed around the blood supply and to tissues and
cells. Distribution is the process by which a drug reversibly leaves the bloodstream and enters the
interstitial or cellular fluid of the body. All the fluid in the body (total body water) in which à drug can
be dissolved may be roughly divided into three compartments: intravascular (blood plasma found
within blood vessels); interstitial/tissue (fluid surrounding cells), and intracellular (fluid within cells,
i.e., cytosol). The distribution of a drug into these compartments is dictated by its physical and
chemical properties.
The volume of distribution (Vd) is a pharmacokinetic parameter representing an individual drug s
propensity to either remain in the plasma or redistribute to other tissue compartments. Vd is a
proportionality constant that relates the total amount of drug in the body to the plasma
concentration of the drug at a given time (check the equation from your pre-reading). The larger the
volume of distribution, the more likely that the drug is found in the tissues of the body. In contrast,
the smaller the distribution volume, the more likely the drug is confined to the circulatory system.
Physiochemical Properties Governing Volume of Distribution
1) Acid-Base Characteristics (ionizability)
Depending on the charge of a drug at physiologic pH, a drug may tend to bind macromolecules
inside or outside the plasma.
 Basic (alkaline) molecules have strong interactions with negatively charged phospholipid
head groups located on phospholipid membranes. The extent of this binding is also dependent
on the overall lipophilicity of the drug. In general, basic molecules will leave the systemic
circulation leading to higher Vd as compared to acidic molecules.
 Acidic molecules have a higher affinity for albumin molecules at lower lipophilicity than
neutral or basic molecules. Therefore, acidic drugs are more likely to bind albumin and remain
in the plasma leading to lower Vd as compared to more basic molecules.

Is it Acidic or Basic? Is it Acidic or Basic?

Why? Why?

Effect on Vd? Effect on Vd?

2) Lipophilicity
In addition to ionic/charge-related interactions between a drug and macromolecules, hydrophobic
interactions also play a similar role.
 Lipophilic molecules are more likely to pass through lipid bilayers and therefore more likely to
leave the bloodstream and distribute to areas with high lipid density (adipose) which is why
they have a higher Vd. However, plasma proteins such as albumin have a high affinity for
lipophilic drugs in which case, the determinant of the extent of plasma protein binding of two
equally lipophilic drugs is the acid/base characteristics.
 Hydrophilic molecules are less likely to pass through lipid bilayers and therefore more likely
to remain in the bloodstream, which is why they have a lower Vd.

Basic: ----------------- Vd Acidic: --------------- Vd

Lipophilic: ------------------ Vd Hydrophilic: ------------------- Vd

3) Tissue Storage of specific drugs

Drugs may accumulate in specific organs or become bound to specific tissue constituents (tissue
storage) depending on their physicochemical properties. Lipophilic compounds may accumulate in
fatty tissues. For instance, thiopental tends to collect in adipose tissues; iodine in the thyroid gland;
calcium and tetracyclines (calcium chelation) in bones and teeth.
Blood Brain Barrier (BBB) penetration
CNS drugs must cross the BBB to enter the brain first, and then exert their pharmacological
effects. Endogenous influx transporters resided at the BBB shuttle nutrients such as
carbohydrates, amino acids, and purine nucleosides to the brain, but the BBB blocks xenobiotics
from entering the brain.

The requirements for CNS drugs’ physicochemical properties are more stringent than other drugs
to cross the BBB via passive diffusion. BBB is largely comprised of lipids thus only lipid-soluble,
unionized drugs penetrate to act on the CNS. and highly polar molecules with strong hydrogen
bonding capacity do not traverse BBB readily.

Lipinski arrived at a rule for CNS penetration. It states that a drug is likely to have good CNS
penetration if it has:
1. Molecular weight ≤ 400
2. Log P = 2-5
3. Hydrogen bond donor ≤ 3
4. Hydrogen bond acceptor ≤ 7
Otherwise, the drug is unlikely to have good BBB penetration.

Conversion of Morphine to the bis-acetyiated derivative Heroin significantly elevated the

lipophilicity as reflected by the boost of the Log p value. The fact that Heroin’s brain penetration is
100-fold greater than that of morphine speaks volumes for the fact that lipophilic drugs are more
likely to penetrate BBB.
In contrast, BBB several'/ limits the entry of non-lipid-soluble, hydrophilic drugs (nominally defined
as drugs with Log p < 0) as exemplified by Amikacin and Gentamicin.

The neurotransmitter dopamine plays an essential role in Parkinson's disease, psychosis,

attention deficit hyperactivity disorder (ADHD), and many other mental disorders. However,
armed with three polar groups, dopamine is too polar to enter the brain via BBB. In contrast, its
precursor levodopa does, thanks to the amino acid transport located on BBB. Once in the brain,
levodopa is metabolized to dopamine.

Levodopa can be considered to be which type of prodrugs?

……………………………………………………………………………………………………
The mechanism of Levodopa transport across the BBB is considered to be
which type of drug absorption
……………………………………………………………………………………………………
Plasma Protein Binding
Drugs can bind to protein macromolecules in the blood, a phenomenon known as plasma-protein
binding (PPB). As shown in this figure, the protein-bound form of the drug must dissociate from
the protein in order to be useful because only unbound compound is available for distribution into
tissues. There are three types of plasma •proteins: human serum albumin (HSA) and a-1 acid
glycoprotein (AAG) are the two more abundant proteins, whereas the third plasma protein,
lipoprotein, is of less importance for PPB.

Human serum albumin (HSA), the most abundant protein in human blood plasma, has more
than six distinctive binding sites including:
- two for long-chain fatty acids;
- one for bilirubin;
- and two for acidic drugs (Site I for warfarin and phenylbutazone, etc., and Site II
for diazepam, ibuprofen, etc.).

Drugs extensively bound to plasma proteins are largely restricted to the vascular compartment
and have low Vd (e.g., warfarin is 99% protein-bound and its Vd is 0.1 L/kg). Drugs sequestrated
in tissues may have Vd values much higher than the total body water or even body mass (e.g.,
propranolol, 3.5 L/kg and digoxin, 6 L/kg) because most of the drugs is present in other tissues,
and the plasma concentration is low. In case of poisoning, drugs with large Vd are not easily
removed by hemodialysis.
Clinical implications of drugs‘ PPB are summarized below:
1. There is an equilibration between the PPB fraction of the drug and the free molecules of the
drug. The PPB fraction is not available for action.
2. The drugs with a high physicochemical affinity for plasma proteins (e.g., aspirin,
sulfonamides, chloramphenicol) can replace the other drugs (e.g., acenocoumarol, warfarin) or
endogenous compounds (bilirubin) with lower affinity.
3. A high degree of protein binding makes the drug long-acting because bound fraction is not
available for metabolism unless it is actively excreted by the liver or kidney tubules.
4. Generally expressed plasma concentrations of the drug refer to bound as well as free drug.
5. In hypoalbuminemia, binding may be reduced, and a high concentration of the free drug may
be attained (e g., phenytoin)

How do you think these implications of PPB will affect drug administration?
With your group, you will explore different structural modification strategies to improve
distribution in one of the following learning activities:
Remember that you can use this online platform ‘ADMETIab 3.0" to calculate LogP and LogD7.4
and pKa for compounds. You can obtain the SMILES for the marketed drugs from PubChem.

Learning Activity (1): Improving Drug Distribution by Changing

AcidZBase Character
Nifedipine (Adalat), a first-generation calcium channel blocker, is a neutral drug with a moderate
Vd of 0.75 L/Kg. It has a short half-life of 2 h, thus must be taken three times a day. In contrast,
amlodipine (Norvasc), a third-generation calcium channel blocker, has a very high Vd of 21 L/Kg
due to its basic center (pKa 9.45), As a result, the elimination phase half-life is 35 h, and it is
administered once a day.

What is the impact of increasing Vd on dosing?

How to increase Vd to elongate half-life?

Erythromycin is a macrolide antibiotic with one basic center. It exhibits a volume of distribution of
4.8 L/kg and a half-life of 3 h. The introduction of a second basic center into the macrolide aglycone
ring in erythromycin yields azithromycin. This change increases the volume of distribution to 62
L/kg and together with a ~ 2- fold reduction in clearance results in a half-life of 18 h. Azithromycin
can therefore be given once a day for a short period compared to multiple administration per day
for erythromycin over a prolonged period.

What is the impact of increasing drug basicity on Vd? And half-life?

Calculate the LogD7.4 of these compounds, and comment on their lipophilicity. How is that
reflected on their Vd values?
Learning Activity (2): Increase Volume of Distribution through Target
Mediated Drug Disposition (TMDD)
When a therapeutic target has a sufficiently high concentration in the body, binding to the target
protein can increase the volume of distribution. Additionally, if the target is expressed at high
levels in particular cells or organs, the drug will be more distributed to these cells or organs than
the rest of the body due to target- mediated drug disposition (TMDD).
TMDD is relatively rare for small molecules. One example is the HSP90 Inhibitors for the
treatment of cancer. HSP90 is one of the most abundant intracellular proteins that promotes
survival, cell proliferation, and anti-apoptosis which is overexpressed in many types of cancers. It
has been shown that the more potent HSP90 inhibitors have larger Vss (~10 L/kg) than the less
potent inhibitors (-2 L/kg). (Vss = volume of distribution at steady state)
In the following compound, fluorine substitution of hydrogen not only improved inhibition potency
(Ki) but also increased Vss. The increase of Vss with enhanced target binding potency appears to
be due to TMDD. This leads to a sustained effect of the drugs on the tumors because they are
extensively distributed to the cancer tissues through high binding to the pharmacological target
(HSP90), this would only work if the target were expressed at high enough levels to bind a
significant portion of the drug that is in the body.

R Vss (L/Kg, Rat) In Vitro HSP90 Inhibition Ki (nM)

H 1.8 6.6
F 10 1.7
Why do you think this fluorine substitution enhanced potency?

Using this reference: An. G. “Concept of Pharmacologic Target-Mediated

Drug Disposition in Large-Molecule and Small-Molecule Compounds".
The Journal of Clinical Pharmacology 60, 149-163 (2020).
https://www.ncbi.nlm.nih..gov/pmc/articles/PMC7472685/

Identify another small molecule whose distribution is affected by TMDD. Draw its structure
and indicate its target.
Learning Activity (3): structural Modifications for Solving PPB Issues
En route to the discovery of venetoclax, acylsulfonamide 1 was identified as a BCL-XL inhibitor
with a Ki of 36 nM. However, its BCL-XL inhibition was nearly obliterated when 1 was tested in
the presence of 10%% human serum it was also deactivated by 69'fold in the presence of 1%
human serum, indicating strong protein binding. It was revealed that the binding to human serum
albumin (HSA) was the main driver for compound Ts serum deactivation.
Structural modifications led to analogs 2 and 3 after installation of the dimethylaminoethyl motif
and replacements of the fluorobenzene moiety. Both 2 and 3 emerged as potent BCL-XL
inhibitors with approximately 1 nM affinity. Furthermore, deactivation from serum binding was
greatly reduced, HSA affinity was reduced by over 2 orders of magnitude.
Calculate the LogP of compounds 1, 2, and 3. How do you correlate between their lipophilicity
and PPB? What is the reason for the strong PPB of 1?

Why did the addition of the dimethylaminoethyl moiety reduce PPB? How do you think their Vd
was affected by this modification?
 Assignment (3)
1) Explain the primary factors that influence the distribution of a drug throughout the body.

2) What roles do tissue permeability and blood flow play in drug distribution?

3) Discuss how the physicochemical properties of a drug can affect its distribution.

4) What are some common structural modifications made to drug molecules to improve their distribution
across biological membranes?
5) How can increasing lipophilicity of a drug molecule enhance its distribution?

6) Explain the key factors that determine whether a drug can cross the blood-brain barrier (BBB).

7) How can structural modifications to a drug enhance its ability to penetrate the BBB?

8) Describe a strategy to increase the BBB permeability ٥‫ أ‬a hydrophilic drug.

9) What is plasma protein binding, and why is it significant in . drug pharmacokinetics?

10) Explain how a drug's binding to plasma proteins can influence its volume of distribution.

11) How can drug interactions affect plasma protein binding and subsequent drug distribution?
 (4) Metabolism
intended Learning Outcomes (ILOs)
1- Understanding drug metabolism through functionalization reactions.
2- Understanding drug metabolism through conjugation reactions.
3- Understanding the significance of studying these types of metabolic reactions.
4- Be able to suggest possible metabolic pathways for drugs based on their structures and
properties.

Pre session Reading

"In any pharmacological intervention, the prescriber must consider how
and when a specific drug is eliminated from the body." - this is a quote
from this page: "Susa ST Hussain A, Preuss CV. Drug Metabolism.
[Updated 2023 Aug 17). In: StatPearls. Treasure Island
(FL): StatPearls Publishing; 2024 Jan. Available from:
https://www.ncbi nlm.nlh.gov/books/NBK442023/”
This article discusses the clinical significance of learning about drug metabolism. Read though it
and record your thoughts on the topic that we are about to explore. You can share your insights
with your group during the session if you would like.
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The metabolism of drugs (biotransformation) and other xenobiotics is often a biphasic process in which
the compound may first undergo a functionalization reaction (phase ‫ ا‬reaction) of oxidation, reduction, or
hydrolysis. This introduces or unveils a functional group, such as a hydroxyl or amino group, suitable for
coupling with an endogenous molecule or moiety in a second metabolic step known as a conjugation
reaction (phase ‫ اا‬reaction). In some cases, phase ‫ ا‬metabolites may be excreted prior to conjugation,
while many xenobiotics can be directly conjugated. Furthermore, reactions of functionalization may follow
some reactions of conjugation (e.g., some conjugates are hydrolyzed and/or oxidized prior to their
excretion).
The major function of xenobiotic metabolism is the elimination of physiologically useless compounds,
some of which may be harmful (metabolism was originally referred to as detoxification). However,
numerous xenobiotics and even a marked number of drugs are known to yield toxic metabolites, a
situation known ‫ ألج‬toxification.
1. Functionalization Reactions
Reactions of functionalization comprise oxidations (electron removal, dehydrogenation, and oxygenation),
reductions (electron addition, hydrogenation, and removal of oxygen), and hydrations/dehydrations
(hydrolysis and addition or removal of water). We will not discuss the enzymes catalyzing these reactions,
but we will cover those functionalization reactions.
1 .٨. Gxidation and Reduction Reactions
These involves C", N-, and S-oxidation & reduction, N-, O-, S-dealkylation, deamination, and certain
dehalogenations. Reduction of several compounds such as polyhalogenated, keto, nitro, and azo
derivatives. We will give few examples here and cover others in a different context.
 1.B٠ Hydration & Hydrolysis Reactions
Hydrolysis and hydration both imply bond breakage with addition of a molecule of HO. The term
"hydrolysis” applies to the cleavage of esters (carboxyl esters, lactones, and inorganic esters), amides
(e.g., carboxamides, sulfamates, phosphamides, and lactams), and glycosides. In contrast, the term
“hydration” will be restricted to epoxides (see carbamazepine hydration introduced above).
2. Conjugation Reactions
Conjugation reactions (also known as phase ‫ اا‬reactions) are of critical significance in the metabolism of
endogenous compounds and biotransformation of xenobiotics. Conjugation generally produces a
metabolite that is more polar, larger in molecular weight (MW), and charged at physiological pH,
characteristics that make the metabolite more amenable for excretion into the urine (more soluble, less
permeable) or bile. Most phase ‫ اا‬conjugates are inactive and nontoxic; however, there are exceptions
where bioactivation or reactive metabolites are formed as a result of conjugation.
Conjugation reactions comprise:
2.٨. Methylation: N-, s- & O-methylation.
2.8. Sulfonation: N- or O-sulfonation occurs to alcohols, phenols, & hydroxylamines.
2.C. Glucuronidation: A major and frequent reaction of conjugation. ‫ا‬٤ includes: O-glucuronidation
(alcohols, phenols, & carboxylic acids), ‫ جالم‬glucuronidation (amides & amines), and S-qlucuronidation
(thiourea).
2.D. Acetylation: of amines, hydrazines, hvdrazides.
2.E. Conjugation with Coenzyme A and subsequent reactions (e.g. conversion of R-ibuprofen to
S-ibuprofen).
2.F. Conjugation Reactions of Glutathione: It plays an important role in detoxification reactions
due to its ability to scavenge free radicals and reactive species (e.g. epoxides & quinoneimines), so it has
a critical role in cellular protection.
2.G. Amino acid conjugation: using glycine, glutamic acid, or taurine.
What is the significance of studying these types of metabolic reactions?
From a medicinal chemistry perspective, metabolic reactions (biotransformation) control many aspects
related to drugs due to the change they introduce to the drugs’ chemical structures, so they can affect
their activation, deactivation, potency, duration of action, absorption, distribution, excretion, toxicity, etc.
Not only are we concerned about the effect of metabolic enzymes on drugs, but also about the effect of
certain drugs on the induction or inhibition of these enzymes thus affecting other drugs metabolism, but
we will not cover this part.
Phase 1 metabolic reactions introduce new polar functional groups or modify the existing ones to produce
one or more of the following changes:
1. Decreased pharmacological activity (deactivation).
2. Increased pharmacological activity (activation).
3. Increased toxicity.
4. Altered pharmacological activity
As a result, biotransformation should be critically examined throughout the drug development process and
the design can be altered to optimize the drug metabolism and thus the drug profile.

Learning Activity (1)

Suggest possible metabolic pathways for the following drugs.
Learning Activity (2)
Case Study
A 42-year-old male patient is taking Valpam (diazepam: sedative, hypnotic & anxiolytic) for sleeping
disorder for a month now, he returned to his physician complaining of somnolence and fatigue that might
persist for a couple of days. The patient is a driver, so he requested a change of his medication, and the
physician shifted him to Ambien (zolpidem, a non BDZ hypnotic) which does not cause significant residual
day-time drowsiness (diazepam ti،2 = 30-56 hr, while zolpidem is 2.5 hr).

Refer to the case study and answer the following questions:

1. Predict the possible metabolites for diazepam.
 2. Although
Assignment diazepam is extensively metabolized, it has a long half-life. Guided by the SAR summary of
(4)
the benzodiazepine sedative-hypnotics provided below, predict the reason for such long half-life?
Several webtools are available for ADMET properties prediction
using computational methods. Use the GLORY" webtool to
predict the possible metabolites of the following drugs.
Drugs: Paracetamol -Diclofenac - Tramadol
(You can get the structures from PubChem)
(‫؟ا‬.‫اال؛ًا‬9‫إ‬٠‫در اج‬، id.univi.e:،‫؛‬٠GLORY (ht٤ps.//ne :‫؛‬Webtoe

1 Diclofenac Tramadol
 (5) Metabolic stability
‫ ا‬Intended Learning outcomes (ILOs)
1- Understanding the advantages of enhancing metabolic stability of drugs.
2- Explore the strategies used to enhance the metabolic stability of drugs.
There is a compelling reason to attempt to modulate metabolism in the drug design process because
incorporating metabolic stability into drug molecules will impart important benefits. First, by controlling
metabolism, formation of reactive intermediates (eg. electrophilic, alkylating metabolites) will be
diminished. Second, reducing metabolism generally simplifies and allows better control of
pharmacokinetics.
Advantages of Enhancing Metabolic stability
1. Increased bioavailability and longer half-life which in turn allow lower and less frequent dosing thus
promoting better patient compliance.
2. Lower patient-to-patient and intra-patient variability in drug levels since this is largely based on
differences in drug metabolic capacity, (e.g. CYP450 and warfarin)
3. By reducing metabolism, the number and significance of active metabolites should be diminished,
lessening the need for further studies on drug metabolites in both animals and man.
4. Last, but certainly not the least, reduction in metabolism diminishes one of the more common
mechanisms of drug interactions. (Grapefruit and CYP450 metabolites)
Thus, while searching for a compound with an optimal intersection of both activity and metabolic stability
(as well as other pharmacokinetic properties) may lengthen the discovery process, this will presumably be
offset by more effective development of the new drug candidate.
Strategies Used to Enhance Metabolic stability
1. Deactivating aromatic rings towards oxidation by substituting them with strongly EWGs (e.g. -CF3, -
SO2NH2, -SO3H).
2. Strategically replace (e.g. amide for ester), block (e.g. fluoro for hydrogen to block a soft spot) or
protect (e.g. N-t-Bu to prevent / dealkylation by steric shielding) the metabolically labile sites or moieties.
3. Th‫ ؟‬use of prodrugs to improve metabolic stability.
4. Constraining the molecule in a conformation which is unfavorable to the metabolic pathway
5. Avoid the phenolic function in any compound intended for oral use, if sterically unhindered, because it
is rapidly glucuronidated.
6. Sometimes the best strategy is to anticipate a likely route of metabolism and prepare the expected
metabolite if it has adequate intrinsic activity. For example, often ‫م‬١‫آلر‬oxides are just as active as the
parent amine but will not undergo further /V-oxidation.
7. Reducing the drug size and lipophilicity.
In principle, attempting to use these strategies to enhance metabolic stability is not easy, as changes in
one parameter can impact other attributes. For instance, attempts to reduce extensive metabolism by
decreasing lipophilicity can result in a negative impact on primary pharmacology (e.g. changes in
selectivity, potency, etc.) and pharmacokinetics (e g. poor oral absorption due to increase in polarity). So,
the desired goal is that the SAR for metabolic liability can be optimized in parallel with the SAR required
for pharmacological activity and other parameters related to ADME and safety. Medicinal chemists will
ultimately strive to achieve an acceptable balance of properties (eg. pharmacokinetics and
pharmacodynamics compatible with an acceptable daily dosing regimen and therapeutic margins for
safety) rather than optimization of each aspect of the molecule.
1. Deactivating Aromatic Rings Towards Oxidation

2. Replacing, Blocking, or Protecting Labile Sites

2.A. Replacing a metabolically labile group with a bioisostere

2,B. Blocking a soft spot

(HLM Human liver microsomes intrinsic clearance)

gc-P oct ioga metabolically labile group by steric shielding
3. Prodrugs
Prodrugs can be used to protect active drugs from first-pass effect by masking a metabolically labile but
pharmacologically essential functional group, to avoid rapid metabolism.

4. Conformation Restriction
5. Avoiding Phenolic Function

6. Prepare Expected Active Metabolite

Drug Report (Read Only!)
Pahperidone is 9-hydroxyrisperidone, the chief active metabolite of risperidone. Although paliperidone
possesses a pharmacological profile very similar to that of its parent compound, it has many different
pharmacokinetic and pharmacodynamic characteristics compared with risperidone. First, paliperidone
does not undergo significant hepatic metabolism, and the drug is predominantly excreted by the kidney as
an unchanged drug; whereas risperidone is extensively metabolized by the CYP2D6 hepatic enzyme.
Second, the off-rate for dissociation from human cloned D2 receptors in tissue culture cells is faster for
paliperidone (60 s) compared with risperidone (27 min). Due to its looser binding to D2 receptors,
paliperidone should be associated with a reduced risk of extrapyramidal side effects compared with its
parent drug. Third, ex vivo studies have indicated that paliperidone injections induce relatively smaller Hl
occupancy levels in the brains of animals when compared with similar dosages of risperidone. This may
contribute to a reduced sedative effect and less weight gain secondary to paliperidone when compared
with risperidone. Finally, paliperidone has no relevant affinity toward muscarinic receptors, resulting in the
absence of anticholinergic side effects; this is another important benefit compared with risperidone.
Therefore, paliperidone would be the drug of choice in young psychotic patients for whom preservation or
improvement of cognitive function is critical. All of these properties might be associated with improved
efficacy and better tolerability of paliperidone compared with risperidone.
7. Reducing Drug Size & Lipophilicity.
Learning Activity (1)
QI. Why substituting hydrogen with fluorine at metabolically labile positions is a common approach to
attenuate metabolism?
Q2. Identify the strategy that had been used to enhance the metabolic stability of the following drugs.
Learning Activity (2)
Case Study (enhancing metabolic stability is not always the target!)
Sertraline (Zoloft) is a selective serotonin reuptake inhibitor (SSRI) that is used for the treatment of
depression. Like many SSRIs, sertraline is a very lipophilic drug with a large Vd and correspondingly long
ti/2. Orally delivered drugs with these properties are readily absorbed from the digestive system and
immediately collect into the first encountered fatty organ, the liver. The drug then slowly distributes from
the liver. As a result, SSRIs require a long time to achieve steady- state concentrations in the body. For
the patient, this means that the drug will only slowly take full effect. Similarly, if a patient quits taking the
drug, therapeutic levels of the drug will remain as the drug clears very slowly. Knowing the problems of
sertraline and other SSRIs, researchers at Pfizer sought to design an SSRI with low Vd and short ti/2.
Such a drug would be expected to have a rapid onset of efficacy. A target ti/2 value was set at 8 hours.
The Pfizer team started with sertraline, a Pfizer product that was already well understood with regard to
which functional groups were required for high activity. Initial efforts focused on the benzo- fused ring of
sertraline. Substitution of a polar sulfonamide group was hoped to decrease both Vd and ti/2. Compound
X showed excellent serotonin reuptake inhibition (ICso = 1 nM) and an estimated ti/2 of 8 h based on
animal model predictions. In humans, however, X underwent phase I metabolism to form a demethylated
compound with a very long ti/2 of 240 h. Attention shifted to opening the aliphatic ring of sertraline. Simple
removal of two CH groups from
sertraline and linking the aromatic rings with an ether provided Y, a
compound with decreased activity (ICso = 25 nM) yet a much lower Vd.

Continued research on ring-opened structures ultimately afforded Zi. In vitro. Zi had excellent predicted
properties: ICso = 5 nM, Vd = 2 L/kg, and ti/2 = 5 h .In vivo, however, compound Zi was demethylated to z
‫؛‬, an active metabolite with a long half-life. Variations on Zi suffered other problems. Replacing the NM62
group with an azetidine (four membered nitrogen-containing ring) led to an unstable compound (23).
Incorporation of a pyrrolidine ring (24) introduced stereochemical complications that were not attractive.

Because metabolism of the amino group was causing problems, the discovery team decided to direct
metabolism to another position of the molecule. Thioethers are known to undergo rapid oxidation, and the
lower half of the molecule was modified to include an SMe group (M). Compound M did show excellent
properties with good activity (Co = 5 nM) and reasonable pharmacokinetics (Vd = 1.5 L/kg, ti/2 = 10 h).
Furthermore, M formed a single, inactive metabolite through phase ‫ ا‬oxidation of the thioether to a
sulfoxide (N). Although compound M was never approved as a marketable drug, its development
highlights complications that drug metabolism can introduce into the drug discovery process. When
metabolites show the same activity as the original drug, then the metabolite is also a part of the full
activity picture of the drug.
Answer the following Questions related to the discussed case study:
1. Enhancing metabolic stability is not always the target, discuss this statement with reference to the
case study.
2. Summarize the different structural modifications of sertraline mentioned in the case study, the reason
behind such design, and how the actual results turned out?
3. Compound M design represents what is known as a soft drug (drug with a soft spot), discuss.
4. Compound H (shown below) was proposed to solve the problem of sertraline. Based on your
knowledge, predict its possible metabolites, and comment on its stability & safety.
‫ ا‬Assignment (5)
Read the assigned paper "tert-Butylphenylthiazoles with an oxadiazole linker: a novel
orally bioavailable class of antibiotics exhibiting antibiofilm activity
(h Its #doi.o.u3L lO3<j/C8RAW‫؛‬i25A١. then answer the following questions:

1. Mention the two design strategies used on compound la in this paper to enhance its
metabolic-stability.

2. From your point of view, suggest other possible reasonable strategies for compound
la to enhance metabolic stability.
3. What is the rationale behind choosing the oxadiazole scaffold specifically to modify la?
4. Based on the compounds list synthesized in this paper, can you deduce the essential feature
required for activity?
(6) Excretion

[ Intended Learning Outcomes (ILOs)

1- - Understanding the different routes of drug excretion.
2- Understanding the physicochemical properties required for each route.
3- Understanding the relationship between drug structure and excretion.
4- Understanding the implications of excretion for drug design.

Pre session Reading

Read this article: Garza AZ, Park SB. Kocz R. Drug Elimination.
[Updated 2023 4 ‫]االل‬. In: StatPearls [Internet], Treasure Island
(FL): StatPearls Publishing. Available from: h DS //WWW
iCi.nlm.nih.t30V/boks/NBK547662/
What is drug elimination? Where is elimination in the ADME
processes?

What is the clinical significance of understanding drug elimination?

Excretion (elimination) is the process by which drugs or metabolites are irreversibly transferred from
internal to external environment through renal or non-renal route.
What is the difference between excret^n and elirnination? ^
Understanding the processes of drug excretion is crucial for medicinal chemistry students, as it directly
impacts the design, efficacy, and safety of pharmaceutical compounds.
Routes of Excretion
The key question here is which drug will be excreted by each pathway. This depends on factors such as
particle size, ionization state, plasma protein binding, and, most importantly, whether the drug is
hydrophilic or lipophilic.
A) Renal Excretion
Renal excretion is the primary route by which most drugs and their metabolites are eliminated from the
body it involves three key processes:
1- Glomerular Filtration:
Glomerular filtration is a non selective process. Both ionized and unionized drugs are filtered except those
bound to plasma proteins. It depends on PPB and renal blood flow It does not depend on the lipid
solubility because all substances (whether water-soluble or lipid-soluble) can cross fenestrated glomerular
membrane. All unbound drug in plasma is filtered in the glomerulus, which is only significant for very polar
compounds with log D <0. Drug that is bound to plasma proteins is not filtered. Drugs eliminated by renal
excretion include aminoglycosides such as streptomycin, p-lactams, sulphonamides, quinolones,
nitrofurans, and polymyxins.
 Acetaminophen Why do you think acetaminophen is readily excreted in urine though glomerular
filtration?
2- "Tubular Re-absorption:
Unionized and lipophilic drugs can undergo passive reabsorption from urine back into the bloodstream
along the nephron, potentially reducing net excretion to zero. This process is especially common for
lipophilic drugs, like diazepam, which prolongs their presence in the body. About 99% of unionized, lipid-
soluble drugs are reabsorbed, while highly ionized and non-lipid-soluble drugs (about 1%) remain in the
urine. Reabsorption occurs after glomerular filtration and throughout the renal tubules, increasing the
drug's half-life.
Why is diazepam re-absorbed in the tubules?
3- Tubular Secretion:
After filtration, some drugs are actively transported from the blood into the renal tubules via specific
transporter proteins. For example, penicillin is actively secreted into the renal tubules, promoting its
elimination. Tubular secretion is independent of lipid solubility and plasma protein binding (PPB).
Separate transporters in the proximal tubules handle acidic and basic drugs: organic acid transport and
organic base transport. Drugs using the same transporter can lead to interactions, such as when
probenecid decreases penicillin excretion but increases uric acid excretion, these pumps eliminate
exogenous substances like penicillin while retaining endogenous ones like uric acid.
Lipid-soluble drugs are more likely to be reabsorbed, reducing excretion, while lipid-
insoluble drugs (hydrophilic) are less likely to be reabsorbed and are more readily
excreted.

B) Non Renal Excretion

While renal excretion is the primary route for many drugs, some drugs are eliminated from the body
through non-renal pathways. Non-renal excretion includes biliary excretion, pulmonary excretion, and
excretion via sweat, saliva, or breast milk.
1- Biliary Excretion:
In this pathway, drugs are secreted by the liver into the bile and then excreted into the intestines. Drugs
that undergo biliary excretion are often reabsorbed through enterohepatic circulation, which can prolong
their half-life. For example, large, polar molecules like the antibiotic ervthromvcin are excreted in the bile.
2- Pulmonary Excretion:
this route involves the elimination of volatile drugs or gases through the lungs. Anaesthetics like isoflurane
are typically excreted through the pulmonary route due to their high volatility and low molecular weight.

3- Excretion through Sweat- Saliva, and Breast Milk:

Some drugs are excreted through alternative pathways, though in smaller quantities. For instance,
nicotine and certain heavy metals can be excreted through sweat, which may lead to side effects like
urticaria. Drugs like morphine can be detected in saliva, causing an unpleasant aftertaste, while
medications such as antibiotics can be excreted into breast milk, an important concern for breastfeeding
mothers.

Linkinq Chemistry to Excretion

The chemical structure of a drug significantly influences its absorption, distribution, metabolism, and
excretion (ADME) as we have discussed before. This part of the section focuses on the relationship
between drug structure and excretion: lipophilicity, size, and protein binding.
A) Hydrophilicity vs Lipophilicity
Hydrophilic Drugs: Generally excreted more efficiently by the kidneys because they dissolve well in water,
which is the primary medium of renal filtration.
Metformin: A biguanide with high hydrophilicity and low plasma protein binding, excreted unchanged in
the urine.
Atenolol: A beta-blocker that is hydrophilic and primarily excreted by the kidneys without significant
metabolism.

Lipophilic Drugs: Typically undergo metabolism to increase their solubility in water before excretion.
(Remember metabolism from an earlier session?)
Diazepam: A benzodiazepine with high lipophilicity, requiring hepatic metabolism before renal excretion.
 Can you suggest metabolic pathways?

Amiodarone: A lipophilic antiarrhythmic drug that undergoes extensive hepatic

metabolism, with metabolites excreted in the bile.

Why is it lipophilic?

B) Size and Molecular Weight

Small Molecules: Easily filtered by the kidneys and often excreted without significant
metabolism.
Caffeine: A small, water-soluble molecule rapidly excreted by the kidneys after hepatic
metabolism.
C) Large Of Protein-Bound Drugs
Require metabolism to become more water-soluble or rely on biliary excretion.
Warfarin: Highly protein-bound, undergoing extensive hepatic metabolism before excretion.

Cyclosporine: A large, lipophilic molecule with poor renal excretion, primarily excreted in bile after
metabolism.

Implications for Drug Design

ri Optimising Excretion Profiles:
Medicinal chemists aim to design drugs that balance efficacy, half-life, and excretion characteristics to
reduce the risk of toxicity and improve patient outcomes.
Structural modification of morphine leads to the production of methadone that is us'd in treatment of
opioid addiction. Methadone has higher plasma protein binding thus prolonged retention time in plasma
and longer half-life.
Prodrug Approach. Valacyclovir: A prodrug of acyclovir, designed to enhance oral bioavailability (how?),
where the active drug is excreted by the kidneys after hepatic metabolism (esterases).

2) Predicting Drug-Drug Interactions:

Understanding excretion pathways aids in anticipating and managing potential interactions, especially in
polypharmacy.
Example: Co-administration of probenecid can delay amoxicillin excretion, as most of the drug is
eliminated unchanged in the urine.
Learning Activity
In the following case studies, you will work with your group to explore clinical aspects of drug excretion,
helping you understand its importance as a pharmacist.
Case 1: Renal Clearance of Polar Drugs
You are provided with digoxin structure. As a clinical pharmacist could you decide whether it is safe to
give it to a patient with renal impairment?

What is the logP of digoxin?

Major route of excretion based on its properties?
Effect of renal impairment on its excretion?
‫ا‬
I
Case 2: Excretion of Lipophilic Drugs and Metabolites
Verapamil is a lipophilic calcium channel blocker, but it undergoes extensive first- pass metabolism.
Based on this, could you suggest the main route of excretion?

Case 3: Drug-Drug Interactions Affecting Excretion

Mr. X was diagnosed with Rheumatoid Arthritis, and the medical team decided to prescribe methotrexate.
Suddenly, he felt unbearable pain for which an NSAID was given. Soon after that, a blood sample was
withdrawn, and an abnormally large concentration of methotrexate was found. Can you justify?

Case 4: Genetic Polymorphism Affecting Excretion

Genetic variations in CYP2D6 can affect the metabolism of codeine to morphine, impacting both efficacy
and excretion. Could you explain why some people develop hallucination after taking codeine for cough
treatment while others do not?
Assignment (6)
Now that you have covered the drug’s journey through the body via the ADME processes, you will apply
your understanding to decipher the pharmacokinetic profile of a newly approved drug. This is a skill you'll
need for lifelong learning as a pharmacist, as new drugs are continuously introduced to the market, and
you will need to quickly familiarize yourself with them. (ILOs: research skills, writing skills, science
communication skills, skills for life)
Check the 2024 FDA approvals here: httDS:#w١vw.fda.ciov/drtcs/novel-driici- aoorovals-fda/novel٠dnjci-
aoDrovals-2Q24
Select a small-molecule drug from the list, and create its pharmacokinetic profile from literature using the
following template:

Pharmacokinetic Profile: Provide drug structure.

A ‫ ج‬route of administration, oral bioavailability
D ‫ ج‬٧d, BBB penetration, PPB
Metabolites? Reactions? 11/2 ? possible drug-drug How these relate to the
interactions? physicochemical properties
E Major route of excretion? possible disease- related dosage (logP, ionizability) and structure
adjustment? of the drug?

Provide the literature references that you used to create the profile.

Extracurricular Activity: Once you’ve prepared the factsheet on drug pharmacokinetics, you can share it on Linkedln
using the hashtag #MedChem_ASU ‫ ا‬his is optional and not part of your assessment. Linkedln is becoming
increasingly popular with employers, and developing your employability and career skills includes familiarizing
yourself with setting up and maintaining an active profile. This assignment is a great opportunity to get started with
Linkedln and begin building your professional presence.
11
(7) Toxicity
Intended Learning Outcomes (ILOs)
1- Understanding the concept of the safety window for drugs.
2- Identify some toxicophores commonly encountered in drug design, and their mechanisms of
toxicity.
3- Explore some structural strategies used to avoid toxicity.
A 2004 article from the Centre for Medicines Research shows that toxicity is the leading cause of failure
of compounds in clinical development. Most safety- related withdrawals (70%) occur pre-clinically
following candidate selection, suggesting that we are still in need of better predictive models of in vivo
toxicity to use earlier in the drug discovery cycle (fail early or fast, fail cheap).
The structure-toxicity relationships for mutagenicity and hepatotoxicity are already well-established and
used to identify common alerting structures or so- called “structure alerts". Identifying structural alerts for
toxicity and performing high-throughput assays for early indicators of toxicity issues in vivo, have become
a normal part of early drug discovery. Regulatory authorities require that these robust assays be run on all
new chemical entities before entering first-in-human trials.
Sometimes, medicinal chemists introduce toxicophores into drug molecules and their reactive nature is
produced or enhanced in vivo during normal metabolic processes. Toxicophores are a chemical structure
or a portion of a structure (a functional group) that is related to the toxic properties of a chemical. They
can act directly or can require metabolic activation.

The Safety Window

All drugs are toxic at some level and so a major challenge in drug discovery is to find a margin of efficacy,
over adverse events, or toxicities, sufficient to provide clinical benefit to patients whilst avoiding putting
them at unnecessary risk. The therapeutic index (Tl) is the ratio between the therapeutic dose and toxic
dose and used to measure the relative safety of drugs. Ideally, it should be around 10- fold or more.

We will cover several common examples of toxicophores, discuss their mechanism of toxicity, and how to
resolve their toxicity. Kindly note that these toxicophores do not necessary indicates drug toxicity, as
many other factors might revoke their effect (e.g. soft spots, low dose, low lipophilicity, additional
substituents that inactivate them, natural detoxification mechanisms), that is why we call them "alerts”.
They should be investigated but should not be a reason for dismissing the drug as a whole
Commonly Encountered Toxicophores
1■ Electrophiles not requiring metabolic activation
During the course of in vitro testing in research programs, certain chemical intermediates and mild
electrophiles find their way into the screening cascade by design or by accident. Despite some of these
being stable chemicals in buffered solution, it must be noted.that in the body, nucleophiles as amines and
thiols will unselectively react with these electrophiles. The toxicity of these functional groups will- in
general be related to their chemical reactivity. These common electrophiles should be avoided unless a
specific drug-protein covalent interaction is the desired goal Examples includes, alkyl halides, epoxides,
0- lactams, aldehydes, Michael acceptors, reactive heterocycles, etc.

Alkyl halide

Epoxide

Aldehyde
 Michael acceptor

2. Quinones, 1,2- and 1,4-diphen0ls or aminophenols

1,2- and 1,4-diphen0ls or aminophenols can be readily oxidized to quinones or quinoneimines. Quinones
& quinoneimines are Michael acceptors, and cellular damage can occur through alkylation of crucial
cellular proteins or DNA. In addition, they are highly redox-active molecules which can redox-cycle with
their hydroquinone (HQ) and semiquinone radical sisters, leading to the formation of reactive oxygen
species (ROS) including superoxide, hydrogen peroxide and hydroxyl radical.
Example
Solution
3■ Nitrobenzenes & Anilines
Nitrobenzenes are reduced to anilines in vivo, which in turn can cause methemoglobinemia,
agranulocytosis, aplastic anemia, hepatotoxicity, skin hypersensitivity and increased risk of mutagenicity.
There are two principal mechanisms of aniline toxicity. The first is oxidation of the aromatic ring ortho or
para to the aniline nitrogen followed by the formation of the highly electrophilic ortho- and para-
iminoquinones mentioned before. The second pathway is oxidation of the aniline nitrogen to
hydroxylamine, nitroso, and related species. The nitroso species is a reactive metabolite in its own right;
the hydroxylamine species undergo acetylation or sulfonation to deliver a good leaving group which leads
to reactive metabolites.
Example
Solution
4. Thiophenes
The thiophene ring is susceptible to hepatic oxidation by CYP and undergoes epoxidation, followed by
epoxide ring opening with nucleophilic biomolecules, to give adducts resulting in toxicity.
Example

5. Furans
In a similar manner to that for thiophenes, furan toxicity occurs via furan epoxidation followed by epoxide
ring opening to a ٧-keto aldehyde which in turn forms adducts with biomolecules and induces toxicity.
Example
Solution

In addition to thiophenes and furans, several heterocycles (pyrrole, thiazole, etc.) and electron-rich
benzenes are susceptible to oxidation to form reactive epoxides. In contrast pyrazoles seem to be the
most stable rings among the five- membered heterocycles.
6. Benzodioxolanes
It is associated with mechanism-based irreversible inhibition or induction of CYPs. In addition, some
benzodioxolanes are associated with hepatotoxicity. Oxidation of the methylene leads to (1) a reactive
carbene intermediate which can form irreversible adducts with the haem of CYPs, and (2) catechol which
is an orthoquinone precursor.
Example

Fortunately, several viable isosteric replacements are available for the benzodioxolane
structure. Replacement of one of the oxygen atoms or blocking the reactive site is
another approach to reverse the toxicity of this group.
Solution
Most of these toxicophores are present in already established and known drugs, and these drugs are not
toxic at their therapeutic dose This is because, as said before, these toxicophores do not necessary
indicate drug toxicity, as many other factors might revoke their effect (soft spots, low dose, low
lipophilicity, additional substituents that inactivate them, natural detoxification mechanisms). And hence,
they get their name “Structural alerts". 0‫ ؛؛‬DO NOT FORGET, they are JUST A RTS.

Learning Activity (1)

Q1. For each of the following examples, identify the toxicophore(s) present and discuss the design
approach to avoid its toxicity?
Q2. Although the alkyne group can act as a toxicophore in vivo via conversion to
reactive oxirane or ketene electrophiles, a drug like ethinyl estradiol does not show
considerable toxicity. What can be the possible reason(s)?
Q3. Based on all the examples discussed today, can you summarize the different strategies that can be
used to resolve metabolism-induced drug toxicity?
‫ إ‬Learning Activity (2)
‫آللل_!لح‬._‫لبل‬.‫لب‬.‫ك_ب‬

Read the assigned paper ' In Vitro Metabolism and Covalent Binding of Enol- Carboxamide Derivatives
and Anti-Inflammatory Agents Sudoxicam and Meloxicam: Insights into the Hepatotoxicity of Sudoxicam"
[You can find it in Appendix (5) at the end of this manual] and answer the following questions:
1. To which class of NSAIDs do these drugs belong to?
2. How do these drugs inhibit cox enzyme although they lack the essential carboxylate group?
3. What is the difference in structure between sudoxicam and meloxicam?
4. Mention the main metabolites identified for sudoxicam, discuss the mechanism of their formation
and correlate to its hepatotoxicity.
5. Mention the main metabolites identified for meloxicam and discuss the mechanism of their
formation.
6. How did the design of meloxicam avoid hepatoxicity although it still has a thiazole ring?
7. What analytical tool was used to separate the resulting metabolites and how were they identified?
Assignment (7)
All along the drug development process, one of the most frequent adverse side effects, leading to the
failure of drugs, is the cardiac arrhythmias. Such failure is mostly related to the capacity of the drug to
inhibit hERG channel activity. The early identification of hERG inhibition properties of biological active
compounds has gained great attention over the years.
Answer the following questions regarding hERG blockers:
(You can use this comprehensive reference: Kalyaanamoorthy, s. & Barakat, K. H. Development of Safe
Drugs: The hERG Challenge. Medicinal Research Reviews 38, 525-555 (2018).
httDS://doi.ora0.1002/med.21445)
1. What are hERG channel blockers and why do they cause cardiac toxicity?
2. What are the physicochemical properties and pharmacophoric features that were identified to
contribute to hERG inhibition?
3. Using the following webtool, predict the hERG liability of the following drug pairs and try to predict the
reason for such variation or lack of?
Drugs: Terfenadine & fexofenadine.
Thioridazine & chlorpromazine.
Grepafloxacin & ciprofloxacin.
Webtool: http://rreclherci.labmol.com.bl/
Hint: Use the SMILES of each drug instead of drawing
the structure.
(8) Drug Design Case Studies
Intended Learning Outcomes (ILOs)
Applying the knowledge gained throughout this course to real-life cases of drug design and discovery.
١١
Pre-session Reading
Read this paper 'Chemists Invent Drugs and Drugs Save Lives", by Donald Weaver, ChemMedChem 19,
6202400074 (2024).
hltDS' chemistrv-euroDe.onlineiibrafv.wilev.com/ doi/10.1002/cmcl(.202400074
This reading is not intended for academic educational purposes but rather to humanize the drug discovery
process and potentially ignite a passion for medicinal chemistry in you.
Write down your thoughts on this paper, and if you would like, share them with your group during the
session.
MMV390048 case study 3
To acquaint you in more practical terms with the concepts covered in this course, we will present a case
study of the compound MMV390048. the first clinical candidate for malaria to be discovered on the
African continent. You can read more about the discovery in the press releases from 2012 here -----------
strong-candidate-for-possible-single-dose-rnalaria-cure). At the time, the compound, which was potent
against a wide panel of resistant strains of the malaria parasite, had the potential to be used as a single-
dose cure for the disease. MMV390048 was active across all stages of the parasite life cycle and had a
novel mechanism of action. Early clinical studies were conducted in Africa with a first-in-human single
rising-dose study completed at the University of Cape Town between 2014 and 2015. In 2017,
MMV390048 reached Phase Ila human clinical trials in Ethiopia.
You will remember that all drug discovery projects start with a screening campaign and hit identification,
in which promising chemical matter is identified as a starting point for a potential new medicine. This is
followed by many rounds of medicinal chemistry (the so-called ‘design-make-test’ cycle) in which, guided
by structure-activity relationships and the screening cascade, compounds are optimised to remove
undesirable features until they fulfil the criteria for a lead. Extensive safety testing of the optimised lead
must take place in preclinical studies before it can be a deemed suitable for human trials.
The MMV390048 story
In 2008, 36,,000 compounds from the BioFocus soft focus kinase library were screened at the Eskitis
Institute for Drug Discovery in Australia. This compound library was designed to provide high-efficiency
hits against a range of targets, specifically kinases (enzymes that transfer a phosphate group to a protein
or a
‫ ساسل سياه ه‬This ‫ ييس‬study ‫ ورا‬been reused from the course “Introduction to Small Molecule Drug Discovery &
Development" by University of Cape Town from Coursera online learning platform.
 lipid). The compounds were screened in phenotypic assays against two strains of the malaria parasite,
the drug-sensitive 3D7 and drug-resistant Dd2 strains. 222 hits were identified with greater than 8O/٥o
inhibition at 1.82 pM against the strains. The hits spanned several compound classes, and some
examples can be seen below.

SFK52 ‫؛‬
Library SK8 pyraoylcarboxy. SFI06 SFKO
aminothiazoles DiaininothieriyF midazopyridazines
Name pyrimidines 2-Aminopyridines

Scaffold

Hit rateai ca. 2 pM: 1% (8 hits)

: »80%

٣
>50% 1 % (5 hits) 15% (85 hits) 1,4%(80 hits)
1 3% (10 hits) 2,3%(118hits) 27% (132 hits)

)1 3 15(
Based on its low cytotoxicity (> 100-fold selectivity against HepG2 cells) and synthetic attractiveness, the
2-aminopyridine (so-called SFK40) series was selected for further development. Some examples of the
hits from the SFK40 series are shown below.

The first priority for scientists at the Holistic Drug Discovery and Development (H3D) Centre at the
University of Cape Town was to validate these hits by re- synthesising them and confirming their activity
against the malaria parasite. The chemical synthesis, shown below, comprised bromination, iodination
and Suzuki cross-coupling chemical reactions to yield 3,5-disubstituted compounds around the 2-
aminopyrdine core.

Once the screening data were verified, a process known as formal hit assessment suggested that this 2-
aminopyridine series was suitable for further development and hence hit-to-lead medicinal chemistry
optimisation was carried out.
Medicinal chemistry optimisation to yield MMV390048
The medicinal chemistry team responsible for the hit-to-lead and lead optimisation of the SFK40 series
was guided by the screening cascade below. A screening cascade always starts with the design and
synthesis of new compounds. These then enter the first tier of the screening cascade in which they are
tested for antiplasmodium activity (Pf EC50) against two strains ofthe malaria parasite, the chloroquine-
sensitive NF54 strain and the multidrug-resistant K1 strain. These compounds are also simultaneously
tested for potential cytotoxicity against two mammalian cell lines and various physicochemical parameters
are measured as well as microsomal metabolic stability.
Compounds that meet the criterion for antiplasmodium activity (< 100 nM) and that have a selectivity
index greater than 100, aqueous solubility greater than 50 pM and good microsomal metabolic stability,
are progressed to the next level of the screening cascade, which involves an in vivo efficacy study against
a Plasmodium berghei mouse model or a p. falciparum humanised severe combined immunodeficient
(SCID) mouse model to test whether the compound is able to cure diseased rodents. If these compounds
are not found to be suitable, the data are fed back to the medicinal chemists to allow them to design new
compounds in the hope of making new molecules that have potential to go all the
Comparing the hit, early lead and late lead
A comparison between the biological properties of the hit, early lead and late lead of the SFK40 series is
shown below. The initial hit compound (MMV010576) has good potency against the two strains of the
malaria parasite with moderate solubility but suboptimal microsomal stability. hERG 1050, a measure of
cardiotoxicity, and cytotoxicity against the L6 cell line for this compound, were not measured. For
MMV017007, the compound defined as the early lead, you will notice that solubility has dramatically
improved as has the compound’s microsomal metabolic stability. hERG and cytotoxicity margins are
good. Finally, for MMV390048 itself (the late lead), potency, microsomal metabolic stability and toxicity
margins are further improved - though at the expense of aqueous solubility.

Compound Solubility Microsomal 1-6

EC nM) liERG
..‫؛؛‬i ‫ا‬/-.، 49 /49 (pH6.5,pM)
30 stability
0.48 - cytotoxicty

٠‫;ل‬.■?■ •:٠‫? ؛‬,--٠: ‫؛‬٦ 51/51 120 026 5.5 >146

'٠١٠ .٠١.
;.:‫مم‬ ٠٠,٠, 29/25 ?.5 <0.07 >11 ?51
٤٠ ١٠١ ٠.٠٠•

These data were published in the Journal of Medicinal Chemistry in 2012. In that publication1 it was also
demonstrated that MMV390048 showed good pharmacokinetics and completely cured p. berghei-infected
mice with a single oral dose.
Antimalarial efficacy across the malaria parasite life cycle
Subsequent life cycle and mechanistic studies were published by H3D scientists and international
colleagues in Science Translational Medicinal in 2017. In that publication, Paquet et al. disclosed the
efficacy of MMV390048 across all stages of the malaria parasite life cycle in a variety of animal models.
Genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite,
phosphatidylinositol 4-kinase (PI4K), as the molecular target of MMV390048.
The blood stage antimalarial efficacy of MMV390048 was assessed in humanised SCID mice infected
with the 3D7 strain of p. falciparum. Efficacy was assessed after a once-daily oral administration of
MMV390048 for four consecutive days, with blood parasitaemia measured by flow cytometry.
MMV390048 achieved a 9O/٥o effective dose (ED90) on the seventh day of 0,57 mg/kg. The rate of in
vivo parasite clearance in this model was comparable to that of the reference drug mefloquine.

Safety, Tolerability, Pharmacokinetics of MMV390048 in Healthy Volunteers

The safety, tolerability, pharmacokinetic profile, and antimalarial activity of MMV390048 were determined
in healthy volunteers in three separate studies. Additionally, a volunteer infection study investigated the
antimalarial activity of MMV39004B using the Plasmodium falciparum induced blood-stage malaria
(IBSM) model. Due to the high pharmacokinetic variability with the powder-in- bottle formulation used in
both of these studies, a third study was undertaken to select a tablet formulation of MMV390048 to take
forward into future studies. MMV390048 was generally well tolerated when administered as a single oral
dose up to 120 mg, with rapid absorption and a long elimination half-life. Twelve adverse events were
considered to be potentially related to MMV390048 in the first-in-human study but with no obvious
correlation between these and MMV390048 dose or exposure. Although antimalarial activity was evident
in the IBSM study, rapid recrudescence occurred in most subjects after treatment with 20 mg
MMV390048, a dose expected to be subtherapeutic. Reformulation of MMV390048 into two tablet
formulations (tartaric acid and Syloid) resulted in significantly reduced intersubject pharmacokinetic
variability. Overall, the results suggest that MMV390048 is well tolerated in humans, and the
pharmacokinetic properties of the compound indicate that it has the potential to be used for antimalarial
prophylaxis or inclusion in a single-dose cure. MMV390048 is currently being tested in a phase Ila study
in Ethiopian adults with acute, uncomplicated falciparum orvivax malaria monoinfection.
j Learning Activity
In this final activity, you will look back at what you have learned so far, and work with your group to write
down two assessment questions for each session (2 questions X 7 sessions) that you think can help
assess your understanding of the topic. These questions can b.e in any format you choose (essay, MCQ,
short answer, matching, right Or wrong, case study, .. etc.).
‫آل‬ ‫آل‬
1

3
 ٠٠٠٠
٠٠
4

Assignment (8)
For this final assignment, you will hand over a copy of your 14 questions from the activity to yourTAduring
the session, along with the names of the group members who participated in the activity. Then, you will
need to submit these same 14 questions and group members' names online in a form (details and link to
the form will be given during the session) to receive the final mark for this assignment.
Appendices
Appendix ('!‫؛‬:,Anexcerp^from (he b٠٥k “Medicinal chemistry for
Pharmacokinetics (ADME)
ADME stands for absorption, distribution, metabolism, and excretion, They are the four pillars that govern the pharmacokinetics of drugs.
When the patient takes a pill, the patient and the drug interact with each other. What the drug does to the body is called
pharmacodynamics (in). The PD of a dnrg could be. at the end. relieving pain, lowering cholesterol level, shrinking mmors. or killing bacteria or
viruses. On the other hand, what the patient's body does to the drug is known as pharmacokinetics (PK). PK is of abundant importance for drag
discovery because no matter how efficacious a drug is, it is still ttseless if it does not reach the targetfs) that causes the disease. For an oral drag, it
goes through four stages in human body: absorption, distribution, metabolism and excretion, which are the focns of this chapter on
pharmacokinetics.
Before 1991. potency was the major tluust of medicinal chemistry. The medicinal chemist's mentality was to make the drag as potent as
possible and then handed it over to the "formulation people" to make it bionvailnble. Regrettably, one cannot make a silk purse out of a sow’s ear.
In fact, in 1991. 40٩0 of drugs in clinical trials failed due to poor PK/bioavailability. That untenable attrition rate sent the message home. The
industry took notice and started paying closer attention to drugs' PK profiles. As a consequence. 10 years later in 2000. the attrition rate from poor
PK in clinical trials was reduced five-fold to merely 8° ٠٠‫؛‬
‫ ا ! ق‬tysicoctui ‫ ا‬let Properties
A drag's ADME is largely influenced by its physicochemical properties. Here we start by discussing lipophilicity, hydrogen bonding, polar
surface area (PSA), and number of rotatable bonds, then the famous rule of 5 (Ro5). followed by ligand efficiency (LE) and lipopltilic figand
efficiency (LIE or LipE).
‫'زا‬،‫دإ‬،١‫ل‬1‫؛‬:‫'ذ‬،‫أل‬. t ‫ أ ن‬.‫ا‬
Lipophilicity is a measure of how greasy a molecule is. It has a profound impact on a drug’s ADME because it is closely associated with drag's
solubility, plasma protein binding (PPB). metabolic clearance, volume of distribution, enzymc.'receptor binding,
.Medicinal Chemistry h'l Piaclilioners. First Edition. Jie Jack Li.
<■ 2,120 Jolin Wiley & Sons. Inc. Published 2020 by John Wiley ‫ نه‬Sons. Inc.
1)111‫ ؛‬moie? A , measure of a molecule’s lipophilicity is its partition coefficient,
p, which is the ratio of the equilibrium concentrations of 1‫ ؛‬dissolved solute in a two- phase system containing two largely immiscible solvents.
Traditionally, the two immiscible solvents are l-octanol (o) 1)111‫ ؛‬water (w). As shown in Figure 3.1. the partition coefficient ‫ م‬is defined in
Eq.•(3.1):

Figure 3.1 Partition of a neutral molecule between !-octanol (o) and water (٦).
(3.1) ٢١١J]/[CO]:: ٠١١٠? : •1
Since the value of partition coefficient ‫ م‬could be unwieldy with a wide range of numbers, log p. defined as Eq. (3.2). is regularly used
instead as a more manageable measure of lipophilicity:
Log p = log[C٠]/(C١>■] (3.2)
Log p impacts nearly all aspects of a drug's pharmacokinetics. As the value of log p increases, binding to targets such as receptors and
enzymes is increased. 1(1 the past, medicinal chemists kept making larger ‫؛‬md larger molecules and were happy to see the potency grow. Indeed,
lipophilicity enhances a drug’s binding as a nonspecific driving force fo’r the partition of the ding into the binding site by raising its free energy
in water. Unfortunately, molecular inflation.-» or molecular obesity and obsession with potency.4 is harmful to drug design because increased
lipophilicity makes the molecules less drug-like with lower bioavailahility. As the log p value increases, the aqueous solubility decreases,
although absorption through the membrane increases. A molecule with a larger log p also tends to have higher binding to CYP450-ntetabolizing
enzymes, thus a higher chance for drug-drug interactions (DDIs). A molecule with a larger log p value ‫؛‬riso is inclined to
!١،١١'،' tighter binding to human ethera-go-go (hERG, Kv 11.11 potassium ion channel and elevate the
cardiovasculri toxicity risk. Last, but not the least, high log /•’ value correlates to high PPB, which has an
impact on efficacious concentration of the drug. It is not all surprising that some argued that log p is the
most consequential property with regard to drugs' attrition. Historical data tend to support this notion as
well.،
Nowadays, computational chemistry is so advanced that calculated log p value of a given
mohx'ule is readily acquired. Clog p. for calculated log p. is a daily vernacular of drug discovery, ten the
simple, ubiquitous Chelidra«■’ program can calculate Clog p ٠ and topological polar surface area (tPSA)
with a click of hutron at Show Chemical ١P,-٥۶،»,"„'٠4 ivindow under 1','ew.
/’ and Clog p are adequate in quantifying a ding’s lipophilicity for neutral molecules. However,
it is more complicated «'hen it cotnes to ionizable acids or bases because their concentrations in octanol
and water vary depending upon the degree of ionization (Figure .3.21. The significance of acid-base
properties in drug discovery has been '.veil documented.0 For acids and bases, distribution coefficient D
is a more appropriate measurement of lipophilicity at a given pH. It is a function of both lipophilicity of
the un-ianized compound and degree of ionization.

Figure 3.2 Partition of an acid or a base between 1-octanol (o) and

water (w,.
For an acid.
I!٨.٠٩ ‫ د‬H٦٧ - A-« (3.3)
(.3) ‫؛‬HAJ„--[A٠J١١]‫؛‬,D---H
 [here are drugs with Wo or more basic nitrogen atoms. Cetirizine (Zyrtec, 9,. an ant ‫؛‬-
histamine for treating allergy, possesses two nitrogen atoms. Sitagliptin phosphate (Januvia. 10). for the
treatment of diabetes mellittis type 2 (I7MT2). has five nitrogen atoms. ١Vhy the prevalence of nitrogen
atoms in many drags?

cetirizine (Zyrtec. 9) sitagliptin pliosphate (danuvia, 10)

In order for a drug to pass through cell membranes, a dichotomy is at play. On the one hand,
the drug shottld be slightly hydrophilic so that it can dissolve in water. On the other hand, it should be
somewhat lipophilic so that it may cross the cell membranes. Amines fit the bill well. Amines' pA'.i
values are in the range of ‫ ة‬to 8. thus they are partially ionized at blood pH 7.45. They can easily
equilibrate between their ionized and nonionized forms with a good balance of the dual requirements of
water and fat solubility'. They can cross cell membrane in the nonionized form. ١٦٠hile the ionized form
gives good water solubility and permits good binding interactions with its target's binding sites. Striking
a balance of lipophilicity is one of the ding design conundrums.

(.‫ثم‬ ‫دزل‬-)')','froge,,
Qomliii■:/
Hydrogen bonding influences interactions between a drug and its target such as a receptor or an enzyme.
The oxygen and nitrogen atoms on the ding serve as hydrogen bond acceptors, while the OH, SH. and
NH groups act as hydrogen bond donors.
Not only is hydrogen bonding ciuciai to a drag’s potency but it also contributes significantly
to its physicochenrical properties as well. For a drag dissolved in water, intermolecular hydrogen bonds ١
١٠ith each other are virtually nonexistent behveen drug molecules themselves, which are
overwhelmingly surrounded by water molecules. To form a hydrogen bond between a donor and an
acceptor, both must first break their
 bonds with surrounding water molecules. Because most oral drugs are absorbed by
transcellular absorption, neutral molecules are favored over solvated molecules. However, desolvation
and formation of a ■■naked" molecule is not favored thermodynamically if the compound forms many
hydrogen and/or ionic bonds with water. As a consequence, clings with too many hydrogen bond donors
and/or acceptors experience difficulty getting from the gut into the blood. In 1988. Young et al.
investigated the role hydrogen bonding played in the penetration of antihistamines into the central
nervous system (CNS).s They concluded that excessive hydrogen bonding prei ented access to the CNS.
With regard to penetrating die cell membrane, carbohydrates, metal ions, neurotransmitters,
and insulin are exceptions to the rule because they are absorbed with the aid of active transports. See
Section 3.2.4 for more details.
Intramolecular hydrogen bonds on drugs are more readily formed ill water since they are much
more favorable entropically. Intramolecular hydrogen bonding frequently boosts cell membrane
penetration. For instance, amido-carbamates 11 and 12 ha١'e identical PSAs, yet compound 12 [P٠A-B٠
= 4.3 nms) is four-times more cell-permeable (Caco-2) than 11 [PapiHA- Bl = 117 nmj by virtue of the
intramolecular hydrogen bond.9 Caco-2 permeability assay is a popular cellular method. The Madin-
Darby canine kidney (MDCK) cell permeability assay is also frequently used.

Another well-known case involving intramolecular hydrogen bonding is cyclosporine A (CsA.

13. molecular weight 1206,. !Measurements of partition coefficient ‫ م‬indicated that the hydrogen bonding
capacity ofCsA( 13) changed dramatically from in an apolar solvent (where it is internally hydrogen
bonded) to ill a polar solvent (where it exposes its hydrogeti bonding groups to the solvent).‘٥'٦
Cyclosporine A (13) is one of the very few macrocycles (including avermection. midccamycin.
and rapamycin) that possess good oral bioavailability. Most macrocycles with many polar groups do not
cross cell membranes because they are too polar, but cyclosporine A (13') does due to the existence of its
four intramolecular hydrogen bonds, ivhich lock its conformation and raise its log p. This phenomenon is
dubbed as chemical chameleon or cyclosporine A chameleon, insinuating that CsA (13). normally a polar
compound, “disguises'■ itself us a greasier molecule CsA (13") by forming intramolecular bonds in order
to cross the cell membrane.
Appendix (2): An excerpt from tlie book “Organic chemistry”, Clayden, G reeves,
and Warren (2012)٠
ACIDITY, BASICtfV. AHO p،,
All isolaioil proton Is extremely reactive—formation of 11,0' in water
tiaseotis H،:lls not an acid at all—it shows IKI tendete) to dissociate into 11 and ( ‫اذ‬٠ as the bond Is stronfi. Bill hydrochloric acid■— that is, a solution
of IK:1 In sealer—is a strong add. Tile diflcrence is that an Isolated prolon II' Is trill unstable to lie encountered under normal conditions, but in waler
Ilie hydrogen of 111:1 Is transferred to a Slater molecule and not released as a free species.
 'Use chloride anion Is the same In both cases: the only difference is that a very unstable naked proton would have to be the other product in the gas
phase but a much more stable II ,‫ ا‬cation would be formed In water. In fact It's even belter than that, as other molecules of water cluster round !'solvatei
the II,،)' cation, stabilizing It with a network of hydrogen bonds.
flial is why H( :1 is an add in waler llul how strong an add Is it? This is where chloride plays 3 role: liydrochlorlc add is a strong add liecause chloride
ion :‫ ؟‬a stable anion. I lie sea Is full of it! Water Is needed to reveal tile addle quality of HCI. and acidity is delennhied In water as tile standard solvent. If
we measure acidity 111 water, what we are really measuring Is flow much our acid transfers a Iiroton to a waler molecule.
HCI transfers its proton almost completely to waler, and is a strong add. But the transfer Ilf protons to waler from carboxylic acids Is only partial. Thal
1‫ ؟‬why carboxylic acids arc weak acids. Unlike the reaction of IICI with water, the reaction below Is an equilibrium.
Hie pl, scale anti pK,
Hie amount of 11,(1 in any solution in water is described using the pH scale. pH is simply a measure of the cmicentrallon ol H,l>' on a logarithmic scale,
and it is characteristic of any aqueous add—it depends not only on what Hie add is (hydrochloric, acetic, etc.) but also on how-concentrated lhe add Is.
« pH Is the negative logarithm of Hie ll٠0" concentration.
pH = -log|H،O٠١
١'ou will already know that neulratily Is pH 7 and that below pH 7 svalcr is increasingly addle while above pH 7 it Is increasingly basic. At lilglicr pH,
there Is little H ،٠(‫ ا‬In the solu- Hon and more hydroxide Ion. but at lower pH there Is more II ,،) and little hydroxide.
 Hie reason Ilia( higher pH means less 11,،)• is because the arbitrary definition ol pH is
tile «‫؛‬Xlffvclogarithm (to the base It), ol Hie II,،)’ concentration. 1'0 summarize in a
‫ ئع’ه‬-‫واح‬، e sii diagram: espia r,'، » □ top al pH‫ ؛ ال؛‬n s ssale
seems‫؛ ؛‬niimliK 1'0 ,.1-1-11‫؛‬٥٤٠
are appio«'mate. Put easy to -'--'-—I'-
 Aciolrv, BASICITY. AND pK,
1،-,، die ،،,Iler ،،,،1,1.1 II،' actinga،،،11 and. Il'،aimi'lng I،، noli،■،• dial hydrogengas،, ‫؛‬
1I،,. ,Ill
Is the coniugale ,Kill ،،( hydride Ion. bill mote 11111)0(1,1111 10 note 111,11 hydroxide ion
IS die conjugal،■ l،a،e ol w-alcr.

. ()11 much •،،‫؛‬ak add and« weak base so we need a ،,,Olig acid like ll،:l tog'١،ater Isa I،،
and a.slmng liase. like hydride ion. to give nuicli hydroxide ion.

،.11’-. ،٥ 111'oniaati٤٤٦‫؛ ب‬
at III - ،no, dm-', bure waler al ١»١w in،te،Ions tn ،cater 1، very l ١٠) Ihe coiweniradon ol 11
c therefore has a ill of 7.0(1. ilydronlum ions In ,lure waler ،-.111 .iris،' only from waler2 ‫؛‬S
protonating land deproionalliig, lisell. One mole ، Ilie of water acts as .1 base,
deprotonating another dial acts a ، an acid, l or every 111،,' ion formed, a hydroxide ion
must also be formed, :111,11،-، ،0 dial In pure waler at ill 7 tile concentrations of If.,،) and
liydroxide Ions must be
٠‫؛‬l(,- nioldni|"IIO|-٠’,'ll,O|

I he product ،.f dies،• Iwo concemraiioih IS know n .١١ Ilie iuliUiitbvi .1'11،'.Ilir (or .1'
die :('Illi
1 constai( in a،i،ieou25 ٥٤ ١. ‫ ا‬111، 1، ‫؛‬١ mbnfi of water. K,.. with .1 ،alue of 1(1-" mol' dm-
(.It,,
olutions. so if we know the hydronluni ion conccnlraiion (Which we can gel by measuring ،
the pill, see also know- die hydroxide concentration sin ،■،• 111،■ produci of the two
concentra- lions always c،,u٥ls IT", what plldoes waler become mostly!،،,' ionsand al what
pllmosily hydrox• ،٥ So. roughly can now add two additional pieces ol infoi malion IO the
approximate char, we ١١٤ ’.Ide tons abolii pl I (,. the concentration ، o( ١‫ا‬. ,،..limosi erilliely
II. gave you lieforc. .\1 pl I 7. water 1١ waler .111111,(1 Ions arc almut 111،- ،.line and ,11
aiutili 1,11 H. die ،-,mccntrailon، of hydroxide ,ar،' about the same ]٦٦٦٤٤ Ion، and
 Th0 ‫؛‬،F, IITIOfi
OFthepK.
o, |>K٠. |1K, i١ the• log Ito til،■ base ten, o، the cciirtlibrum, constant lor dissociation ol
the adcl. lor ,11, .,،'ill 11A tills i١:

I he concentration ol ,rater is ignored II, the definition because it is also constant (al 25 ‫ا‬
٠)٠ liecause of the minus sign in th ،• definition III'‫ ؛‬there too in the definition of pill the lose i is water. Ore‫؛‬،t،٠:HO.'،
c٠c«n sate: has a trass
the pKj Ihe larger the equilibrium constant and the stronger the acid. You ma‫ '؛‬lind the ,sa‫'؛‬
UM0‫ ؛‬٠ male
sre Introduced pK, more helpful as a'concept for visualizing pK,: an‫ •؛‬acid Is hall
dissociated In a solution srhose pH matches the add's pK,. Al a pl l abos'c Ihe pK ، the add ollSgaad occupies 18 cm'.
exists largely as its coniugale base <A’I but at a pH belo,,' the pA the add largely exists as So. in I dm-‘, there ،re
lOUDlS ٠ SSSfr mol. Wat،!
HA.
tvitli pK, ١١e can put figures to the relative strengths of hydrochloric and acetic acid ,re 1، a 55.56 mol dm-'
introduced earlier. 11(1 Is a much stronger add than acetic add: the pA ol H(.'l Is around -7 solufcr, ol water, io',ale:
compared to 3.76 for acetic add. ■fills tells US that in solution A for hjdrogcn chloride Is It»:
mol dm-,. I his is an enormous number: only one molecule in 111,(IO().()()(! Is not dissoci-
ated, so It Is essenllally fully dissociated. Bui A for acetic add is only H)-'»1.74 ‫؛‬٠'٠ X !(,'،mol
dm", so it is hardly dissociated at all: only a fetv molecules in ever ‫ ’؛‬million of acetic add
are ,,resent as the acetate ion.

What about the ‫ ردب‬ol water? ١’ou know the figures ahead‫•؛‬: A for water Is 111,0'1 X
1110'17
0,( see why water Isn't really quite‫ ؛‬ow'١. .15.7-٠ logli() 1*755.51 - ٥ 55.5. So pA/٠'-«,l ٠ 11.01‫؛‬
hall dissociated at pH 14—the concentration of water in the equation means that the two
ends of the scale on p. 168 are noi al () and 14, but at -1.7 and 15.7.
A yraphic I deccription 0، 41,0 of acids anti bases
for boll, cases, aclustlng the‫ أزا‬l alters the proportions of the add form and of the coniugate
base. I he graph plots the concentration of the free acid All (green curve) and the ionized
coniugate b.sse ٨- tred c urve) as percentages of the total concentration as the ‫ ااار‬Is
varied. At low pH the compound exists entirely as ١11 and at high pH entirely as A". Al the
‫ د'د(ا‬the con- cenlrallon of each species, ،٦11 and A', Is the same. ٥١1 pHs near lie pA Ihe
compound exists as a mixture of the t VO forms.

Mow ١٠٤ have established wli‫ ■؛‬you need Io understand acids and bases. SVC must
move on to consider why some adds arc stronger 11,,,1, other adds and some bases
stronger than other bases lodolhistscniust be able tocstiniatethepAol common classes of
organic compounds.
 ACIDIIY,
QASICIIYiAfJO
pKj

Nitroyen compounds as acids and base.‫؟‬

I ire no, Important organic nitrogen compoumts nr،' mines and amides. Inline nitrogens
،tin lie joined to alkyl or aryl groups (In which ،'،IS،' Hie amines tire called anilines). They
all have lone pairs on nitrogen and may have hydrogen atoms on nitrogen loo. As nitrogen
Is less ،'leclronegatlve than oxygen, you should expect amines lo be less acidic and more
basic than alcohols. And they are. The pK, values for the protonated amines are about 10
this value Is about () for ivater and alcohol) and the pK. values lor amines acting as acids
are very high, something like 35 (compared svith about 15 for an alcohol). So ammonium
salts are about as acidic as phenols and amines ssill be protonated at 1,11 7 in water. This
Is why amino acids 1)1.1,7) exist as zwitterions in svater.

Removing a proton Irom an amine Is very difficult as the anion (unfortunately called an
amide' anion) Is very unstableand very basic, rhe only way to succeed Is to use a very
strong base, usually an alkylllthlum. The 'anion' then has a N-l.i bond and is soluble In
organic sol- vents. This example, known as 1.1 A. Is commonly used as a strong lias ،■ In
organic chemistry.
■file basicity of amines as neutral compounds Is measured by the pKj of their conjugate
adds—so, for example, the PX associated ,،ilhihe protonation of Irlethylamlne.acominonly
used tertiary amine. Is 1.0).
lli٠'|)ff١s' of bases
Chemists often say tilings like the pK, of trielliylamlne Is about It).' (It's actually 11.0 bill 10
is a good number to remember for typical amines). This may surprise you as triethylamine
has no acidic hydrogens. What they mean is of course this: 'the pK, of the conjugate acid
of triethylamine Is about IO." Another way to put this Is to write 'the pK.,„ of triethylamine
is about 10.' ■file subscript 'all' refers 10 the conjugate acid.
Ils OK 1« say the pK.
٠( trtethylamina Is atout 10' as
long as you undorsland ’.hot
When a molecule Is linth acidic and basic. as for example aniline, it Is Imporlanl lowork
out which ph',, is meant as again chemistswiial is really
will loosely refermeant ISof'the
to'the pA", pX,Is !.6
aniline
when they mean 'thepA', of the conjugate acid of aniline is 4.6." Aniline is much less basic
of the Irtolhyiammonluin ion IS
than ammonia or triethylamine because Ihe lone pair on nitrogen Is conjugaied Into the
ring and less avail- able for prolonation. oboul 10'. villici' can also bo
expressed Idus: the pK٠H of
trielhylamlne is about 10'
Appendix (3): An excerpt from the paper ،' Acidic and basic drugs in medicinal
chemistry: A perspective”, Charifson & Walters, ،‫ ل‬Med Chern (201 4)٠
Journal of Chemistry
Medicinal mmary: Effects of Ionization State on DMPK
،’able i. Properties
Literature St, key observation, f،un١ ref، reviewed
paramete - Ionized fonn of a drug Is considerably mere water-
r soluble with adds generally more soluble than
solubili/ bases, possibly due to an overall greater extent of
rp efflux‫'؛‬li,y/P Ionization for most acids at physiological pH.
reviewed
4٧‫؛‬،p،n،i - Solubility affects almost all aspects ٠f drug behavior
«٠٢,
٠ and characterization from in vitro assay
J. 2. ٤.6
or,,l reproducibility to bioavailabiEty and formulatability
1,5,6,
atnarpiiou/ (sec Form/Formulatability discussion).
16,18, r
bivavulalutty - Ionized molecules at physiological pH tend to
t. 5. ٥٠.٩٩
du interact with ne‫؟‬tì٧y charged lipid membranes
1.5. ،. 24
' ٠ generally resulting in alow permeability: neutrals >
٠٩٥ ,
vclunte ٥,٠ 1٦‫لد‬،‫ ) ه‬zwitterions > acids.
4,5,29
JklrtbuHon - Increasing lipophilicity may increase permeability,
l, 4-6,29.50
£‫؛‬١i.prvfcls especially for adds, bases, and zwitterions.
‫ج‬.٦.H, .،,،,J
t>u٠ - p٠gp efflux ١vas influenced to some extent in the
9-J6
dull . following order: zivittcriono neutral ‫ تي‬bases >
،,‫؛‬.‫؛‬.‫؛؛‬
‫'خف‬-‫ا‬. adds: the number of HBD (as sveli as MW) might
1.
metabolism be the predominant factor.
- Adds generally have higher oral bloava ،lability than
bases in spile of poorer permeability, possibly
because of better solubility and lower clearance.
- Aridic and neutral drags may tolerate greater
lipophilicity; a potentially interesting measure of
lipophilicity for ionized species with regard to oral
absorption by passive diffusion ‫ ئ‬the distribution

7‫ا‬ ‫اد‬٠.‫رالعذ‬07-97‫ا‬
coefficient (log D) at pH 6.5, which is the

‫اإ‬،‫راد„م‬. ‫ثم'ع‬٠‫؛ و‬6,٠٠٦

‫‪٠١0‬سدا‪4‬‬
Appendix (4):dournal of
An excerpt Chemistry
from ٠. , to
the book "An Introduction - - . . m
’!'able
Medkinall. key obkffution» front ,of» mie١«٠،٠d Iff»
Medicinal Chemistry”,
continued Graham
ironwl Patrick (2013).
٠ In the ،aw ofN-oxidation. when‫ د‬molecule possesses morethan one
l»»r٠n١c١c
r basic nitrogen atom,agents
Antibacterial the preferred site cell
which inhibit is generali),
wall the most basic
٨ center. synthesis ‫؛؛رم‬
٠ CYP2D6 tenth (o prefer basic substrates; basic compounds tended
to also be the most potent inhibitors, followed by zwitterions, neutral
molecules, .,nd ،inally acidic molecules.
٠ cyi>2(;9•prefers neutral aitd acidic substrates, and these same
o-Al»A is now produced more the greater the nctivit), but the
ctlicicntly by hydrolys- ing penicillin ( greater the instability of the
‫ ت‬or penicillin ١" ١١٠itb an enzyme molecule to other factors;
‫ مج)ذ‬ionized at pH 7.4. It should be noted, channel, etc.) presents an opportunity to
(pcnicil- lin acylase) (fig. 19.21), or by . the acylamino side chain is
honvever, that even a relatively small examine the relation* ship between
‫ د‬chemical method that allows the essential;
degree of unionized species may, in some protonation state and target class. An
hydrolysis of the side chain In the ٠ sulphur is usual btn Itot essential
case،, be responsible for observed analysis of Inactivity data from the
presence of the highly strained ،‫؛‬- (see section 19.5.3);
btolugical/pharmacologkai effects, thus ChEMBL database with a reported IC*»
lactam ring. Ihc latter procedure IS - the stereochemistry of the bicyclic
complicating some of the following EC or K٠ value was performed, wherein
described in more detail In section ring with respect to the acylamino
analyses. each compound was classified as an
19.5.2.2. where it is used to side chain is important.
We used the pK, plugin in version 6.2 of acid, base, neutral, or 2١٩ittenon.
hydrolyse the side chain from Ihc results of this analysis led to
ChcmAxcn's cxcak program" to (?Jculate Figure .‫ أ‬shows the distribution of
cephalosporins. the inevitable conch،- sion that very
ph values. ٦٦١٠sc pF،', values were tlicn protonation states across all target
١١’e have emphasized the drive to little variation is tolerated by the
used to eakulik- percent bnizatiun and classes as well as the four most
m.١ke penicillin analogues with penicil- lln nucleus and that any
classify molecule as aciilx, bajic, neutral, or populated classes. It was found that
varying acyl side chains, but what Is variations are restricted to the
zwitterionic. A recent pul licatJon there were a greater relative proportion
so special about the acyl side chain? acylamino side chai،,.
by .Settimo : evaluated the accuracy of the of basic compounds among membrane
Could changes not be made 19b.J.hPenici!fa١ai١l٠e5
ChcmAxon pK، predictor against ‫ د‬number receptor and transporter targets while
el.sewherc in the molecule? In order In this section we consider the
of !)harwaceutreally relevant data s،٠K Ilk' neutral compounds predominated among
to answer these (]ue.slions we need penicillin analogues which proved
authors reported ‫ د‬median absolute enzyme and ion channel targets. Io
to look at the structure-activity successful In tackling the problems
deviation ensure that this observation ‫ الم‬not
relationships (SARs) of penicillins. of acid sensitivity, ،‫■؛‬lactamase
‫ دحرء الد‬Wc'١١ .nd 0.31-064 for bases‫؛‬، M\ strongly, biased by intrinsic activity, we
،‫’ًاأت‬٧‫اا‬٠‫ ك‬٢‫’ع‬،‫األ‬0:٦‫ جزا;ا؟خ‬sensitivity, and limited breadth of
D) of 033-0.37 for acids) plotted the data using two different cutoff
‫> ؛‬١)SllbCltH ‫ان ن ون‬. activity.
prediction methods are 'unpcrfvrt, we values, one allowing only highly active
believe that the reported resolution is compounds (less than 100 nM, 361 104
٠7Q،a(٠?١۶«97٥ .sz .‫ا‬0>:‫دؤ‬،‫أمن هألدر‬١ ‫اهد‬٠
‫مهانم‬٠٢‫ال‬١.٠‫ها‬1٠‫ح‬1‫ؤم‬،‫قاا‬0
 Chapter 19 A’١tibacrci٠٠،
٦f agents

i٦it،tu»i٧><١١ rini' under acidic ٠,،٠٠,,١١, the p ،٠،

Ring-opening

R ١٤ ١ "۴ JR. ‫م‬ R 8

To, tia ٩٠ ‫ه‬٠٠ ٠٢ “•NR? ٦ ٠‫غ‬
٥,n:de 4“

Me
‫ه صح‬ ‘، ‫المحنح ■■جح‬
Folded ring structure ‫شهتزه‬
٠10‫>د‬
.I gloups ١i١١،c٠١rb,l.ii١ ،,l‫؟‬٠|
amide .,I,d ٠١,،>’،/,CompariMU, ot re
more electrophilic »!١٠١,١ one would Other penicillin ،,,,.,logues with an
expect tor ‫؛‬١ ter- ti٠,ry amide. clc<tfon٠٠٠١٦th
full• limili (neighbouring group ،,،٦١•‫ ؛‬,,on the ،،carbon oftlic side chat (٠٤
‫ ا‬٠١‫و‬٠٠‫اامم‬٠,'،,biffi . Illg ،>،il١stitu،m \،١.dr
the *demonstrates li(i .‫ذذ‬ ١١ ١is.١ly،to AC,،( l١ydr ١ist٠u٢،'.١١
19 .participation): fig ٠١ ٤,١٠١cti١ely prwed ١،١ve a١s،h ١9 25١ ig-‘١
participate group can ‫ؤاد‬٠‫ ل‬.,19.29 .en orally (e.g ainpiclttin: see
neighbouring coi-,‫ ؛‬,mechanism lo I ig’and can be gi١ conclude, the
open up the betam ring. Ihus self- problent of acid sensitivity is fairly ٠,
destruct mechanism l١uilt into it.s ٦. l١. ٠٠ elcctRmwithdrawmg group ٠١,١
٦s ,) ١،,c,lt Ciwily sotted by having
structure. ,ide chain،‫ ؛‬cyl,‫ ؛‬on the
١t countering acid se sill'.ity is a dirti.‫؛‬It the problen, of pia،tan١ases (or
can be seen tl‫ا‬١١١١ ‫ اآلل‬١ be done penicillinases! become critical in
 Antibacterial agenls which inhibit celt wall
synthesis

,of penicillins ‫؛‬Inlluence Ilf ,11 acyl side ،11 Ilin on

Ilie acid sin licit 1 ‫؛‬،!‫ ا ا درب‬,:‫؛‬

.(٠B١\-٠-،) group ٦١ ،|٧‫ء‬1٢‫ال‬٠‫ا‬-٠١-‫اا‬1‫الل‬٢‫د‬٦١-‫ال‬٦‫ه‬Hh an roup putlicipailou‫ ؛؛؛‬Kduvthm ol

neiglibnuring ‫<ًا؛‬.‫ تأي‬riSURE

to bo done tn find die Ideal shield— Fortunately, shields Here found

one large enough to ivard off the which could make that
lactamase enzyme, but sufficiently discrimination. Methicillin (Fig.
small to alloivthe penicillin to bind to 111.27) was the first effective scmi-

of steli، shields to blocking penicillin from reaching the (!■lactamase active site -،.‫؛‬
11.2? ’Ilie It ‫ا ك؛|اا‬,Hi
Appendix (S): An excerpt from the paper “In Vitro Metabolism and Covalent Binding of
Enol-Carboxamide Derivatives” (2008).
Chnn. Res. Toxicol. 2008,?/. 18901 899

In Vitro Metabolism mid Covalent Biding of Enol-Carboxamide

Derivatives and Anti-Inflammatory Agents Sudoxicam and
Meloxicam: Insights into the Hepatotoxicity of Sudoxicam
R. Scott Obach,* Amit s. Knlgulkar, 'fini F. Ryder, and Gregory s. Walker
PhafmucoWncfics. Dwamlcs, autl M،tfl١١٥١u‫؛‬n Deparuucnt, Pfizer Glubal Research n٠١o Develapiuei ‫ا‬
Groton. Connecticut
R،c،t„«t May ?008
Sudoxleam ami meloxicam arc nonsteroidal anti-inflammatory dmgs (NSAIDs) from the enol- carboxamide class. While the only slnictural difference
between tltc two NSAIDs Is the presence of a mclbyl group on the C5-position of the 2-carboxamidolliiazole motif in meloxicam, a marked difference in
their toxicological profile in humans has been discerned. In clinical trials, sudoxicam was associated witli several cases of severe hepatotoxicity that
led to its discontinuation, while meloxicam has been in the market for over a decade and is devoid of hepatotoxicity. In an attempt to understand the
biochemical basis for the differences in safety profile, an in uitro investigation of the metabolic pathways and c ovalent binding of the two NSAIDs was
conducted in NADPH suppleincntcd human liver microsomes. Both compounds demonstrated NADPH-dcpendent covalent binding to human liver
microsomes; however, the extent of binding of (,4c !-meloxicam was ■٧2-fold greater than that of ('Csdoxicam. While inclusion of glutathione (GSH) in
microsomal incubations resulted in a decrease in covalent binding for both NSAIDs. the reduction in binding was more pronounced for meloxicam.
Metabolite identification studies on ['4Ci-sudoxicam in NADPH-supplctncntcd human liver microsomes indicated that the primary route of metabolism
involved a P450-n٦ediated thiazole ring scission to the corresponding acylthiourea melnbolite (S3), a well-established pro-toxin. The mechanism of
formation of S3 presumably proceeds via (a) epoxidation of the C4—C5-thiazole ring double bond, (b) epoxide hydrolysis to the corresponding
tltlazolc- 4,5-d!hydrodiol derivative, which was observed US a stable metabolite (S2). (c) ring opening of the thinzole- 4,5-dihydódiol to an 2-
oxoethylidene thiourea intennediate, and (d) hydrolysis of tltc imine bond within this intermediate to yield S3. In the cnse of meloxicam, the
corresponding acylthiourea metabolite M3 was also observed, but to a lesser extent; the main route of meloxicam metabolism involved hydroxylation
of the 5'-methyl group, a finding that is consistent with the known metabolic fate of this NSAID. Inclusion of GSH led to a decrease in the formation of
M3 with ths concomitant formation of an unusual two- electron reduction product (metabolite M7). The formation of M7 is proposed to arise via
reduction of the imine bond in 2-oxopropylidenc thiourea, an intermediate in the thinzole ring scission pathway in meloxicam. In conclusion, the
results of out analysis suggest that if the covalent binding of the two NSAIDs is important to the overall hepatotoxicity risk, the differences in
metabolism (differential preponderance of fonnaticn of the acyllhiourea relative to total metabolism), differential effects of GSH on covalent binding,
and finally differences in daily doses of the two NSAIDs may serve as a plausible explanation for the marked differences in toxicity.

Introduction ,he presence of the

linol-carboxamidcs are a well- 4٠hydroxy٠2٠n١ethyl٠2f/-!٠2٠aiylthiazine-
established class of nonsteroidal anti 3٠ carboxamide !.! dioxide motif.
inflammatory drugs (NSAIDs1) with Mechanistic studies designed to explore
several marketed representatives the molecular bash of cox inhibition by
including piroxicam, tenoxicam, enol, carboxamides suggest that the
tomoxicam and meloxicam (Figure 1). The weakly acidic keto enol group (pjfca 5.2)
biochemical basis for anti-inflam- ntatory at position C4 functions as a carboxylic
action of enol-carboxamldcs involves acid bioisostere and inhibits cox activity
reversible inhibi-
t(».l02l/rx800l85b tion of ©cyclooxygenase
CCC: 340.75 2003 Americanby bindingSociety
Chemical to active site on
Published amino acid
٦٧٤ 08,10*2008
Ill Vitro Metabolism and Covalent Hindiitg Studies Chern. Kes. Toxicol.. Vol. 21, Nt). 9. 2008 1893

Figure 1. Structures ofcnol-uirboxainidc

observations
class 0٢ NSAlDs. on sudoxicam
to the vastly
hepatotoxicity superior safety profile of
are in stark contrast
meloxicam (4hydroxy٠2٠niethyl٠A/٠(5-
l,2٠bcnzothiazine٠3carboxamide٠l,!
is commonly prescribed as an anti-
humans and veterinary ajjplications.
1995, reports of dniR-induccd liver
meloxicam have been extremely rare
Ill many cases, an in depth
basis for toxicity has been u.scful in
of a chemiccl class effect with
chemical peculiarity. With sudoxicam
.structural difference is the presence of
5-F١ticn of the thiazole ring in
However, the minor change in structure
the met bolic liofile of the two drugs.
metabolism of sudoxicam m animals
foimation of acylthiourea metaholite(s)
ring scission (Figure 1) (7). Fairly strong
thiazole ring bioactivation to
in preclinical species and/or humans
numerous thiazolc-cont lining
Following enzymatic thiazole ring
susceptible to further .oxidation (0
sulfinic acid derivatives capable of
molecules or depleting glutathione
mechanism of 5' oxidation has also
for the hepatotoxicity associated with
the basis of these studies, it is possible
derived from thiazole ring opening in
role in the clinical hepatotoxicity.
ing this proposal stems from the
metabolism ill humans, which
thiazole nng scission; instead, oxidation
methyl group to an alcohol and
(Figure 1) IS the principal metabolic fate
humans (14). While this hypothesis
experimental evidence to support It; all
circumstantial. Consequently, we
1 y side comparison of the in vitro
Ill Vitro Metabolism and Covalent Hindiitg Studies Chern. Kes. Toxicol.. Vol. 21, Nt). 9. 2008 1893

Figure 2. Radiometric ،•htomaioxrams of NADPH-supplemcnted human liver microsomal incubation mixture‫؛‬، of

sucloxicam and mcloxicinn. Key: SI =3 ttnehnnged MKloxicanr. S2 rr sudoxicamdthydiodiok S3 ‫ تع‬thiourea
meltibi)lite;S4 = oxidized thioureametabolite; Ml ‫ اح‬unchanged meloxicam: M2 ٥ 5'٠h)d roxynteloxicam; M3 ٥ thiourea
metabolite; M4 ‫ ع‬oxidized thiouiea metabolite; M5 — cyclized metabolite; M() ‫ ئ‬oxidized meloxicam rnctjibolitc; M7 ٥
reduced open-ringed nteloxicam metabolite.
 1894 Chcm. Hex Toxicol., Vol. ‫د‬,, No. 9. 2008 Obach Ct al.

Figure .١. CH) .spectra of Midoxiciim nnd S2. ٠ Figure s. CID spectra for metabolite S2 containing ”o (mlz

٩.١٠and S4 metabolites. 372). S4/M4 generated from S3ZM3 m ocid and containing
IMO (ink 330‫؛‬. ،M7 containing deutenum (369 ‫لرهم‬/٠:
generated in deuterated water), and M? after reduction
with NaBD، (ink 371).

Figure 4. CID spectra of ineloxiciun and M2, M5. M6, onci

M7 metabolites.
In Vitro Metabolism and Covalent Rindinst Studies Chem. Res. Toxicol.. Vol. ‫ذو‬, No. 9. 2003 1895

Figure ،5. Radiometric chromatograms of S3 incubated with acid. base, and in a liver microsomal incubation.

Table 2. n NMR Chemical Shifts (ppm) for Mcloxlcum, M2, and M7

proton، meloxicam M2 M7
٨rH(٥) 8 02 (7.8 ‫ ه‬٠/٥ Hz. (111 .7.89 (d, 2 = 8.3 8.01 (7.8 = 2 ‫ه‬
bcd 11‫را‬ Hz (،،1 .7.45 (d, 2 = Hz. HI)
cf (٤1! .7.7، (m 7.8 Hz 7.16 (،n, IH) 7.73 (m. til)
£ 7.6، (m. 2H, 7.14(m.JH) (311 .2.75 7.64 (m. 211)
(،!‫ل‬.،)2.74 (Hi ,١) (s (11 .،)11.8 14.5(.،. 2.73 (s, 3H) not
1896 Chan. Krr. Towol.. Vol 2،. ,Vo. 9. K Obath « ‫'ال‬

^'Igure 7. Propense(! mcloboli، p3ihv..٦<١ for

MMloxicnm in h٠٠n liver ‫اله‬،٠‫د‬.

Hgnrt X. Proposed inelabolic pc٠.h١٧ays 1*0( ine ١٠

١lc ۵ m in hunun liver m٠Kr٠،$٥itK>٠
bl Vitro Metabolism mid Covalent Binding Studies Chein. Kes. Toxicol., Vol. 2/, No. 9, 2008 1899

Discussion
Studies addressing (he biochemical basis for the hepatotoxicity and/or renal toxicity of ?.-amino- and/or 2-catl xamidothiazolc- containing
xcnobiotics have provided .strong evidence for the bioactivation of the thiazole ring system as a contributing factor in the undcilying toxicilies
associated with these compounds (9 ‫)ه‬. In a manner .similar to other 5-mcmbeted ring hetero- cycles such as thiophenes and furans (/ ٥), thiazole ring
bioactivation involves oxidation on the C4-C5 ring double bond to generate an unstable epoxide: spontaneous hydrolysis of which leads to the
corresponding dihydrodiol intermediate. Ring scission of the dihydrodiol then occurs, resulting in th( ‫ ؛‬liberation of glyoxal and the corresponding
thiourea and/or acylthiourca as metabolites. Once formed, thiourea and acylthiourea me- tabolites can undergo . ‫؟‬-oxidation to electrophilic sulfcnic
and/ or sulfmic acid intermediates that can covalently modify or oxidize critical proteins leading to toxicity (8. 9. 12). A similar mechanism of toxicity
has also been postulated for thiobenza- rnides (/.?, /7). Thus, it appeared reasonable to speculate that the idiosyncratic hepatotoxicity ofsudoxicain in
humans arose from mciabolism of its thiazole ring to fonn reactive intermedi- ates. The argument becomes even more compelling since meloxicam is
not hepatotoxic, a feature which appears to be consistent with the lack of thiazole ring opening as a metabolic fate in humans.
However, to our sttrprise Iroth sudoxicam and meloxicam demonstrated metabolism-dependent covalent binding to liver microsomes. The extent of
microsomal covalent binding by lite two NSAlD.s even after incubation at 50 / ،M was much lower than that reported for prototypic hepatotoxins
(typically >50 pmol/mg of protein at substrate concentrations of - 10 However, it is noteworthy to point out that covalent binding rates estimatcxl in
these studies need to be ,»laced within context of total metabolism of the (iiug. If a drug is extensively but slowly metabolized then standard in vilro
covalent binding experiments in liver microsomes may yield low levels of covalent binding. Consistent with the observed low clearance of 0.2
raUmin/kg (estimated hepatic extraction ratio of ‫ اج‬% bused on human hepatic blood flow of 20 mI7min/kg) for nteloxic m in humans (/4). Ill viivo
human liver microsomal incubations assessing ،netabolic stability of meloxicam and sudoxicam .show veiy little depletion of these NSAlDs (in film I ،6
« ‫ )(در‬min). While the measured clearance of meloxicam in humans is very low, mass balance studies reveal extensive metabolism of this ‫ ل‬tug with
only trace levels of parent NSAID detectable in urine and feces after 6 days (14). In contrast, even a small fraction of metabolism proceeding t ١١iougl١
a bioacti. vation pathway will yield higher rates for in vitro covalent binding for high clearance drags prone to very rapid metabolism. This phenomenon
was demonstrated in our recent studies O1١ the side-by-side comparison of covalent binding by hepatotoxins and nonhepatotoxins (/8).
In the absence of GSH. meloxicam exhibited greater binding in liver microsomes than sudoxicam, but the trend was reversed when GSH was
included. The greater binding demonstrated by meloxicam when compared with sudoxicam probably reflects a subtle increase in the rate of P45O-
catalyzed metabolism of meloxtcani While the finding that GSH attenuates covalent binding (O hepatic tissue is usually consistent with the scavrng-
ing of reactive metabolites by the sulfydryl nucleophile, in the
bl Vitro Metabolism mid Covalent Binding Studies Chein. Kes. Toxicol., Vol. 2/, No. 9, 2008 1899

present case, no GSIl conjugates derived carboxy«، acid) constitutes the major route
from sudoxicam and/ or mcloxicant were of clearance of meloxicam in lilt/ in
olserred in NADPH-suppleniented liver animals and humans accounting for >7O‫؟‬f
microsomal Incubations, l or many of meloxicam metabolism (/4, ‫ )وثم‬which
thioureas dial covaicnily bind to may divert much of the do.se of
microsomes, addition ot as» abolishes meloxicam away from reactive
covalent binding via reduction 0٢ Ihe intermediates. Meloxicam epoxidation
sulfcnic acid metabolites and Ilie appears (0 follow (he SIC rearrange- men(
concomitant formation of GSSG (/9). Thu pathway to the acylthiourea, but in the
OSH can be involved in the detoxication of presence of GSH reduction of (he imine
thioureas as a cellular reductant for their intermediate appears Io occur, a
reactive intermediates. It has been difference from what was observed in the
speculated that depletion of cellular GSIl metabolism of sudoxicam. Il IS not
pools in mammals via continuous presently known if this reduction is
exposure of thiourea intermediates chemical or enzyme• catalyzed. While not
contributes to toxicity (20). The finding that reported in humans, ring opened acylth•
the toxicity of 2-am‫؛‬noh azoles is iourea metabolites of meloxicam similar (0
exacerbated in mice depleted of GSIl by M7 have observed in rat in w (15). Finally,
treatment with buthionine sulfoxtmine the acylthiotrca carbonyl is positioned
supports the protective role of the sulfydryl such that attack by (lie ,,-hydroxy group of
nucleophile against the toxico- logical the MmcthyM^benzothiozin 1,!•dioxide ,‫؟‬
response (27. 22). In the present study, ng occurs (sec Figure S). ،Such ring
two changes, however, were observed in formation was readily observed for
the metabolic profile of meloxicam in meloxicam (i.e., metiibolite MS), hut was
human liver microsomes supplemented only seen in race amounts in sudoxicam
with NADPH and GSIl. First, metabolite incubations. A corresjionding cyclized
Md, which is derived front oxidation of the pyrimidine• diane (hydrolysis of the
acyllhiourca metabolite (M3) was no longer thiourea to a urea) metabolite of
present and a new metabolite (M7) that IS meloxicam has been reported in vivo in
proposed to arise via a GSH- dependent die rat (15). It is of interest to note that
reduction pathway was observed. For such a cyclization has never been
sudoxicam. Ihe generation of metabolite reported with sudoxicam. Reasons for this
bl Vitro Metabolism mid Covalent Binding Studies Chein. Kes. Toxicol., Vol. 2/, No. 9, 2008 1899

an acetaminophen analog. ١vhi،i١ ،lc.١pi‫؛‬e mcthylpropyhthiouic، species

covalently binding to microsomal ptolems, comparison and identification of a novel
is devoid of the hepaloloxic effects thiocorbantidc.ghrtathio.nc adduct. Cluni.
associated with acetaminophen (2.2‫؟‬, ٥). Res. Toxicol. 10. 733-741. (‫ا‬.٩) Ji. T..
Therefore, an under- standing of adducted Ikehata, K.. Koen. Y. M.. Esch. s. w
macromolecular targets and their role.‫ ؟‬in William،. T. L).. and
modulating essential cellular processes Hanzlik, R. p. (2007) Covalent
may provide greater insight into modification of microsomal lipids !١٠٧
mechanisms of toxicity of sÉìcam. fhlobcnzomidc metabolites in vivo. cium.
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Goodwin. 1). c., Katcuikj■. ،٩ G.. Kaxchkc, .‫؟‬.. and Kummer. M. (1995)
‫ا‬-Arachidonic ."،id oxygenation by COX ( Mcloxicam. Phanmicokinciics and
‫ )وووا‬٨ .and Lfinzo. c metabolic pattern after intravenous
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and inhibition.‫ د‬Biol. Chern. subjects. Drug Metnb Dispox. 27, 1206-
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(2)Rowlinson, s w., Kirfcr. L R., Prusakicwlcz, Schmid, 7.. Busch. ‫نا‬.. Tramutìi!/, c,.,
‫ل‬. ‫ل‬, Pasvlitz, 7. L. Kpzak, K. R . Prox, A.. Kaschke. s., and Wachsmuth, H.
Kalgulkar. A. Shillings ١v. C-, Kuruinbail. R. (1995) Mcloxicam: metabolic profile and
0., ‫؛اد‬،‫ مًاحل‬met t, L. I. (2003) ٩ novel mcch١biotrans- formation product, tn the rat.
٠٠;k١n١ of cyclooxygenase-, Xcnoblotica 25. ,2،9 1236.